EP4720630A1

EP4720630A1 - Means and methods for connectomic screening

Info

Publication number: EP4720630A1
Application number: EP25734260.0A
Authority: EP
Inventors: Moritz Helmstaedter; Smaro SOWORKA
Original assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Current assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Priority date: 2024-06-14
Filing date: 2025-06-13
Publication date: 2026-04-08
Also published as: CN121844194A; WO2025176917A1

Abstract

The present invention relates to means and methods for connectomic screening using 3D electron microscopy. In particular, the invention relates to a chaperone block for holding one or more biological tissue samples to be analyzed, imaged and/or screened (herein also referred to as "tissue sample of interest"), wherein the chaperone block comprises at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities. The "chaperone block" is a three dimensional structure for holding the biological tissue samples of interest during analysis, imaging and/or screening, particularly using 3D EM. The "chaperon sample" is also a biological tissue sample in which the biological tissue samples of interest will be embedded ("loaded") for analysis, imaging and/or screening, particularly using 3D EM. The invention also relates to a screening sample that can be loaded into a chaperone sample as well as an apparatus for loading a chaperone block with a screening sample. The "screening sample" is the biological tissue sample of interest that has been modified (incl. embedded in a resin) so that it can be loaded into the chaperone sample on the chaperone block for EM. Furthermore, the invention relates to methods to produce a chaperone block, methods to produce a screening sample and methods for preparing a tissue sample for connectomic screening.

Description

Means and methods for connectomic screening

The present invention relates to means and methods for connectomic screening using 3D electron microscopy. In particular, the invention relates to a chaperone block for holding one or more biological tissue samples to be analyzed, imaged and/or screened (herein also referred to as “tissue sample of interest”), wherein the chaperone block comprises at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities. The “chaperone block” is a three dimensional structure for holding the biological tissue samples of interest during analysis, imaging and/or screening, particularly using 3D EM. The “chaperon sample” is also a biological tissue sample in which the biological tissue samples of interest will be embedded (“loaded”) for analysis, imaging and/or screening, particularly using 3D EM. The invention also relates to a screening sample that can be loaded into a chaperone sample as well as an apparatus for loading a chaperone block with a screening sample. The “screening sample” is the biological tissue sample of interest that has been modified (incl. embedded in a resin) so that it can be loaded into the chaperone sample on the chaperone block for EM. Furthermore, the invention relates to methods to produce a chaperone block, methods to produce a screening sample and methods for preparing a tissue sample for connectomic screening.

The connectomic analysis of brain samples has yielded surprising insights into the structure of neuronal circuits across various species and brain regions. However, these insights have so far been obtained from relatively small sets of experimental samples, ranging from n=l to about 12. For the systematic screening of connectomic variability across many relevant axes of variation, however, such as postnatal development, aging, pathological change, behavioral variability, disease states and pharmacological and other interventions, efficient data acquisition from ideally hundreds of volume electron microscopy (EM) samples is essential.

The effort to obtain connectomic volume EM samples at a scale required for neuronal circuit analysis has so far impeded large-scale connectomic screening efforts. Most connectomic studies have so far been conducted on a single sample (N=l) (Bock et al., 2011; Briggman et al., 2011; Helmstaedter et al., 2013; Hildebrand et al., 2017; Kornfeld et al., 2017; Lee et al., 2016; Shapson- Coe et al., 2024; Svara et al., 2022; Winding et al., 2023; Zheng et al., 2018) or on up to three samples (n=2..3) (Motta et al., 2019; Schmidt et al., 2017), or in some rare cases on up to 8 to 13 samples (Gour et al., 2021; Loomba et al., 2022; Witvliet et al., 2021).

For example, Hildebrandt et al. 2017 embedded a single sample from larval zebrafish in a resin surrounded by mouse cerebral cortex as a support tissue to stabilize sectioning. While some of these studies have provided fundamental insights about neuronal circuit structure, the relationship of connectomic properties to important parameters of variation or relevant events of brain formation cannot be systematically screened. For such investigations, screening at a scale of n=10² to 10³ samples is required.

The effort to sample and process a single 3D EM sample of the size 100 pm³ is currently about 4 weeks of combined imaging and cutting on a single-beam EM setup (or alternatively about 36 hours of imaging time or a total time of 77 hours including overhead time for a 100pm³ sample using automated tape-collecting ultramicrotome (ATUM) and a scanning electron microscope like a Zeiss multiSEM). Evidently, scaling this to 100 samples would take in the order of 10 years of imaging time for single beam setups (and about 150 days for imaging time or a total time of 322 days including overhead time for a 100pm³ sample in multi -beam setups). Thus, the full scalability into this range of connectomic screening is not feasible.

At the same time the imaging of large tissue blocks at sized one to two millimeters in-plane has become realistic with the multiSEM imaging approach (Loomba et al., 2022; Shapson-Coe et al., 2024; Sievers et al., 2024). Here, within about 6 weeks a volume of 2 mm by 2 mm by about 100 pm can realistically be imaged/acquired.

A further key impediment of single sample repeated data acquisition is both the difficulty of cutting and handling very small samples, collecting sections reliably at small size and the overhead of sample preparation and movement during imaging.

Given the above-mentioned shortcomings in the prior art for large-scale connectomic screening resulting in months or years for analyzing complex samples, there is a need for improved means and methods for connectomic screening.

Accordingly, the technical problem underlying the present invention is the provision of means and methods for improved connectomic screening, particularly using 3D electron microscopy (EM).

The technical problem is solved by provision of the embodiments characterized in the claims and as provided herein below. Specifically, the technical problem is solved, and the above-mentioned difficulties are overcome by the provision of a chaperone block comprising at least two layers of resin and at least one layer of a chaperone sample. In particular, the above-mentioned problem is solved by a chaperone block comprising at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities.

The inventors developed an approach for the efficient sample preparation and imaging of order - of-magnitude exemplary shown for 101 and 106 connectomic samples. By embedding a large number of screening samples into cavities of a chaperone block, long-series ultrathin cuttability and artefact-free large-scale EM imaging could be obtained. The inventors demonstrated the usability of this approach with 101 tissue samples from mouse cortex and with 106 tissue samples of mouse cortex. These tissue samples can be imaged and analyzed in a single section of the loaded chaperone block thereby reducing the imaging time and the overhead time. With this chaperone block, connectomic screening at an exemplary scale of 10² to the 10³ samples is made possible, enabling connectomic analyses of unprecedented magnitude. The chaperone block of the invention improves connectomic screening by allowing to cut and detect many samples simultaneously. The chaperone block of the invention is particularly advantageous since it provides structural integrity as well as homogenizes the electrical conductivity and surface of one or more screening sample. Homogenizing the surface reduces mechanical stress when cutting the chaperone block e.g. when loaded with a tissue sample of interest, thereby allowing cutting of the chaperone block in slices as thin as 35 nm. This in turn enables high throughput connectomic screening of a multitude of samples in parallel via EM imaging. Furthermore, homogenizing the electrical conductivity allows for high-quality imaging of the slices of the loaded chaperone block without damaging the sample or introducing artifacts due to charge accumulation. Thus, the chaperone block of the invention speeds up sample processing by a factor of 16-26 compared to sample processing in the prior art, such as single sample processing with the Multi SEM and by a factor of 60 regarding only the imaging time for serial block-face scanning electron microscopy (SBEM) (Gour et al., 2021; Loomba et al., 2022; Motta et al., 2019; Shapson-Coe et al., 2024; Sievers et al., 2024).

The above-mentioned problem is also solved by the provision of a method for producing a chaperone block, e.g. the chaperone block of the present invention, the method comprising:

(a) embedding a chaperone sample onto a lower layer of resin,

(b) adding a top layer of resin on top of the chaperone sample,

(c) introducing a multitude of cavities penetrating the top layer of resin and at least partially penetrating the chaperone sample.

The method for producing a chaperone block solves the above stated problem since the thereby produced chaperone block can be used to speed up sample processing for connectomic screening. Accordingly, the inventive concept explained above in context of the chaperone block of the invention applies mutatis mutandis to the method for producing a chaperone block.

Furthermore, the above-mentioned problem is solved by the provision of a screening sample comprising a tissue sample of interest, a predetermined breaking point and a base, wherein the screening sample is configured to be loaded into a chaperone block, e.g. a chaperone block of the present invention.

By embedding a large number of screening samples into cavities of a chaperone block, long-series ultrathin cuttability and artefact-free large-scale EM imaging could be obtained. The screening sample of the invention can be loaded into a chaperone block. By loading a high number of screening samples into cavities of a chaperone block, long-series ultrathin cuttability and artefact- free large-scale EM imaging can be obtained. This has been demonstrated in Example 1. Thus, the screening sample configured to be loaded into a chaperone block improves connectomic screening by allowing to cut and detect many screening samples simultaneously. This speeds up sample processing by a factor of 16-26 compared to sample processing in the prior art, such as single sample processing with the multi SEM and by a factor of 60 regarding only the imaging time for SBEM.

The chaperone block of the present invention and the screening sample complement each other, i.e. are interrelated products. Although they are different entities, they work together, e.g. when the screening sample is loaded into the chaperone block. Insofar, both lead to the advantageous effects of the present invention described herein, which are further amplified in combination, e.g. when screening samples are loaded into the chaperone block.

Furthermore, the above-mentioned problem is solved by the provision of a method for producing a screening sample, e.g. the screening sample of the present invention, the method comprising:

(a) embedding a tissue sample of interest in a resin,

(b) milling the embedded tissue sample of interest into a shape comprising the tissue sample of interest, a resin layer and a resin base,

(c) introducing a predetermined breaking point into the resin layer between the tissue sample of interest and the resin base.

The method for producing a screening sample solves the above stated problem since the thereby produced screening sample can be used to speed up sample processing for connectomic screening. Accordingly, the inventive concept explained above in context of the screening sample of the invention applies mutatis mutandis to the method for producing a screening sample.

Furthermore, the above-mentioned problem is solved by the provision of an apparatus for loading a chaperone block, e.g. the chaperone block of the present invention, with a screening sample, e.g. the screening sample of the present invention, comprising:

(a) a first holder for holding the chaperone block,

(b) a second holder for holding the screening sample, wherein the apparatus is configured to move the screening sample by means of the second holder into a cavity of the chaperone block and to break of the screening sample from the second holder, thereby loading the screening sample into the chaperone block.

As mentioned above, the screening sample may be loaded into the chaperone block thereby resulting in advantageous effects provided herein. To achieve this loading of the chaperone block, the inventors have developed an apparatus that can load the screening samples into the cavities of the chaperone block. The loaded chaperone block can then be cut, and the slices can be imaged thereby improving connectomic screening speed by allowing to cut and detect many samples simultaneously. Accordingly, the apparatus for loading a chaperone block with a screening sample provided herein solves the above-mentioned problem by improving the speed of connectomic screening via loading screening samples into the chaperone block.

The chaperone block, the screening sample and the apparatus of the present invention complement each other, i.e. are interrelated products. Although they are different entities, they work together, e.g. when the apparatus is used to load the screening samples into the chaperone block. Insofar, all three entities lead to the advantageous effects of the present invention described herein, which are further amplified in their combination, e.g. when screening samples are loaded into the chaperone block via the apparatus of the invention.

Furthermore, the above-mentioned problem is solved by the provision of a method for preparing a sample for connectomic screening, the method comprising:

(a) providing one or more screening sample, e.g. one or more screening sample of the present invention,

(b) providing a chaperone block, e.g. a chaperone block of the present invention, or producing a chaperone block according to the present invention,

(c) loading the one or more screening sample(s) into the chaperone block with the apparatus according to the present invention.

The method for preparing a sample for connectomic screening has been demonstrated in Example 1. This method combines the interrelated entities of the present invention, i.e. the screening sample, the chaperone block, or the methods to produce the same and the apparatus. Accordingly, the inventive concept explained above in relation to the screening sample, the chaperone block, or the methods to produce the same and the apparatus of the present invention applies mutatis mutandis to the method for preparing a sample for connectomic screening. The sample prepared by the method can cut, and the slices can be imaged thereby improving connectomic screening speed by allowing to cut and detect many samples simultaneously. The method thus improves the speed at which samples can be analyzed in connectomics and thereby solves the above-mentioned problem.

Furthermore, the above-mentioned problem is solved by the provision of a batch sample comprising a multitude of tissue samples embedded in a resin, wherein the tissue samples are configured to provide structural integrity to each other and thereby to the batch sample, and wherein the tissue samples are configured to homogenize the electrical conductivity and surface of the batch sample.

The inventors have found that tissue samples of interest, e.g. tissue sample punched through biopsy, can be arranged into a batch comprising a multitude of samples. This has been achieved by arranging tissue samples selected to be analyzed, e.g. in a pyramid shape or in a hexagonal shape, so that the tissue samples are in direct contact to each other. The batch sample is further stabilized by infiltration with a resin and then embedded into a resin to fill open space between individual tissue samples. The individual samples and the resin provide structural integrity to each other. Furthermore, the multitude of tissue samples leads to a homogenized electrical conductivity and surface of the batch sample. The batch sample can then be cut, and the slices can be imaged thereby improving connectomic screening speed by allowing to cut and detect many samples simultaneously. The batch sample thus improves the speed at which samples can be analyzed in connectomics and thereby solves the above-mentioned problem. Preferred embodiments of the present invention are defined in the dependent claims.

The invention provides the following items:

1. A chaperone block for holding one or more (biological) tissue sample(s) of interest, particularly for electron microscopy (EM), wherein the chaperone block comprises at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities.

2. The chaperone block of item 1, wherein the chaperone block is configured to provide structural integrity to one or more screening sample(s) which comprise said tissue sample(s) of interest, and wherein the chaperone block is further configured to homogenize the electrical conductivity and surface of the one or more screening sample(s).

3. The chaperone block of item 1 or 2, wherein the chaperone sample is arranged in between the at least two layers of resin.

4. The chaperone block of any one of items 1 to 3, wherein the chaperone block comprises a lower layer of resin, a middle layer of chaperone sample, and a top layer of resin.

5. The chaperone block of any one of items 1 to 4, wherein the resin is Epon 812.

6. The chaperone block of any one of items 1 to 5, wherein the cavities are configured to be loaded with screening samples.

7. The chaperone block of any one of items 1 to 6, wherein the cavities are drill holes.

8. The chaperone block of any one of items 1 to 7, wherein the cavities have a diameter of about 70-2000pm, preferably of about 100-150pm.

9. The chaperone block of any one of items 1 to 8, comprising about 2-2500 cavities, preferably comprising about 80-120 cavities.

10. The chaperone block of any one of items 1 to 9, wherein each of the cavity is arranged about 20-1000pm, preferably at least 20pm, apart from each other cavity.

11. The chaperone block of any one of items 1 to 10, wherein the cavities at least partially penetrate the chaperone sample.

12. The chaperone block of item 11, wherein the cavities penetrate the top layer of resin and at least partially penetrate the chaperone sample, preferably wherein the cavities penetrate the chaperone sample. The chaperone block of any one of items 1 to 12, wherein the cavities are approximately cylindrical or in the form of a polyhedron. The chaperone block of any one of items 1 to 13, wherein the chaperone sample is a tissue sample. The chaperone block of item 14, wherein the chaperone sample is a tissue sample of a mammal, reptile, fish, or bird, preferably a tissue sample of a mouse brain or a human brain. The chaperone block of items 14 or 15, wherein the chaperone sample and the screening sample comprise tissue samples originating from the same type of organ having similar or same structural and conductive properties. The chaperone block of any one of items 1 to 16, wherein the chaperone sample and/or the screening sample is at least 100pm to 5000pm, preferably at least 500pm thick. The chaperone block of any one of items 1 to 17, comprising a screening sample in one or more of the multitude of cavities, preferably comprising a screening sample in each cavity. The chaperone block of any one of items 1 to 18, wherein the chaperone block has a polygonal/polyhedric shape, preferably a hexagonal shape. A method for producing the chaperone block of any one of items 1 to 19, the method comprising:

(a) embedding a chaperone sample onto a lower layer of resin,

(b) adding a top layer of resin on top of the chaperone sample,

(c) introducing a multitude of cavities penetrating the top layer of resin and at least partially penetrating the chaperone sample. The method of item 20, wherein the lower layer of resin is placed onto an aluminum pin. The method of item 20 or 21, further comprising staining the chaperone sample. The method of any one of items 20 to 22, further comprising dehydrating and/or infiltrating the chaperone sample with a resin. The method of any one of items 21 to 23, further comprising milling an alignment plane into the aluminum pin. The method of any one of items 20 to 24, further comprising milling the top layer of resin, so that the top layer of resin has a thickness of about 200 pm to 2000 pm, preferably 500 pm. The method of any one of items 20 to 25, further comprising milling the chaperone block into a polygonal/polyhedric shape, preferably a hexagonal shape. The method of any one of items 20 to 26, further comprising washing of the chaperone block in an ultra-sonic bath. The method of any one of items 20 to 27, further comprising step (d) loading one or more screening sample into the multitude of cavities. The method of item 28, wherein a screening sample is loaded into each cavity of the chaperone block. A screening sample comprising a tissue sample of interest, a resin layer comprising a predetermined breaking point and a base, wherein the screening sample is configured to be loaded into a chaperone block of any one of items 1 to 19. The screening sample of item 30, wherein the tissues sample of interest is cylindrical or in the form of a polyhedron The screening sample of item 30 or 31, wherein the tissue sample of interest is at least 100 pm to 5000 pm, preferably at least 500 pm thick. The screening sample of any one of items 30 to 32, wherein the resin layer comprising the predetermined breaking point is arranged between the tissue sample of interest and the base. The screening sample of any one of items 30 to 33, wherein the basis is made of resin. The screening sample of any one of items 30 to 34, wherein the tissue sample of interest and/or the resin layer comprising the predetermined breaking point have a diameter of about 70-2000 pm, preferably, preferably 100-150 pm. The screening sample of any one of items 30 to 35, wherein the base has a width of about 500-3000 pm and a height of about 500-5000 mm. The screening sample of any one of items 30 to 36, wherein the tissue sample of interest comprises a sample of a mammalian, reptile, fish, or bird tissue, preferably a tissue of a mouse brain or a human brain. The screening sample of any one of items 30 to 36, wherein the tissue sample of interest comprises a tissue originating from the same type of organ having similar or same structural and conductive properties as a tissue of the chaperone sample in the chaperone block of any one of items 1 to 19. A method for producing the screening sample of any one of items 30 to 36, the method comprising:

(a) embedding a tissue sample of interest in a resin,

(b) milling the embedded tissue sample of interest into a shape comprising the tissue sample of interest, a resin layer and a base,

(c) introducing a predetermined breaking point into the resin layer between the tissue sample of interest and the base. The method of item 39, further comprising staining the tissue sample The method of item 39, further comprising dehydrating and/or infiltrating the tissue sample of interest with a resin. The method of item 39, further comprising treating the tissue sample of interest with a heavy metal treatment. The method of item 39, further comprising milling an alignment plane into the resin base, preferably wherein the base is a resin base. An apparatus for loading a chaperone block of any one of items 1 to 19 with a screening sample comprising:

(a) a first holder for holding the chaperone block,

(b) a second holder for holding the screening sample, wherein the apparatus is configured to move the screening sample by means of the second holder into a cavity of the chaperone block and to break of the tissue sample of interest from the base, thereby loading the tissue sample of interest into the chaperone block. The apparatus of item 44, wherein the first holder and the second holder are attached to an air table. The apparatus of item 44 or 45, wherein the second holder is configured to move the screening sample in three dimensions. The apparatus of item 46, wherein the second holder further comprises a micromanipulator. The apparatus of item 47, wherein the micromanipulator is attached to a breadboard, e.g. an aluminum breadboard, optionally wherein the breadboard is attached to the air table via a height adjuster. The apparatus of any one of items 44 to 48, wherein the first holder and the second holder are arranged at an angle of 30° to the horizontal, with the chaperone block and the screening sample facing each other. The apparatus of any one of items 44 to 49, wherein the first holder and the second holder further comprise fastening means to reversibly fasten the chaperone block and the screening sample. The apparatus of item 50, wherein the first holder further comprises screws and screw sockets to reversibly fasten the chaperone block to the first holder. The apparatus of item 50 or 51, wherein the second holder further comprises a pneumatic gripper to reversibly collect, hold and/or discard the screening sample. A method for preparing a sample for connectomic screening, the method comprising:

(a) providing one or more screening sample,

(b) providing a chaperone block according to any one of items 1 to 19, or producing a chaperone block according to any one of items 20 to 29,

(c) loading the one or more screening sample into the chaperone block with the apparatus according to any one of items 44 to 52. The method of item 53, wherein the one or more screening sample is configured to be loaded into the cavities of the chaperone block. The method of item 53 or 54, wherein one screening sample is loaded into each cavity of the chaperone block. The method of any one of items 53 to 55, wherein the screening sample is loaded into the chaperone block by inserting the screening sample into a cavity of the chaperone block followed by breaking off the screening sample at a predetermined breaking point from its base hold by the second holder, thereby loading the chaperone block with the screening sample. The method of item 56, wherein the screening sample is broken off at a predetermined breaking point while being inserted into the cavity of the chaperone block by rapidly moving the second holder. The method of any one of items 53 to 56, further comprising removing the loaded chaperone block from the first holder. The method of any one of items 53 to 58, further comprising adding resin on top of the chaperone block thereby filling open spaces in the loaded cavities between the screening samples and the cavity walls. The method of any one of items 53 to 59, further comprising milling the top of the chaperone block to expose a continuous layer of chaperone sample and screening samples. The method of any one of items 53 to 60, further comprising slicing the chaperone sample loaded with the screening sample. A batch sample comprising a multitude of tissue samples embedded in a resin, wherein the tissue samples are configured to provide structural integrity to each other and thereby to the batch sample, and wherein the tissue samples are configured to homogenize the electrical conductivity and surface of the batch sample. The batch sample of item 62, wherein the multitude of tissue samples is arranged in the shape of a pyramid, square, rectangle, triangle and the like such that the tissue samples are piled up like logs. The batch sample of item 62 or 63, wherein a tissue sample (in the multitude of tissue samples) comprises a sample of a mammalian, reptile, fish, or bird tissue, preferably a sample of humans, more preferably a tissue of a mouse brain or a human brain. The batch sample of any one of items 62 to 64, wherein a tissue sample (in the multitude of tissue samples) is collected from whole brain, hemispheres, cortical and/or subcortical volumes, spinal cords and peripheral tissues containing nerve endings and the like. 66. The batch sample of any one of items 62 to 65, wherein a tissue sample (in the multitude of tissue samples) is a neuronal tissue sample, preferably a neuronal structure of several hundred micrometers in diameter, such as barrels in the mouse SI cortex, subnuclei in the thalamus, or directional preference columns in the visual cortex.

67. The batch sample of any one of items 62 to 66, wherein a tissue sample (in the multitude of tissue samples) and/or the resin layer (comprising the predetermined breaking point) have a diameter of about 70-2000 pm, preferably 100-150 pm.

Connectomics

Brains are unique not only in the number of cells they comprise (about 85 billion neurons in the case of the human brain) but also in the extent of the direct and specific communication between their cells via synaptic connections (each neuron has on the order of 1,000 synaptically coupled partner neurons). Mapping the resulting complex connectivity graph is the goal of connectomics. As used herein “connectomics” or “connectomic screening” and the like refers to the study of the connectome, which is a comprehensive map of neural connections in the brain. Connectomics encompasses the mapping and analysis of neural networks at different scales, ranging from the level of individual neurons and their synaptic connections to larger-scale networks that connect different regions of the brain. The goal of connectomics is to understand the structure and function of the brain's complex wiring in both health and disease, which can provide insights into how information is processed and how various brain functions are implemented. This is crucial for advancing the understanding of neuroscience, neurobiology, and for the development of treatments for neurological disorders. Accordingly, the aim of the products and methods provided herein is to improve connectomic screening, and in particular, to accelerate screening of connectomes in brain tissue.

Chaperone block

A chaperone block in the sense of the present invention comprises at least two layers of resin and at least one layer of a chaperone sample. As used herein “chaperone block” relates to the entity comprising at least two layer of resin and at least one layer of a chaperone sample. Accordingly, the term “chaperone block” comprises at least all three of said elements. A chaperone block is not limited to any specific form or dimensions, the skilled person rather is capable of selecting feasible forms depending, e.g. on the size and form of the chaperone sample and/or depending on the machines processing the chaperone block. Thus, a chaperone block does not necessarily have to be a geometrical block but can be in any form.

Accordingly, the invention provides a chaperone block comprising at least two layers of resin and at least one layer of a chaperone sample. The chaperone block of the invention is useful insofar it can provide structural integrity as well as homogenize the electrical conductivity and surface of one or more screening sample(s). This is useful for speeding up connectomic screening since it allows to cut and analyze many samples simultaneously. Structural integrity can be crucial to obtain high quality slices as thin as 35 nm and to avoid disintegration of a slice during cutting. Furthermore, homogenization of the electrical conductivity can be crucial when imaging slices of the loaded chaperone block with an electron microscope due to the nature of the imaging process. Electron microscopes use a beam of electrons instead of light to form an image. Since electrons have a much shorter wavelength than light, electron microscopes can achieve much higher resolution, allowing for the visualization of structures at the nanometer scale. A homogenous electrical conductivity of the samples is important since low- conductivity materials, such as biological tissues, can accumulate electrical charge when exposed to the electron beam. This charge accumulation can deflect the electron beam, leading to image distortions, reduced resolution, and even damage to the sample. Conductive materials, or a tissue with a homogenized electrical conductivity on the other hand, allow the charge to dissipate, preventing these issues. Electrical conductivity of the sample can further affect the image quality. Samples with a homogenous electrical conductivity provide better contrast and resolution in the resulting images. Since the interaction between the electrons and the sample is more controlled, the image formed on the detector is clearer and more detailed. Samples with inhomogeneous electrical conductivity may be more susceptible to damage from the electron beam, e.g. when imaging tissue sample of interests with surrounding chaperone sample at the same time. The energy from the electrons can cause heating and other destructive effects. Samples with a homogenous electrical conductivity can better spread out and dissipate this energy, reducing the risk of damage. Any of the chaperone samples and/or the tissue sample of interests may be coated with a thin layer of conductive material, such as gold, platinum, or carbon to improve and homogenize the electrical conductivity, e.g. of non-conductive tissue. Such a coating can help to prevent charging and improve the quality of the resulting electron microscope images. Thus a homogenous electrical conductivity of a chaperone sample and a tissue sample of interest, e.g. when the chaperone block is loaded with tissue sample of interests can be important to ensure that high-quality EM images are obtained without damaging the sample or introducing artifacts due to charge accumulation.

As used herein “homogenizing electrical conductivity” or “homogenous (electrical) conductivity” also includes increasing the electrical conductivity of a sample, e.g. increasing the electrical conductivity of screening samples and/or the chaperone sample to a homogenous level. This is advantageous to achieve the above-described effects resulting to improved imaging. For example, a tissue samples that are cuttable by a diamond knife may have low electrical conductivity and thereby may be difficult to image in EM. The tissue sample can then be embedded in a material that has sufficient electrical conductivity while providing homogenous cuttability i.e. homogeneous stiffness, shear resistance, etc. Thus, the electrical conductivity of the screening sample may be increased via treatment/embedding of the sample with or in a material with sufficient electrical conductivity. This may increase the electrical conductivity of the sample but at the same time homogenize the electrical conductivity of the chaperone sample and the other screening samples to be loaded.

Accordingly, the invention provides a chaperone block comprising at least two layers of resin and at least one layer of a chaperone sample, wherein the chaperone block is configured to provide structural integrity to a one or more screening sample(s), and wherein the chaperone block is configured to homogenize the electrical conductivity and surface of one or more screening sample.

In general, the chaperone sample can be arranged in between the at least two layers of resin, so that the chaperone block comprises a lower layer of resin, a middle layer of chaperone sample, and a top layer of resin. Furthermore, the chaperone sample in the middle layer may additionally comprise a resin, e.g. can be embedded into resin and/or infiltrated/fixated by resin such as to provide structural integrity to the chaperone sample within the chaperone block. The chaperone sample can for example be embedded between a lower layer and a top layer of resin.

Accordingly, the invention provides a chaperone block comprising at least two layers of resin and at least one layer of a chaperone sample, wherein the chaperone sample is arranged in between the at least two layers of resin.

In a further aspect, the invention provides a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample, and a top layer of resin.

In a further aspect, the invention provides a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample, and a top layer of resin, wherein the chaperone sample is embedded into a resin and/or wherein the chaperone sample is infiltrated/fixated with a resin.

The lower layer of resin and top layer of resin are exchangeable. The arrangement of top and lower layer may only depend on the orientation of the chaperone block, e.g. when the chaperone block is placed onto a pin for introducing cavities and to be loaded with a screening sample. In general, the resin layer comprising the cavities is referred herein as the top layer of resin. As long as the chaperone sample is covered in two layers of resin, their orientation, i.e. top, bottom, left or right does not matter. Accordingly, the position of the resin layer may be exchanged in the chaperone block of the present invention, e.g. between top and bottom layer. The resin layer comprising the cavities reaching through the layer and into the chaperone sample may be referred herein as top layer of resin or top resin layer, irrespective of orientation of the chaperone block, e.g. when the block is turned. When the chaperone block of the present invention is placed on a pin, e.g. an aluminum pin, the resin layer in contact with the pin may be the lower layer of resin, followed by the chaperone sample, followed by the top layer of resin in ascending order away from the pin.

The chaperone block may also comprise at least one layer of resin, such as a single layer of resin a chaperone sample. Although it may be advantageous to comprise at least two layers of resin, such as a lower layer of resin and a top layer of resin, the chaperone sample may also directly placed upon a pin or holder and the like without a layer of resin separating the chaperone sample from the surface to which it is attached. Accordingly, the invention also provides a chaperone block comprising at least one layer of resin, such as one layer of resin, and at least one layer of a chaperone sample, such as one layer of chaperone sample.

The chaperone block in the sense of the present invention may further comprise a multitude of cavities. As used herein a cavity is an open space introduced into the chaperone block. As shown in Example 1, the cavities allow for parallel processing of a multitude of screening samples, when being loaded with screening samples, thereby speeding up connectomic screening since the multitude of cavities allow to cut and analyze many samples simultaneously.

Accordingly, the invention provides a chaperone block comprising at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities.

The invention also provides a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample and a top layer of resin, wherein the chaperone block comprises a multitude of cavities.

The invention also provides a chaperone block comprising a lower layer of resin, a middle layer of a chaperone sample, a top layer of resin and a multitude of cavities.

The invention also provides a chaperone block comprising at least one layer of resin, such as one layer of resin, at least one layer of a chaperone sample, such as one layer of chaperone sample and a multitude of cavities.

In general, the cavities can be loaded with screening samples. For this, the cavities of the chaperone block are filled with individual screening samples comprising tissue of interest to be analyzed, e.g. for connectomic screening. Thus, the cavities are configured to be loaded with a screening sample. This means, that the cavities may have any form and/or dimensions, as long as they are capable of being loaded with screening samples. A cavity is typically loaded with a single screening sample. Accordingly, the invention provides a chaperone block comprising at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities, wherein the cavities are configured to be loaded with screening samples.

The invention also provides a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample and a top layer of resin, wherein the chaperone block comprises a multitude of cavities, wherein the cavities are configured to be loaded with screening samples.

The skilled person is aware how to introduce cavities into a chaperone block. For example, cavities may be introduced into the chaperone block via drilling, hammering, screwing, milling, hollowing out, carving, etching, melting, punching, pricking, grating, piercing, perforating, puncturing, spiking and the like. In a preferred embodiment, the cavities may be introduced by a drill, or by drilling.

Accordingly, the invention provides in one aspect a chaperone block comprising at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities, wherein the cavities are drill holes. The invention also provides a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample and a top layer of resin, wherein the chaperone block comprises a multitude of cavities, wherein the cavities are drill holes.

The cavities may be of any diameter, shape and size as long as they are suitable to be loaded with a screening sample and/or have an appropriate size and shape corresponding to the size and shape of the tissue sample, such as the screening sample. If the cavities are introduced by drilling as in the Example, the shape will be cylindric. But the shape of the cavities may also be polyhedric, rectangular, cubic, pyramidic, cone shaped, prism shaped, spheric or the like. In particular, cavities of a polyhedric shape, such as a cuboidal shape may be preferred over a cylindric shape since this allows for less movement of the screening samples in the cavities and can aid the separation of the tissue in the screening sample from its base via breaking of the predetermined breaking point. Thus, in one embodiment, the cavities are approximately polyhedric, rectangular, cubic, pyramidic, cone shaped, prism shaped, or spheric. In a preferred embodiment, the cavities are approximately polyhedric, such as cuboidal. In a preferred embodiment, the cavities are approximately cylindrical. In a preferred embodiment, the cavities are polyhedric, such as cuboidal. In a preferred embodiment, the cavities are cylindrical. As mentioned above, the chaperone block of the present invention can comprise cavities of any shape. Accordingly, in some embodiments, the chaperone block of the present invention can comprise approximately polyhedron cavities. In some embodiments, the chaperone block of the present invention can comprise approximately rectangular cavities. In some embodiments, the chaperone block of the present invention can comprise approximately cylindrical cavities. In some embodiments, the chaperone block of the present invention can comprise approximately cubic cavities. In some embodiments, the chaperone block of the present invention can comprise approximately pyramidic cavities. In some embodiments, the chaperone block of the present invention can comprise approximately cone shaped cavities. In some embodiments, the chaperone block of the present invention can comprise approximately prism shaped cavities. In some embodiments, the chaperone block of the present invention can comprise approximately spherical cavities.

Accordingly, the invention provides in one aspect a chaperone block comprising at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities, wherein the cavities are approximately cylindrical or polyhedric, such as cuboidal.

The invention also provides a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample and a top layer of resin, wherein the chaperone block comprises a multitude of cavities, wherein the cavities are approximately cylindrical or polyhedric, such as cuboidal.

As mentioned above, the chaperone block of the present invention can comprise cavities of any diameter. The diameter of the cavities may depend on the diameter of the screening sample. For example, it is advantageous, that the diameter of the cavities is slightly larger than that of a screening sample to be loaded into the cavities. This is to enable loading of the screening sample into the chaperone block. Accordingly, in some embodiments, the cavities in the chaperone block and the screening sample have approximately the same diameter. In some embodiments, the cavities in the chaperone block and the screening sample have substantially the same diameter, as long as the screening sample can still be loaded into the cavities of the chaperone block. In some embodiments, the diameter of the screening sample is smaller than that of the cavities of the chaperone block. Accordingly, in some embodiments, the cavities have a diameter of about 70- 2000 pm, such as about 70pm, about 80pm, about 90pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, about 300 pm, about 310 pm, about 320 pm, about 330 pm, about 340 pm, about 350 pm, about 360 pm, about 370 pm, about 380 pm, about 390 pm, about 400 pm, about 410 pm, about 420 pm, about 430 pm, about 440 pm, about 450 pm, about 460 pm, about 470 pm, about 480 pm, about 490 pm, about 500 pm, about 510 pm, about 520 pm, about 530 pm, about 540 pm, about 550 pm, about 560 pm, about 570 pm, about 580 pm, about 590 pm, about 600 pm, about 610 pm, about 620 pm, about 630 pm, about 640 pm, about 650 pm, about 660 pm, about 670 pm, about 680 pm, about 690 pm, about 700 pm, about 710 pm, about 720 pm, about 730 pm, about 740 pm, about 750 pm, about 760 pm, about 770 pm, about 780 pm, about 790 pm, about 800 pm, about 810 pm, about 820 pm, about 830 pm, about 840 pm, about 850 pm, about 860 pm, about 870 pm, about 880 pm, about 890 pm, about 900 pm, about 910 pm, about 920 pm, about 930 pm, about 940 pm, about 950 pm, about 960 pm, about 970 pm, about 980 pm, about 990 pm, about 1000 pm, about 1010 pm, about 1020 pm, about 1030 pm, about 1040 pm, about 1050 pm, about 1060 pm, about 1070 pm, about 1080 pm, about 1090 pm, about 1100 pm, about 1110 pm, about 1120 pm, about 1130 pm, about 1140 pm, about 1150 pm, about 1160 pm, about 1170 pm, about 1180 pm, about 1190 pm, about 1200 pm, about 1210 pm, about 1220 pm, about 1230 pm, about 1240 pm, about 1250 pm, about 1260 pm, about 1270 pm, about 1280 pm, about 1290 pm, about 1300 pm, about 1310 pm, about 1320 pm, about 1330 pm, about 1340 pm, about 1350 pm, about 1360 pm, about 1370 pm, about 1380 pm, about 1390 pm, about 1400 pm, about 1410 pm, about 1420 pm, about 1430 pm, about 1440 pm, about 1450 pm, about 1460 pm, about 1470 pm, about 1480 pm, about 1490 pm, about 1500 pm, about 1510 pm, about 1520 pm, about 1530 pm, about 1540 pm, about 1550 pm, about 1560 pm, about 1570 pm, about 1580 pm, about 1590 pm, about 1600 pm, about 1610 pm, about 1620 pm, about 1630 pm, about 1640 pm, about 1650 pm, about 1660 pm, about 1670 pm, about 1680 pm, about 1690 pm, about 1700 pm, about 1710 pm, about 1720 pm, about 1730 pm, about 1740 pm, about 1750 pm, about 1760 pm, about 1770 pm, about 1780 pm, about 1790 pm, about 1800 pm, about 1810 pm, about 1820 pm, about 1830 pm, about 1840 pm, about 1850 pm, about 1860 pm, about 1870 pm, about 1880 pm, about 1890 pm, about 1900 pm, about 1910 pm, about 1920 pm, about 1930 pm, about 1940 pm, about 1950 pm, about 1960 pm, about 1970 pm, about 1980 pm, about 1990 pm, or about 2000 pm.

In a preferred embodiment, the cavities have a diameter of about 70-500 pm, such as about 70pm, about 80pm, about 90pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, about 300 pm, about 310 pm, about 320 pm, about 330 pm, about 340 pm, about 350 pm, about 360 pm, about 370 pm, about 380 pm, about 390 pm, about 400 pm, about 410 pm, about 420 pm, about 430 pm, about 440 pm, about 450 pm, about 460 pm, about 470 pm, about 480 pm, about 490 pm, or about 500 pm. In a preferred embodiment, the cavities have a diameter of about 70-150 pm, such as about 70pm, about 80pm, about 90pm, about 100pm, about 110pm, about 120pm, about 130pm, about 140pm, or about 150pm. In more a preferred embodiment, the chaperone block of the present invention can comprise cavities with a diameter of about 120 pm.

In some embodiments, the chaperone block of the present invention comprises cavities with a diameter of about 1 mm to 3 mm.

Accordingly, the invention provides in one aspect a chaperone block comprising at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities, wherein the cavities have a diameter of about 70-2000 pm.

The invention also provides a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample and a top layer of resin, wherein the chaperone block comprises a multitude of cavities, wherein the cavities have a diameter of about 70-2000 pm.

In general, the chaperone block of the invention is not limited to any specific number of cavities. The exact number of cavities in the chaperone block of the present invention may depend on several factors, such as on the size, shape, and/or number of screening sample, which are to be analyzed, on the application of the chaperone block or the like. In principle, it is advantageous to include as many cavities in the chaperone block as possible. This is restricted by the area of the chaperone sample in the chaperone block. Accordingly, it is preferred to optimize the cavity number to the area of the chaperone sample, thereby optimizing the speed of connectomic screening. The area of the chaperone sample is used as efficiently as possible. As many cavities as possible are created with the least distance between them as necessary, but so that the partitions remain stable even after the screening samples are loaded. In Example 1 it has been shown that this allows cutting the chaperone sample loaded with 10² to 10³ screening samples at a thickness of 35 nm. The minimum distance between cavities can be determined to provide a homogeneous cut surface despite the many individual screening samples and guarantees homogeneous imaging due to sufficient electrical conductivity.

As mentioned above, the chaperone block of the present invention can comprise any number of cavities. Accordingly, in some embodiments, the chaperone block comprises about 2 to 2500, such as about 1, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 600, about 610, about 620, about 630, about 640, about 650, about 660, about 670, about 680, about 690, about 700, about 710, about 720, about 730, about 740, about 750, about 760, about 770, about 780, about 790, about 800, about 810, about 820, about 830, about 840, about 850, about 860, about 870, about 880, about 890, about 900, about 910, about 920, about 930, about 940, about 950, about 960, about 970, about 980, about 990, about 1000, about 1050, about 1100, about

1150, about 1200, about 1250, about 1300, about 1350, about 1400, about 1450, about 1500, about

1550, about 1600, about 1650, about 1700, about 1750, about 1800, about 1850, about 1900, about

1950, about 2000, about 2050, about 2100, about 2150, about 2200, about 2250, about 2300, about

2350, about 2400, about 2450, or about 2500 cavities. In some embodiments, the chaperone block comprises about 2 to 1000 cavities. In some embodiments, the chaperone block comprises about 10 to 1000 cavities. In some embodiments, the chaperone block comprises 500 to 1000 cavities. In some embodiments, the chaperone block comprises about 50 to 500 cavities. In some embodiments, the chaperone block comprises about 100 to 500 cavities. In some embodiments, the chaperone block comprises about 100 to 250 cavities. In a preferred embodiment, the chaperone block comprises about 80 to 120 cavities. In some embodiments, the chaperone block comprises about 2 to 10000 cavities or more. In general, the chaperone block of the invention may comprise as many cavities as can possibly be fitted into the area of the chaperone sample without compromising its structural integrity either in a loaded or unloaded state.

Accordingly, the invention provides in one aspect a chaperone block comprising at least two layers of resin, at least one layer of a chaperone sample and about 1 to 1000 cavities, preferably about 2 to 1000 cavities, most preferably about 80 to 120 cavities.

The invention also provides a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample and a top layer of resin, wherein the chaperone block about 1 to 1000 cavities, preferably about 2 to 1000 cavities, most preferably about 80 to 120 cavities.

The spacing of the cavities is such that the cavities are arranged with offset and with rectangular surrounds. The cavities can be separated by walls of chaperone sample that are arranged with offset thereby improving identification of the samples afterwards. As used herein “offset” means the distance from one cavity to another cavity. The offset is measured from the outline of the cavity in the cross-section of the chaperon block, i.e. that the offset is measured from the outline of the cavity to the outline of the next cavity. For example, if the cavities are cylindrical, the outline of the cavities will appear as circles in the cross-section of the chaperone block (see e.g. Figures 2E and 4A. The offset will be measured from the outline of one circle, to the outline of the next circle, and always relate to the minimum of chaperone tissue in between the two outlines.

The offset is chosen to maximize the number of cavities in the chaperone block and at the same time to obtain a reasonable tradeoff regarding structural integrity. When the offset is too low, the structural integrity may be lost and the chaperone sample loaded with screening samples may disintegrate when cut for, e.g. EM imaging. If the offset is too low, cavities may also break during the loading process of a screening sample into the cavity of the chaperone block. Thus, there is always a tradeoff between maximizing the number of cavities and thereby loaded screening samples and structural integrity. Accordingly, defining an offset may depend on the exact experimental setup and may differ between the desired application of the chaperone sample. The skilled person is capable without undue burden to determine which offset is acceptable for the desired application of the herein, one sample, e.g. by testing chaperone samples comprising a multitude of cavities with different offsets and assessing the quality of the sample when cut an imaged. The terms “offset”, “arranged apart from each other”, “spaced”, “arranged with offset”, and the like are used interchangeably herein and all mean the distance between one cavity to any other cavity as described above. In general, the smaller the offset, the better, since a smaller offset fits more tissue sample of interests.

Accordingly, in some embodiments of the chaperone block of the invention each cavity is arranged about 20-1000 pm, such as about 20 pm, about 30 pm, about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, about 300 pm, about 310 pm, about 320 pm, about 330 pm, about 340 pm, about 350 pm, about 360 pm, about 370 pm, about 380 pm, about 390 pm, about 400 pm, about 410 pm, about 420 pm, about 430 pm, about 440 pm, about 450 pm, about 460 pm, about 470 pm, about 480 pm, about 490 pm, about 500 pm, about 510 pm, about 520 pm, about 530 pm, about 540 pm, about 550 pm, about 560 pm, about 570 pm, about 580 pm, about 590 pm, about 600 pm, about 610 pm, about 620 pm, about 630 pm, about 640 pm, about 650 pm, about 660 pm, about 670 pm, about 680 pm, about 690 pm, about 700 pm, about 710 pm, about 720 pm, about 730 pm, about 740 pm, about 750 pm, about 760 pm, about 770 pm, about 780 pm, about 790 pm, about 800 pm, about 810 pm, about 820 pm, about 830 pm, about 840 pm, about 850 pm, about 860 pm, about 870 pm, about 880 pm, about 890 pm, about 900 pm, about 910 pm, about 920 pm, about 930 pm, about 940 pm, about 950 pm, about 960 pm, about 970 pm, about 980 pm, about 990 pm, or about 1000 pm apart from each other cavity.

In some embodiments of the chaperone block of the invention each cavity is arranged about 20- 500 pm, such as about 20 pm, about 30 pm, about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, about 300 pm, about 310 pm, about 320 pm, about 330 pm, about 340 pm, about 350 pm, about 360 pm, about 370 pm, about 380 pm, about 390 pm, about 400 pm, about 410 pm, about 420 pm, about 430 pm, about 440 pm, about 450 pm, about 460 pm, about 470 pm, about 480 pm, about 490 pm, or about 500 pm apart from each other cavity. In some embodiments of the chaperone block of the invention each cavity is arranged about 20-150 pm, such as about 20 pm, about 30 pm, about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, or about 150 pm apart from each other cavity. In some embodiments of the chaperone block of the invention each cavity is arranged about 20-100 pm, such as about 20 pm, about 30 pm, about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, or about 100 pm apart from each other cavity. In some embodiments of the chaperone block of the invention each cavity is arranged with an offset to each other cavity. In some embodiments, the chaperone block comprises a multitude of cavities, wherein the cavities are arranged within rectangular surrounds. In a preferred embodiment, of the chaperone block of the invention each cavity is arranged about at least 50 pm or 50 pm apart from each other cavity.

As mentioned above, the cavities are configured to be loaded with screening samples. This can be realized by introducing the cavities such that they at least partially penetrate the chaperone sample. This allows to introduce screening samples into the cavities of the chaperone sample, thereby allowing successful loading.

Accordingly, the invention provides in one aspect a chaperone block comprising at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities, wherein the cavities at least partially penetrate the chaperone sample.

The invention also provides a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample and a top layer of resin, wherein the chaperone block comprises a multitude of cavities, wherein the cavities at least partially penetrate the chaperone sample.

In general, to at least partially penetrate the chaperone sample, the resin layer on top may be penetrated as well by the cavities. The cavities of the present invention may extend through the top layer of resin. The cavities of the present invention may partially or fully penetrate the chaperone sample or the tissue sample, which is configured to provide stability for the screening sample. It may be preferred herein that the cavities fully penetrate the chaperone sample, i.e. penetrate the chaperone sample though its entire thickness. This is preferred herein since it maximizes the space to be loaded with a screening sample and allows for loading with thicker screening samples, thereby allowing to cut more sections of the loaded chaperone block resulting in a more efficient imaging and analysis of the screening samples. In some embodiments, the chaperone block of the present invention comprises a multitude of cavities, wherein the cavities at least partially penetrate the chaperone sample. In a preferred embodiment, the chaperone block further comprises a multitude of cavities, wherein the cavities penetrate the chaperone sample. In some embodiments, the chaperone block comprises a multitude of cavities, wherein the cavities penetrate the top layer of resin and at least partially the chaperone sample, preferably wherein the cavities penetrate the chaperone sample.

Accordingly, the invention provides in one aspect a chaperone block comprising at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities, wherein the cavities penetrate the top layer of resin and at least partially penetrate the chaperone sample, preferably wherein the cavities penetrate the chaperone sample.

The invention also provides a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample and a top layer of resin, wherein the chaperone block comprises a multitude of cavities, wherein the cavities penetrate the top layer of resin and at least partially penetrate the chaperone sample, preferably wherein the cavities penetrate the chaperone sample.

As used herein “chaperone sample” in general refers to a sample of tissue or tissue -derived sample that is arranged with at least two layers of resin. Accordingly, the “chaperone sample” may be a tissue-derived sample or comprise a tissue-derived sample. Accordingly, “chaperone sample” may be interchangeably used with “chaperone tissue sample” in the context of the chaperone block of the invention. This is not to be confused with the tissue sample of interest that may be comprised in the screening sample of the present invention. The tissue sample of interest comprised in the screening sample of the present invention is described in detail herein below. In general, both can be distinguished in so far that the chaperone sample will not be analyzed but provides structural integrity and homogenized electrical conductivity to the tissue sample of interests, wherein the tissue samples of interest will be analyzed by cutting and imaging. Accordingly, the chaperone sample can be seen as kind of a framework tissue that helps achieving the efficient analysis of the tissue samples of interest leading to the advantageous effects described in detail above.

Preferably, the chaperone sample is arranged in between the at least two layers of resin, so that all three form a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample, which optionally may be embedded and/or infiltrated by a resin and a top layer of resin. The chaperone sample may be arranged like a layer, e.g. similar to a resin layer, since it can be embedded and/or infiltrated by resin. Such an exemplary chaperone sample is shown in Figure 2C, note that the chaperone block is illustrated on top of an aluminum pin.

Accordingly, the invention provides in one aspect a chaperone block comprising at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities, wherein the chaperone sample comprises a tissue sample.

The invention also provides a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample and a top layer of resin, wherein the chaperone block comprises a multitude of cavities, wherein the chaperone sample comprises a tissue-derived sample which is not to be confused with the “tissue sample of interest”. However, the possible origins and types (as defined herein below) of the “tissue-derived sample” which is used as a chaperone sample and the “tissue sample of interest” which is used for preparing the “screening sample” are the same.

The chaperone sample may comprise any tissue. Tissue in the sense of the present invention is preferably biological tissue, e.g. from an organ. Any biological tissue with sufficiently dense membranes (that can be stained) can be used in the sense of the present invention for the chaperone sample. It is advantageous to use biological tissue with a sufficient membrane density. In some embodiments, the chaperone sample may comprise a meshed homogenate of a biological tissue. In general, chaperone sample comprises tissue of a similar type of that of the tissue sample of interest. The similarity of the chaperone tissue-derived sample and the tissue sample of interest results in a homogenized surface and avoids any differences in electrical conductivity within the loaded chaperone block, e.g. when cutting the block and imaging/analyzing the tissue samples of interest. For example, choosing a tissue with similar structural properties allows for cutting the loaded chaperone sample without mayor differences in density and rigidity between the chaperone tissue sample and the tissue sample of interest. This allows for uniform cutting of the loaded chaperone block since differences in the density and rigidity can lead to breaking of the slice during cutting. Homogenizing the structural properties by choosing similar tissue for the chaperone tissue sample and the tissue sample of interest reduces mechanical stress when cutting the chaperone block, thereby allowing cutting of the chaperon block in slices as thin as 35nm. This in turn enables high throughput connectomic screening of a multitude of samples in parallel via EM imaging. Furthermore, choosing a tissue with similar conductive properties, thereby homogenizing the electrical conductivity between the chaperone sample and the tissue sample of interest, allows for high-quality imaging of the slices of the loaded chaperone block without damaging the sample or introducing artifacts due to charge accumulation. Accordingly, the invention is not limited to a specific tissue type, the skilled person is rather capable of choosing a chaperone tissue sample with similar tissue properties to the tissue sample of interest to be analyzed, thereby resulting in the above-described effects. In general, the chaperone sample and the tissue sample of interest may be from similar tissue, however, can be from different species.

Typically, the chaperone sample to be used in the present invention is a biological tissue-derived sample. In general, such a tissue-derived sample may comprise any tissue from any organism. In a preferred embodiment of the chaperone block of the invention the chaperone sample comprises a tissue sample comprising neurons. In some embodiment of the chaperone block of the invention the chaperone sample comprises a tissue-derived sample of a mammal, reptile, fish, or bird. In some embodiments, the chaperone sample comprises a tissue-derived sample of a rodent or a primate. In some embodiment the chaperone sample comprises a tissue-derived sample of a mouse, macaque or a human. In a preferred embodiment, the chaperone sample comprises a tissue-derived sample of a mouse brain, macaque brain or a human brain. When a ventricle is present in the chaperone sample it can negatively affect the cuttability of the loaded chaperone block. Accordingly, in some embodiments, the chaperone sample comprises a tissue-derived sample, wherein the tissue-derived sample does not comprise a ventricle.

In the context of the present invention, a chaperone sample comprising neurons is preferred. The neuronal tissue-of-interest may be a structure of several hundred micrometers in diameter, such as barrels in the mouse SI cortex, subnuclei in the thalamus, or directional preference columns in the visual cortex.

In the context of the present invention, a chaperone sample can comprise a tissue-derived sample of the whole brain, hemispheres, cortical and/or subcortical volumes, spinal cords and peripheral tissues containing nerve endings and the like of any of the above-mentioned species. The chaperone sample may also comprise a tissue-derived sample of the cortex such as the somatosensory cortex, parietal cortex and the like of any of the above-mentioned species. The chaperone sample may comprise an intact tissue-derived sample, meaning a tissue-derived sample that has maintained its native structural integrity, or may be a tissue-derived sample that has been homogenized, e.g. has been milled/blended into a homogenous sample. Herein, the chaperone sample can comprise any type of biological tissue-derived sample, e.g. a biological tissue-derived sample of a living or dead subject/animal.

Accordingly, the invention provides in one aspect a chaperone block comprising at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities, wherein the chaperone sample comprises a tissue-derived sample.

The invention also provides a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample and a top layer of resin, wherein the chaperone block comprises a multitude of cavities, wherein the chaperone sample comprises a tissue sample.

The invention also provides a chaperone block comprising at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities, wherein the chaperone sample comprises a tissue-derived sample of a rodent or other mammal, reptile, fish, or bird, preferably a tissue sample of a mouse brain, a human brain or a macaque brain.

The invention also provides a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample and a top layer of resin, wherein the chaperone block comprises a multitude of cavities, wherein the chaperone sample comprises a tissue-derived sample of a mammal, reptile, fish, or bird or a rodent, preferably a tissue-derived sample of a mouse brain, a human brain or a macaque brain.

As discussed above, it is advantageous that the chaperone sample and the tissue sample of interest that may be loaded into the chaperone block have similar structural and conductive properties. Thus, the chaperone sample and/or the tissue sample of interest may comprise the same or similar tissue, e.g. from a same or similar type of organ having same or similar structural and conductive properties. As mentioned above, the chaperone sample may comprise a tissue-derived sample having same or similar structural and/or conductive properties as a tissue sample of interest even if the chaperone sample and the tissue sample of interest comprise a tissue from different species. For example, the chaperone sample may be a tissue-derived sample from a rodent while the loaded tissue samples of interest are a sample from a macaque and vice versa, as long as the tissues have similar or same structural and/or conductive properties, thereby ensuring the homogenization of the chaperone sample surface when loaded with the tissue sample of interests and the electrical conductivity of both samples.

Accordingly, the invention provides in one aspect a chaperone block comprising at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities, wherein the chaperone sample and the tissue sample of interest comprise tissue originating from the same or similar type of organ having same or similar structural and conductive properties.

The invention also provides a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample and a top layer of resin, wherein the chaperone block comprises a multitude of cavities, wherein the chaperone sample and the tissue sample of interest comprise tissue originating from the same or similar type of organ having same or similar structural and conductive properties.

The size or shape of the chaperone sample is not particularly limited. In particular, size or shape of the chaperone sample may depend on the intended use of the sample. For example, a large sized chaperone sample may be advantageous for accommodating a multitude of cavities. In general, the dimensions of the chaperon sample are not particular limited and depend on the size of the tissue area of interest. To facilitate full insertion of a screening sample into a cavity penetrating the chaperone sample, the chaperone sample may be at least as thick as the tissue sample of interest. A particular shape of the chaperone sample may be beneficial for efficient cutting. The chaperone sample may have or be in a hexagonal shape. This can be advantageous for cutting the loaded chaperone sample since the shape reduces stress on the sample while being cut. An exemplary chaperone sample in hexagonal shape is shown in Figure 4A and 4C. In some embodiments of the chaperone block of the invention, the chaperone block and/or the chaperone sample may be in the form of a polygon. In some embodiments of the chaperone block of the invention, the chaperone block and/or the chaperone sample may be in the form of a, or may have the form of a circle, ellipse, triangle, such as a equilateral triangle, isosceles triangle, scalene triangle, right triangle, square, rectangle, parallelogram, rhombus (or diamond), trapezoid (or trapezium), pentagon, hexagon, heptagon (or septagon), octagon, nonagon (or enneagon), decagon, or dodecagon and the like.

Accordingly, the invention provides in one preferred aspect a chaperone block comprising at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities, wherein the chaperone block and/or the chaperone sample has a hexagonal shape.

The invention also provides in a preferred aspect a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample and a top layer of resin, wherein the chaperone block comprises a multitude of cavities, wherein the chaperone block and/or the chaperone sample has a hexagonal shape.

The chaperone block of the present invention may have or be in the same shape as the chaperone sample as defined above. Accordingly, features defined above in context of the shape of the chaperone sample apply to the chaperone block mutatis mutandis. Accordingly, in some embodiments, the chaperone block has a hexagonal shape.

The chaperone block and/or the chaperone sample may have any size. In Example 1, a chaperone block has been produced in a size of approximately about 2 mm times 2 mm, i.e. 2 mm². In general, the chaperone block and/or chaperone sample can have any size/area that is desired, in particular, the size/area may depend on the number of cavities comprised in the chaperone block. As used herein “size” refers to the size of the chaperone block and/or chaperone sample in the x- and y- axis. As used herein “thickness” refers to the size of a chaperone block, chaperone sample, screening sample, tissue sample of interest, and/or resin layer in the z-axis. This can be seen in Figure 4A, where the top left panel shows the x- and y-axis and the top right and bottom left panel show the z-axis. In general, the cavities are introduced along the z-axis of the chaperone block.

Accordingly, in some embodiments the chaperone block and/or the chaperone sample may have a size of about 1mm to 10cm in the x-axis and a size of about 1mm to 10cm in the y axis. Preferably, the chaperone block and/or the chaperone sample may have a size of about 5cm to 10cm in the x- axis and a size of about 5cm to 10cm in the y axis. In some embodiments, the chaperone block and/or the chaperone sample may have a size of about 1mm to 1cm in the x-axis and a size of about 1mm to 1cm in the y axis. In some embodiments, the chaperone block and/or the chaperone sample may have a size of about 2mm in the x-axis and a size of about 2mm in the y axis and a thickness of about 500pm to about 1500pm.

In some embodiments, the chaperone block and/or the chaperone sample may have an area of about 1mm² to about 10cm², wherein the area is the area defined by the x- and y- axis. In some embodiments, the chaperone block and/or the chaperone sample may have an area of about 5cm² to about 10cm², wherein the area is the area defined by the x- and y- axis. In some embodiments, the chaperone block and/or the chaperone sample may have an area of about 1mm² to about 1cm², wherein the area is the area defined by the x- and y- axis.

Since the chaperone sample will ultimately be cut, the thickness of the chaperone sample determines the number of sections that can be obtained from the chaperone block. At the same time, the thickness of the chaperone sample determines the maximum of thickness of a tissue sample of interest that may be loaded into the chaperone block. Accordingly, the thickness of the chaperone sample and the tissue sample of interest may be increased or decreased to modify the number of sections that can be obtained from a single chaperone block. In general, the chaperone sample and the tissue sample of interest may be the same or substantially same thickness. This is because only the part of tissue sample of interest that has been entirely loaded into the chaperone sample can be cut and subsequently analyzed. Accordingly, in one embodiment, the chaperone sample and the tissue sample of interest are the same thickness. The thickness of the chaperone and/or the tissue sample of interest may be scaled depending on the desired application. Accordingly, in one embodiment the thickness of the chaperone sample and/or the tissue sample of interest is at least about 100 pm to 5000 pm, such as about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, about 300 pm, about 310 pm, about 320 pm, about 330 pm, about 340 pm, about 350 pm, about 360 pm, about 370 pm, about 380 pm, about 390 pm, about 400 pm, about 410 pm, about 420 pm, about 430 pm, about 440 pm, about 450 pm, about 460 pm, about 470 pm, about 480 pm, about 490 pm, about 500 pm, about 510 pm, about 520 pm, about 530 pm, about 540 pm, about 550 pm, about 560 pm, about 570 pm, about 580 pm, about 590 pm, about 600 pm, about 610 pm, about 620 pm, about 630 pm, about 640 pm, about 650 pm, about 660 pm, about 670 pm, about 680 pm, about 690 pm, about 700 pm, about 710 pm, about 720 pm, about 730 pm, about 740 pm, about 750 pm, about 760 pm, about 770 pm, about 780 pm, about 790 pm, about 800 pm, about 810 pm, about 820 pm, about 830 pm, about 840 pm, about 850 pm, about 860 pm, about 870 pm, about 880 pm, about 890 pm, about 900 pm, about 910 pm, about 920 pm, about 930 pm, about 940 pm, about 950 pm, about 960 pm, about 970 pm, about 980 pm, about 990 pm, about 1000 pm, about 1010 pm, about 1020 pm, about 1030 pm, about 1040 pm, about 1050 pm, about 1060 pm, about 1070 pm, about 1080 pm, about 1090 pm, about 1100 pm, about 1110 pm, about 1120 pm, about 1130 pm, about 1140 pm, about 1150 pm, about 1160 pm, about 1170 pm, about 1180 pm, about 1190 pm, about 1200 pm, about 1210 pm, about 1220 pm, about 1230 pm, about 1240 pm, about 1250 pm, about 1260 pm, about 1270 pm, about 1280 pm, about 1290 pm, about 1300 pm, about 1310 pm, about 1320 pm, about 1330 pm, about 1340 pm, about 1350 pm, about 1360 pm, about 1370 pm, about 1380 pm, about 1390 pm, about 1400 pm, about 1410 pm, about 1420 pm, about 1430 pm, about 1440 pm, about 1450 pm, about 1460 pm, about 1470 pm, about 1480 pm, about 1490 pm, about 1500 pm, about 1510 pm, about 1520 pm, about 1530 pm, about 1540 pm, about 1550 pm, about 1560 pm, about 1570 pm, about 1580 pm, about 1590 pm, about 1600 pm, about 1610 pm, about 1620 pm, about 1630 pm, about 1640 pm, about 1650 pm, about 1660 pm, about 1670 pm, about 1680 pm, about 1690 pm, about 1700 pm, about 1710 pm, about 1720 pm, about 1730 pm, about 1740 pm, about 1750 pm, about 1760 pm, about 1770 pm, about 1780 pm, about 1790 pm, about 1800 pm, about 1810 pm, about 1820 pm, about 1830 pm, about 1840 pm, about 1850 pm, about 1860 pm, about 1870 pm, about 1880 pm, about 1890 pm, about 1900 pm, about 1910 pm, about 1920 pm, about 1930 pm, about 1940 pm, about 1950 pm, about 1960 pm, about 1970 pm, about 1980 pm, about 1990 pm, about 2000 pm, about 2100 pm, about 2200 pm, about 2300 pm, about 2400 pm, about 2500 pm, about 2600 pm, about 2700 pm, about 2800 pm, about 2900 pm, about 3000 pm, about 3100 pm, about 3200 pm, about 3300 pm, about 3400 pm, about 3500 pm, about 3600 pm, about 3700 pm, about 3800 pm, about 3900 pm, about 4000 pm, about 4100 pm, about 4200 pm, about 4300 pm, about 4400 pm, about 4500 pm, about 4600 pm, about 4700 pm, about 4800 pm, about 4900 pm, or about 5000 pm. In a preferred embodiment, the thickness of the chaperone sample and/or the tissue sample of interest is about 500 pm.

Accordingly, the invention provides in one preferred aspect a chaperone block comprising at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities, wherein the chaperone sample and/or the tissue sample of interest is at least about 100 pm to 5000 pm, preferably at least about 500 pm, more preferably about 500 pm thick.

The invention also provides in a preferred aspect a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample and a top layer of resin, wherein the chaperone block comprises a multitude of cavities, wherein the chaperone sample and/or the tissue sample of interest is at least about 100 pm to 5000 pm, preferably at least about 500 pm, more preferably about 500 pm thick.

As mentioned above, the dimensions of the chaperon sample are of variable size. The chaperon sample and tissue sample of interest may be the same or substantially same thickness. Accordingly, in some embodiments, the chaperone block of the present invention comprises the chaperone sample, wherein the chaperone sample is between about 100 and 2000 pm thick. In some embodiments, the chaperone block of the present invention comprises the tissue sample of interest, wherein the tissue sample of interest is between about 100 and 2000 pm thick. In some embodiments, the chaperone block of the present invention comprises the chaperone sample, wherein the chaperone sample is between about 100 and 1000 pm thick. In some embodiments, the chaperone block of the present invention comprises the tissue sample of interest, wherein the tissue sample of interest is between about 100 and 1000 pm thick. In some embodiments, the chaperone block of the present invention comprises the chaperone sample and the tissue sample of interest, wherein the chaperone sample and/or the tissue sample of interest is/are between about 100 and 1000 pm thick. In some embodiments, the chaperone block of the present invention comprises the chaperone sample, wherein the chaperone sample is about 500 pm thick. In some embodiments, the chaperone block of the present invention comprises the tissue sample of interest, wherein the tissue sample of interest is about 500 pm thick. In some embodiments, the chaperone block of the present invention comprises the chaperone sample and the tissue sample of interest, wherein the chaperone sample and/or the tissue sample of interest is/are about 500 pm thick. In some embodiments, the chaperone block of the present invention comprises the chaperone sample and the tissue sample of interest, wherein the chaperone sample and the tissue sample of interest are the same or substantially same thickness. In a preferred embodiment, the chaperone block of the present invention comprises the chaperone sample and the tissue sample of interest, wherein the chaperone sample and the tissue sample of interest are about 500 pm thick and wherein the chaperone sample and the tissue sample of interest have the same thickness.

As used herein a “layer of resin” or a “resin layer” in general is a layer of resin arranged in a block, e.g. chaperone block or screening sample of the present invention. In general, the invention is not limited to any kind of resin, the skilled person is rather capable of choosing a feasible resin composition to securely embed a chaperone sample. The resin provides structural integrity to the chaperone sample allowing for later cutting of the sample, e.g. when loaded with screening samples. A resin is initially viscous when mixed and used. After the resin has been in an oven, e.g. at 60°C, it becomes hard, so that a defined amount can be trimmed away e.g. from the top, with a cutter/milling cutter, e.g. a diamond cutter.

In the context of the present invention, resin refers to a natural or synthetic organic compound, which can be used for embedding samples, such as biological tissue samples. Embedding tissue samples in resin allows the preservation of tissue and intracellular structures. In the context of the present invention, the term “resin infiltration” or “infiltration” refers to treating a tissue sample, such as a chaperone sample and/or a tissue sample of interest with a resin that penetrates throughout the tissue to the molecular level, subsequently hardens and finally solidifies thereby rendering the sample rigid. The resin used herein may be used to form one or more separate layers. In general, the size, shape, and/or thickness of the resin and/or the resin layers are not particularly limited. The skilled person is aware that size, shape and/or thickness may depend on the specific application of the chaperone block, e.g. when a machine for processing requires certain dimensions. A lower layer of resin may refer to a layer of resin that is located underneath a tissue sample, such as a chaperone sample. A lower layer of resin may have any thickness that is suitable to prevent direct contact of a tissue sample, such as a chaperone sample, with the environment below the tissue sample. In some embodiments, the lower layer of resin may be positioned on or may be placed onto a pin, e.g. aluminum pin. A top layer of resin may refer to a layer or resin that is located on top of a tissue sample, such as a chaperone sample. A top layer of resin may facilitate insertion of a screening sample into the chaperone block comprising a chaperone sample. A top layer of resin may further facilitate breaking of a sample, which is inserted into the chaperone block, at a predetermined breaking point. An example of such a sample, is a screening sample, which has been milled to a predefined shape and inserted into the chaperone block. Breaking occurs in such a way that the tissue sample of interest of the screening sample is not broken but loaded into the chaperone sample.

Resins used herein are preferably epoxy resins and include but are not limited to commercially available epoxy resins such as Epon 812 substitute resin, resin prepared according to Spurr, and the like. The resin according to Spurr (Spurr’ s resin, 10 g) consists of 3,4-epoxycyclohexylmethyl 3,4-epoxycyclo-hexaneca (ERL 4221, 4.1 g), diglycidyl ether of polypropylene glycol (DER 736, 0.95 g), nonenyl succinic anhydride (NSA, 5.9 g) and dimethylaminoethanol (DMAE, preferably 11 to 113 pl, more preferably 55 to 113 pl, even more preferably 113 pl). According to the present invention, the Epon 812 substitute resin (Epon 812, 10 g) consist of tri-glycidyl ether of glycerol (5.9 g), Methyl nadic anhydride (MNA, 3.7 g), Dodecenylsuccinic anhydride (DDSA, 2.25 g), 2,4,6-Tris(dimethylaminomethyl)phenol (DMP, 20 to 205 pl, more preferably 100 to 205 pl, even more preferably 205 pl). In order to achieve a homogenous resin infiltration, the methods of the present invention relate to the use of a low viscosity resin infiltration method, wherein the concentration of the resin is gradually increased. The resin may be diluted by any suitable means known in the art, including organic solvents such as alcohols, ketones and the like. The resin may be dissolved in acetone.

In some embodiments, the resin may comprise metal. In some embodiments, the resin may be a mixture of one or more resins.

The present invention provides in one aspect, a chaperone block comprising at least two layers of resin and at least one layer of a chaperone sample. In some embodiments, the chaperone block comprises the at least one layer of a chaperone sample which is arranged on top of the lower layer of the resin. In some embodiments, the chaperone block comprises two layers of resin, wherein the chaperone sample is arranged in between the two layers of resin. In some embodiments, the chaperone block comprises a lower layer of resin, a middle layer of a chaperone sample, and a top layer of resin. In a preferred embodiment, the resin is Epon 812.

Accordingly, the invention provides in one preferred aspect a chaperone block comprising at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities, wherein the resin is Epon 812.

The invention also provides in a preferred aspect a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample and a top layer of resin, wherein the chaperone block comprises a multitude of cavities, wherein the resin is Epon 812.

The size or shape of the resin layers is not particularly limited. In particular, size or shape of the resin layers may depend on the intended use of the sample. For example, a large sized chaperone sample may require similarly large sized resin layers to accommodate the chaperone sample. Thus, the dimensions of the resin layers should at least be configured to accommodate the chaperone sample, e.g. the resin layers should be sized so that the chaperone sample can be embedded. In general, the dimensions of the resin layers are not particular limited and may depend on the size of the chaperone sample and other considerations, e.g. particular dimensions of processing machines. The skilled person is capable of choosing appropriate dimensions of the resin layers.

In general, the lower layer of resin has dimensions that allow for introducing cavities penetrating the chaperone sample but not any material that may be beneath the lower layer of resin. For example, the lower layer of resin may be placed onto an aluminum pin to avoid that when cavities are introduced in the chaperone sample, the aluminum pin is damaged. Thus, the lower layer of resin may serve as a buffer between e.g. a pin onto which the chaperone sample is placed. Furthermore, dimensions of the lower layer of resin may be configured to provide a buffer when cutting the chaperone sample loaded with the tissue sample of interests to that the chaperone sample can be cut throughout its entire thickness without reaching the bottom of the chaperone block. Accordingly, in some embodiments the thickness of the lower layer of resin is at least about 200 pm to 2000 pm, such as about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, about 300 pm, about 310 pm, about 320 pm, about 330 pm, about 340 pm, about 350 pm, about 360 pm, about 370 pm, about 380 pm, about 390 pm, about 400 pm, about 400 pm, about 410 pm, about 420 pm, about 430 pm, about 440 pm, about 450 pm, about 460 pm, about 470 pm, about 480 pm, about 490 pm, about 500 pm, about 510 pm, about 520 pm, about 530 pm, about 540 pm, about 550 pm, about 560 pm, about 570 pm, about 580 pm, about 590 pm, about 600 pm, about 610 pm, about 620 pm, about 630 pm, about 640 pm, about 650 pm, about 660 pm, about 670 pm, about 680 pm, about 690 pm, about 700 pm, about 710 pm, about 720 pm, about 730 pm, about 740 pm, about 750 pm, about 760 pm, about 770 pm, about 780 pm, about 790 pm, about 800 pm, about 810 pm, about 820 pm, about 830 pm, about 840 pm, about 850 pm, about 860 pm, about 870 pm, about 880 pm, about 890 pm, about 900 pm, about 910 pm, about 920 pm, about 930 pm, about 940 pm, about 950 pm, about 960 pm, about 970 pm, about 980 pm, about 990 pm, about 1000 pm, about 1010 pm, about 1020 pm, about 1030 pm, about 1040 pm, about 1050 pm, about 1060 pm, about 1070 pm, about 1080 pm, about 1090 pm, about 1100 pm, about

1110 pm, about 1120 pm, about 1130 pm, about 1140 pm, about 1150 pm, about 1160 pm, about

1170 pm, about 1180 pm, about 1190 pm, about 1200 pm, about 1210 pm, about 1220 pm, about

1230 pm, about 1240 pm, about 1250 pm, about 1260 pm, about 1270 pm, about 1280 pm, about 1290 pm, about 1300 pm, about 1310 pm, about 1320 pm, about 1330 pm, about 1340 pm, about

1350 pm, about 1360 pm, about 1370 pm, about 1380 pm, about 1390 pm, about 1400 pm, about

1410 pm, about 1420 pm, about 1430 pm, about 1440 pm, about 1450 pm, about 1460 pm, about

1470 pm, about 1480 pm, about 1490 pm, about 1500 pm, about 1510 pm, about 1520 pm, about

1530 pm, about 1540 pm, about 1550 pm, about 1560 pm, about 1570 pm, about 1580 pm, about

1590 pm, about 1600 pm, about 1610 pm, about 1620 pm, about 1630 pm, about 1640 pm, about

1650 pm, about 1660 pm, about 1670 pm, about 1680 pm, about 1690 pm, about 1700 pm, about

1710 pm, about 1720 pm, about 1730 pm, about 1740 pm, about 1750 pm, about 1760 pm, about

1770 pm, about 1780 pm, about 1790 pm, about 1800 pm, about 1810 pm, about 1820 pm, about

1830 pm, about 1840 pm, about 1850 pm, about 1860 pm, about 1870 pm, about 1880 pm, about

1890 pm, about 1900 pm, about 1910 pm, about 1920 pm, about 1930 pm, about 1940 pm, about

1950 pm, about 1960 pm, about 1970 pm, about 1980 pm, about 1990 pm, or about 2000 pm. In a preferred embodiment, the thickness of the lower layer of resin is about 200 pm to 500 pm, such as about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, about 300 pm, about 310 pm, about 320 pm, about 330 pm, about 340 pm, about 350 pm, about 360 pm, about 370 pm, about 380 pm, about 390 pm, about 400 pm, about 410 pm, about 420 pm, about 430 pm, about 440 pm, about 450 pm, about 460 pm, about 470 pm, about 480 pm, about 490 pm, or about 500 pm. In a further preferred embodiment, the thickness of the lower layer of resin is about 500 pm.

In general, the top layer of resin may be configured to provide structural support to a screening sample when loaded into the chaperone block. The loading procedure is explained below in further detail. In brief, when loading the chaperone block, the tissue sample of interest will be introduced into the cavity of the chaperone sample, whereas the resin layer of the screening sample will be introduced into the cavity of the top layer of resin of the chaperone block. This is shown for example in Figure 3B. This ensures that the screening sample breaks of at the predetermined breaking point and allows loading of the tissue sample of interest into the chaperone sample. Thus, it is advantageous, that the chaperone sample and the tissue sample of interest are the same or substantially the same thickness. Furthermore, it is advantageous that the top layer of resin and the layer of resin of the screening sample are the same or substantially the same thickness. This way, the chaperone sample and the tissue sample of interest will overlap in the loaded chaperone block. Furthermore, the top layer of resin will overlap with the layer of resin of the screening sample. Accordingly, in some embodiments, the top layer of resin and the layer of resin of the screening sample are the same or substantially the same thickness. Accordingly, in some embodiments the thickness of the top layer of resin is at least about 200 pm to 2000 pm, such as about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, about 300 pm, about 310 pm, about 320 pm, about 330 pm, about 340 pm, about 350 pm, about 360 pm, about 370 pm, about 380 pm, about 390 pm, about 400 pm, about 400 pm, about 410 pm, about 420 pm, about 430 pm, about 440 pm, about 450 pm, about 460 pm, about 470 pm, about 480 pm, about 490 pm, about 500 pm, about 510 pm, about 520 pm, about 530 pm, about 540 pm, about 550 pm, about 560 pm, about 570 pm, about 580 pm, about 590 pm, about 600 pm, about 610 pm, about 620 pm, about 630 pm, about 640 pm, about 650 pm, about 660 pm, about 670 pm, about 680 pm, about 690 pm, about 700 pm, about 710 pm, about 720 pm, about 730 pm, about 740 pm, about 750 pm, about 760 pm, about 770 pm, about 780 pm, about 790 pm, about 800 pm, about 810 pm, about 820 pm, about 830 pm, about 840 pm, about 850 pm, about 860 pm, about 870 pm, about 880 pm, about 890 pm, about 900 pm, about 910 pm, about 920 pm, about 930 pm, about 940 pm, about 950 pm, about 960 pm, about 970 pm, about 980 pm, about 990 pm, about 1000 pm, about 1010 pm, about 1020 pm, about 1030 pm, about 1040 pm, about 1050 pm, about 1060 pm, about 1070 pm, about

1080 pm, about 1090 pm, about 1100 pm, about 1110 pm, about 1120 pm, about 1130 pm, about

1140 pm, about 1150 pm, about 1160 pm, about 1170 pm, about 1180 pm, about 1190 pm, about

1200 pm, about 1210 pm, about 1220 pm, about 1230 pm, about 1240 pm, about 1250 pm, about

1260 pm, about 1270 pm, about 1280 pm, about 1290 pm, about 1300 pm, about 1310 pm, about

1320 pm, about 1330 pm, about 1340 pm, about 1350 pm, about 1360 pm, about 1370 pm, about

1380 pm, about 1390 pm, about 1400 pm, about 1410 pm, about 1420 pm, about 1430 pm, about

1440 pm, about 1450 pm, about 1460 pm, about 1470 pm, about 1480 pm, about 1490 pm, about

1500 pm, about 1510 pm, about 1520 pm, about 1530 pm, about 1540 pm, about 1550 pm, about

1560 pm, about 1570 pm, about 1580 pm, about 1590 pm, about 1600 pm, about 1610 pm, about

1620 pm, about 1630 pm, about 1640 pm, about 1650 pm, about 1660 pm, about 1670 pm, about

1680 pm, about 1690 pm, about 1700 pm, about 1710 pm, about 1720 pm, about 1730 pm, about

1740 pm, about 1750 pm, about 1760 pm, about 1770 pm, about 1780 pm, about 1790 pm, about

1800 pm, about 1810 pm, about 1820 pm, about 1830 pm, about 1840 pm, about 1850 pm, about

1860 pm, about 1870 pm, about 1880 pm, about 1890 pm, about 1900 pm, about 1910 pm, about

1920 pm, about 1930 pm, about 1940 pm, about 1950 pm, about 1960 pm, about 1970 pm, about

1980 pm, about 1990 pm, or about 2000 pm.

In a preferred embodiment, the thickness of the top layer of resin is about 200 pm to 500pm, such as about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, about 300 pm, about 310 pm, about 320 pm, about 330 pm, about 340 pm, about 350 pm, about 360 pm, about 370 pm, about 380 pm, about 390 pm, about 400 pm, about 410 pm, about 420 pm, about 430 pm, about 440 pm, about 450 pm, about 460 pm, about 470 pm, about 480 pm, about 490 pm, or about 500 pm. In a further preferred embodiment, the thickness of the top layer of resin is about 500 pm.

A particular shape of the resin layers may be beneficial for efficient cutting. In general, the resin layers may be in the same or substantially same shape as the chaperone sample as described herein above.

Accordingly, the invention provides in one preferred aspect a chaperone block comprising at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities, wherein the chaperone sample and/or the tissue sample of interest is at least about 100 pm to 5000 pm, preferably at least about 500 pm, more preferably about 500 pm thick, and wherein the at least two layers of resin are at least about 100 pm to 5000pm, preferably about 500 pm thick.

The invention also provides in a preferred aspect a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample and a top layer of resin, wherein the chaperone block comprises a multitude of cavities, wherein the chaperone sample and/or the tissue sample of interest is at least about 100 pm to 5000 pm, preferably at least about 500 pm, more preferably about 500 pm thick, and wherein the lower and top layer of resin are at least about 100 pm to 5000 pm, preferably about 500 pm thick.

In some embodiments, the chaperone block of the present invention comprises a chaperone sample and a top layer of resin, wherein the chaperone sample and the top layer of resin are the same thickness. In some embodiments, the chaperone block of the present invention comprises a chaperone sample and a top layer of resin, wherein the chaperone sample and the top layer of resin do not have the same thickness. In a preferred embodiment, the chaperone block of the present invention comprises a chaperone sample and a top layer of resin, wherein the chaperone sample and the top layer of resin are each about 500 pm thick.

As mentioned herein above, the chaperone can be loaded with screening samples or the part of the screening sample comprising the tissue samples of interest. This can be realized by loading part of a screening sample, e.g. the part comprising the tissue into a cavity of the chaperone block. The loading of the chaperone block is explained below in further detail in the context of the apparatus of the present invention. Accordingly, the chaperone block of the present invention may also be loaded with one or more screening samples. Preferably, a screening sample is loaded into each of the cavities of the chaperone block, thereby optimizing the number of samples that can be analyzed using a single chaperone block. However, the skilled person is aware that due to technical restraints it may not be possible to load all cavities of the chaperone block, e.g. because a cavity is block by debris or a screening sample that may have not been properly loaded can block a cavity. Thus, in one embodiment the chaperone block comprises a screening sample in one or more of the multitude of cavities. In a preferred embodiment, the chaperone block comprises a screening sample in at least about 50 % to 100 %, such as about 50 %, about 55 %, about 60 %, about 65 %, about 70 %, about 75 %, about 80 %, about 85 %, about 90 %, about 90 %, about 91 %, about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 %, or about 100 % of cavities. In a further preferred embodiment, the chaperone block comprises a screening sample in each cavity.

Accordingly, the invention provides in one preferred aspect a chaperone block comprising at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities, wherein the chaperone block comprises a screening sample in one or more of the multitude of cavities, preferably comprising a screening sample in each cavity.

The invention also provides in a preferred aspect a chaperone block comprising a lower layer of resin, a middle layer of chaperone sample and a top layer of resin, wherein the chaperone block comprises a multitude of cavities, wherein the chaperone block comprises a screening sample in one or more of the multitude of cavities, preferably comprising a screening sample in each cavity. In some embodiments, the chaperone block of the present invention does not comprise a screening sample in any cavity. In some embodiments, the chaperone block comprises a screening sample in some cavities. In a preferred embodiment, the chaperone block comprises a screening sample in each cavity.

The chaperone sample may be an unprocessed or a processed tissue sample. As used herein, “processed tissue sample” refers to laboratory and preparatory methods known to a person skilled in the art, in particular to methods for preparing a tissue sample for EM imaging (Gour et al., 2021; Loomba et al., 2022; Motta et al., 2019; Shapson-Coe et al., 2024; Sievers et al., 2024). Such methods include for example tissue staining, fixation, embedding, infiltration, dehydration, washing, heavy metal treatment and the like. This is also explained in detail in the section regarding a method to produce a chaperone block of the present invention below.

The chaperone block may be configured to hold one or more screening samples for parallel imaging and processing of said screening samples for 3 -dimensional electron microscopy -based connectomic screening. The chaperone block homogenizes the cutting surface of the tissue sample of interest and confers a homogenous electrical conductivity, leading to improved stability of the screening sample and to improved overhead reduction. Thus, by using a chaperone sample a magnitude of screening samples can be analyzed in parallel, resulting in a fast, reliable, and reproducible method for 3 -dimensional electron microscopy -based connectomic screening. In general, the shape, thickness and size of the chaperone block is not particularly limited. However, some shapes have the advantage that slicing or cutting of the chaperone block is facilitated.

In some embodiments, the chaperone block of the present invention comprises the chaperone sample, wherein the chaperone sample comprises a tissue sample. In some embodiments, the chaperone block of the present invention comprises the screening sample, wherein the screening sample comprises a tissue sample. In some embodiments, the chaperone block of the present invention comprises the chaperone sample and the screening sample, wherein the chaperone sample and/or the screening sample comprise(s) a tissue-derived sample (i.e. the “tissue sample of interest” in the case of the screening sample). In some embodiments, the chaperone block of the present invention comprises the chaperone sample and the screening sample each comprising a tissue-derived sample, wherein the tissue-derived samples are tissues of a mammal, reptile, fish, or bird, preferably tissues of a mouse, macaque, human, rat, ferret, rabbit, marmoset, Komodo dragon, turtle, salamander, zebrafish, zebra finch, or pigeon, more preferably tissues of a mouse brain or of a macaque brain or human brain. In some embodiments, the chaperone block of the present invention comprises the chaperone sample and the screening sample, wherein the chaperone sample and the screening sample comprise tissues from mouse brain. In some embodiments, the chaperone block of the present invention comprises the chaperone sample and the screening sample, wherein the chaperone sample and the screening sample comprise tissues from macaque brain. In some embodiments, the chaperone block of the present invention comprises the chaperone sample and the screening sample, wherein the chaperone sample and the screening sample comprise tissues from human brain. In some embodiments, the chaperone block of the present invention comprises the chaperone sample and the screening sample, wherein the chaperone sample and the screening sample comprise tissues district from each other. In some embodiments, the chaperone block of the present invention comprises the chaperone sample and the screening sample, wherein the chaperone sample comprises tissue of mouse brain and the screening sample comprises tissue of a macaque brain. In some embodiments, the chaperone block of the present invention comprises the chaperone sample and the screening sample, wherein the chaperone sample comprises tissue of macaque brain and the screening sample comprises tissue of a mouse brain.

Method for producing a chaperone block

The invention also provides a method for producing a chaperone block, e.g. the chaperone block of the invention as defined herein. An exemplary method for producing a chaperone block of the present invention is described in detail in Example 1.

Accordingly, the invention provides in one aspect a method for producing the chaperone block of the invention, the method comprising:

(a) embedding a chaperone sample onto a lower layer of resin,

(b) adding a top layer of resin on top of the chaperone sample,

The chaperone block produced by the method can comprise any of the features described herein in the context of the chaperone block of the invention. Accordingly, the above-described advantages apply mutatis mutandis to the method for producing a chaperone block. Furthermore, any of the features described in the context of the chaperone block as a product herein, apply mutatis mutandis to the method for producing a chaperone block with the provision that said features are formulated as method steps rather than product features.

The method for producing a chaperone block can further comprises one or more of the following steps:

• Trimming of the top layer of resin from top so that the top layer of resin is about 500pm thick

• Trimming of an alignment plane onto a pin, e.g. an aluminum pin comprising the chaperone block for orientation of the block in subsequent steps

• Putting the pin, e.g. aluminum pin, in a 3D printed holder

• Take a picture of the chaperone block with binocular from top

• Defining the chaperone sample position, sample shape and cavities in CAD software • Putting the chaperone block into a milling machine

• Milling the chaperone block into a pre-defined shape, e.g. using a CAM software

• Introducing cavities, e.g. with a high performance drill

• Washing the chaperone block in an ultrasonic bath to remove debris, e.g. dust. Chaperone block may be washed for several seconds.

• Performing a micro computed tomography scan (pCT) to check the cavities and their offset.

The method for producing a chaperone block according to the present invention preferably comprises one or more of the following steps, preferably in chronological order:

Provision of biological tissue from a region of interest, e.g. from a brain of a mammal, reptile, fish, or bird. It is desirable for the tissue to comprise neurons. The mammal can be any mammal, such as a mouse, a human, or a macaque. The tissue serves as the chaperone sample.

Fixing the chaperone sample using an appropriate fixative according to methods known to the person skilled in the art to preserve the intracellular structures of the tissue. Appropriate fixatives include PFA 4%-40%, Osmium 2-4%, Ethanol 50-10%, Glutaraldehyde 50-2.5%, or 2-Propanol 50-10%. In general, any of the fixatives used in Sivers et al. 2024 and Loomba et al 2022, which are incorporated herein in their entirety, can be used in the sense of the sense of the present invention. A fixative in the sense of the present invention is preferably paraformaldehyde and/or glutaraldehyde. A fixative is preferably used comprised in a cacodylate buffer. An alternative fixative may be osmium.

Coronal or sagittal sections of the chaperone sample with an appropriate size are prepared e.g. using a vibratome or the like. The sections may preferably be about 500 pm thick.

Tissue sections are cut to an appropriate size, e.g. using razor blades or the like. Preferably, the tissue-derived sample does not comprise ventricles. As used herein, this tissue-derived sample is referred to as chaperone sample.

Staining of the chaperone sample, e.g. to facilitate microscopy -based image acquiring. The chaperone sample can be imaged using electron microscopy. To image tissue samples with an electron microscope, the tissue can be stained using heavy -metal staining e.g. to increase the contrast. Staining methods are known to a skilled person. The chaperone sample may be stained according to the protocol of Hua et al. 2015 or Song et al., 2023, which are incorporated herein by reference in its entirety.

Dehydrating the stained chaperone sample.

Infiltrating the chaperone sample with a resin. The resin may be a resin that penetrates throughout the tissue to the molecular level, subsequently hardens and finally solidifies thereby rendering the sample rigid.

Providing a metal pin. The metal of the pin is preferably a metal that is not magnetic, does not dissolve when in contact with resin, can withstand temperatures above 60°C, and is suitable to be milled. A suitable metal may be aluminum. Accordingly, in some embodiments a pin or a metal pin of the present invention is an aluminum pin, also referred herein “alupin”. The preparation of the aluminum pin may comprise treating the surface of the aluminum pin with sandpaper, placing a tube on the aluminum pin, filling the tube with resin, curing in the oven for 1-3 days, removing the tube, trimming the surface so that only a defined thickness of the resin layer remains, wherein a suitable thickness of the resin layer is preferably about 500 pm, treating the surface with sandpaper, and placing a tube on the aluminum metal pin with the resin layer. The layer of resin which is on top of the aluminum pin is referred to as lower layer of resin in the context of the present invention.

Embedding the chaperone sample on the lower layer of resin of the metal pin, e.g. aluminum pin. This step may comprise embedding the chaperone sample in resin on top of the lower layer of resin.

Adding a top layer of resin on top of the chaperone sample. Thus, the chaperone sample is embedded in resin on the metal pin, e.g. aluminum pin, as a middle layer between the lower layer and the top layer of resin. As used herein, the lower level of resin, the middle layer of the chaperone sample, and the top layer of resin are comprised in the chaperone block of the present invention.

Curing the chaperone block in an oven for about 3 days.

Trimming the top layer of resin to an appropriate thickness. A preferred thickness of the top layer of resin is about 500 pm. The top layer may be trimmed to any thickness as defined above in the context of the chaperone block. Trimming may, for example, be performed by using a diamond head milling system. The top layer of resin may be trimmed to the same or substantially same thickness as the resin layer of the screening sample.

Trimming an alignment plane into the metal pin, e.g. aluminum pin. The trimming may, for example be done by milling using a 6 mm-diameter aluminum milling cutter on a milling machine. The alignment plane on the aluminum pin helps to maintain a steady position of the chaperone block in an apparatus/machine for further processing of the chaperone block. Thus, the alignment plane improves orientation.

Placing the chaperone block together with the metal pin, e.g. aluminum pin into an appropriate holder. This may be for example a custom-made 3D-printed holder or mold. By placing chaperone block with the metal pin into such an appropriate holder imaging is facilitated. Imaging may be performed with any suitable method, for example at a magnification of l.Ox by using a microscope which is connected to a camera. This is to layout cavities and offset of the chaperone block.

Imaging the chaperone block. Based on this image the exact position of the chaperone sample, the shape of the chaperone sample, the position of the cavities, and the shape of the cavities can be defined using a suitable software, for example a CAD software.

Milling the chaperone block into a pre-defined shape, e.g. using a suitable software, such as a CAM software, and a suitable milling cutter, such as a 6mm-diameter aluminum milling cutter. The shape may be any shape defined herein for the chaperone sample and/or the chaperone block. In a preferred embodiment, the chaperone block is milled into a hexagonal shape.

Optionally milling outlines of a multitude of cavities into the chaperone block. The form of the cavities may be any of the forms defined herein in context of the chaperone block.

Introducing a multitude of cavities into the chaperone block. The location of the cavities may be indicated by the milled outlines of the cavities. The one or more cavities may, for example be introduced by drilling, hammering, screwing, milling, hollowing out, carving, etching, melting, punching, pricking, grating, piercing, perforating, puncturing, spiking and the like. The cavities fully penetrate the top layer of resin. The cavities may either partially or fully penetrate the chaperone sample. The cavities may be of any suitable shape as defined above. The diameter of the cavities may correspond to the diameter of the outlines of the cavities. The number of the cavities may correspond to the number of outlines of the cavities. The arrangement of the cavities may correspond to the arrangement of the outlines of the cavities. Washing the chaperone block to remove debris. The washing step may be performed by putting the chaperone block into an ultrasonic bath to remove debris, e.g. dust. The chaperone block may be washed for several seconds. This is advantageous for proper loading of screening samples into the chaperone block.

Assessing the quality of the cavities and the offset walls by acquiring an image, such as a pCT image. As used herein a high quality of cavities are cavities that are uniform and have the desired shape, size, arrangement/offset, and location. As used herein, checking the quality of the offset means that the offset walls of the chaperone sample separating each cavity from each other are intact and that the offset walls have the desired, pre-defined thickness.

Accordingly, the present invention provides in one aspect a method for producing a chaperone block preferably the chaperone block of the present invention, the method comprising:

(a) embedding a chaperone sample onto a lower layer of resin,

(b) adding a top layer of resin on top of the chaperone sample,

The method may further comprise playing the lower layer of resin onto a metal pin, e.g. an aluminum pin. This can be advantageous for processing the chaperone block, e.g. when introducing the cavities.

Accordingly, the invention provides a method for producing a chaperone block, the method comprising:

(a) embedding a chaperone sample onto a lower layer of resin which is paced onto an aluminum pin,

(b) adding a top layer of resin on top of the chaperone sample,

(c) introducing a multitude of cavities penetrating the top layer of resin and at least partially penetrating the chaperone sample, optionally further comprising milling an alignment plane into the aluminum pin.

The invention also provides a method for producing a chaperone block, the method comprising:

(a) embedding a chaperone sample onto a lower layer of resin,

(b) adding a top layer of resin on top of the chaperone sample,

(c) introducing a multitude of cavities penetrating the top layer of resin and at least partially penetrating the chaperone sample, further comprising staining the chaperone sample, e.g. with a heavy metal treatment.

(a) embedding a chaperone sample onto a lower layer of resin,

(b) adding a top layer of resin on top of the chaperone sample,

(c) introducing a multitude of cavities penetrating the top layer of resin and at least partially penetrating the chaperone sample, further comprising dehydrating and/or infiltrating the chaperone sample with a resin.

(a) embedding a chaperone sample onto a lower layer of resin,

(b) adding a top layer of resin on top of the chaperone sample,

(c) introducing a multitude of cavities penetrating the top layer of resin and at least partially penetrating the chaperone sample, further comprising milling the top layer of resin, so that the top layer of resin has a thickness of about 500 pm.

(a) embedding a chaperone sample onto a lower layer of resin,

(b) adding a top layer of resin on top of the chaperone sample,

(c) introducing a multitude of cavities penetrating the top layer of resin and at least partially penetrating the chaperone sample, further comprising milling the chaperone block into a hexagonal shape.

(a) embedding a chaperone sample onto a lower layer of resin,

(b) adding a top layer of resin on top of the chaperone sample,

(c) introducing a multitude of cavities penetrating the top layer of resin and at least partially penetrating the chaperone sample, further comprising washing of the chaperone block in an ultra-sonic bath.

(a) embedding a chaperone sample onto a lower layer of resin,

(b) adding a top layer of resin on top of the chaperone sample,

(c) introducing a multitude of cavities penetrating the top layer of resin and at least partially penetrating the chaperone sample,

(d) loading one or more screening sample into the multitude of cavities. The invention also provides a method for producing a chaperone block, the method comprising:

(a) embedding a chaperone sample onto a lower layer of resin,

(b) adding a top layer of resin on top of the chaperone sample,

(d) loading one or more screening sample into the multitude of cavities, wherein a screening sample is loaded into each cavity of the chaperone block.

In some embodiments of the method for producing a chaperone block provided herein the multitude of cavities penetrate the top layer of resin and the chaperone sample.

In some embodiments, the method for producing a chaperone block of the present invention further comprises milling the top layer of resin, so that the top layer of resin has a thickness of about 200- 500pm, such as a thickness of 200pm, 210pm, 220pm, 230pm, 240pm, 250pm, 260pm, 270pm, 280pm, 290pm, 300pm, 310pm, 320pm, 330pm, 340pm, 350pm, 360pm, 370pm, 380pm, 390pm, 400pm, 410pm, 420pm, 430pm, 440pm, 450pm, 460pm, 470pm, 480pm, 490pm, or 500pm. In a preferred embodiment, the chaperone block of the present invention further comprises milling the top layer of resin, so that the top layer of resin has a thickness of about 500pm.

In any of the embodiments of the method for producing a chaperone block provided herein, the chaperone block may be the chaperone block of the present invention.

In some embodiments the method for producing a chaperone block, e.g. the chaperone block of the present invention, does not comprise a step of obtaining a tissue sample of a subject/organism.

The collection of the tissue comprised in the chaperone sample and/or tissue sample of interest may include biopsies or autopsies for example. According to the present invention, the chaperone sample is preferably collected from a mammal, reptile, fish, or bird. More preferably, the chaperone sample is collected from mice. In the context of the present invention, a chaperone sample containing neurons is preferred. The neuronal tissue-of-interest may be a structure of several hundred micrometers in diameter, such as barrels in the mouse SI cortex, subnuclei in the thalamus, or directional preference columns in the visual cortex.

In the context of the present invention, a chaperone sample may be collected from whole brain, hemispheres, cortical and/or subcortical volumes, spinal cords and peripheral tissues containing nerve endings and the like. Also included as defined herein is the cortex such as the somatosensory cortex, parietal cortex and the like. The chaperone sample can be an unprocessed or a processed biological tissue sample.

In some embodiments, the method for producing a chaperone block of the present invention further comprises milling the chaperone block into a pre-defined shape, such as a hexagonal shape, a cube shape, a pyramid shape, a triangular shape, a rectangular shape or the like. In a preferred embodiment, the method for producing a chaperone block of the present invention further comprises milling the chaperone block into a hexagonal shape.

The chaperone sample may be prepared for EM imaging by methods known in the art. This may include any of the following procedures:

Fixation of the chaperone sample to preserve the tissue in as close to its natural state as possible by using a fixative. Chemical fixatives such as glutaraldehyde or osmium tetroxide are commonly used because they cross-link proteins and stabilize the tissue’s structure, preventing enzymatic degradation and autolysis. The chaperone sample may be immersed in the fixative for a specified period, which can vary depending on the tissue type and size. A fixative may also be delivered to the chaperone sample by transcranial perfusion.

After fixation, the chaperone sample may be washed with a buffer solution to remove excess fixative and any byproducts that may interfere with further processing of the chaperone sample. The chaperone sample may undergo a second fixation step, e.g. with osmium tetroxide, which provides additional fixation and stains the tissue, enhancing contrast by reacting with lipids to create electron-dense areas.

The chaperone sample may be dehydrated through a graded series of e.g. ethanol or acetone solutions, starting with a lower concentration and gradually increasing to 100% to remove all water from the sample. This step can be advantageous because the embedding media, e.g. resin, may be hydrophobic and water would prevent proper infiltration.

After dehydration, the chaperone sample may be gradually infiltrated with a liquid embedding medium, such as any resin defined herein. The infiltration process may start with a mixture of the dehydrating agent and the resin and then transition to pure resin. The chaperone sample can be left in the resin for it to completely permeate. In the sense of the present invention the chaperone sample may be infiltrated by a graded acetone-resin mixture, wherein the amount of resin increases throughout the grade. Finally, the chaperone sample can be infiltrated with pure resin, i.e. without acetone.

Once the chaperone sample is infiltrated, it can be placed in a mold, e.g. custom made mold adapted to the needs of the individual application, with fresh resin and polymerized (hardened) in an oven at a controlled temperature, e.g. 60°C. The result is a solid block with the chaperone sample embedded within it. This block may form the middle layer comprising the chaperone sample comprised in the chaperone block of the invention.

Using an ultramicrotome, thin sections of the tissue are cut from the loaded chaperone block. The thickness of these sections is typically around 50-100 nanometers for transmission electron microscopy (TEM) and can be thicker for scanning electron microscopy (SEM). As explained herein, the chaperone block of the present invention is particularly advantageous since it allows for sections as thin as 35 nm. The sections may be collected on grids made of metal, such as copper or nickel, or on carbon coated tape or silicon wafers.

To enhance contrast, the sections may be stained with heavy metals such as uranyl acetate and lead citrate, which bind to different cellular components and scatter electrons to varying degrees, thereby enhancing the visibility of different structures under the electron microscope.

The prepared and stained sections can be placed into the electron microscope for examination. The electron beam interacts with the sample, and the resulting images are captured, on a digital camera system. The images can then be analyzed e.g. for connectomics.

Screening sample

The invention also provides a “screening sample”. A screening sample refers to an entity comprising a tissue sample of interest, which is a tissue sample that is to be imaged and analyzed, e.g. for connectomic screening (therefore the tissue sample of interest is herein also referred to as a “tissue sample to be analyzed, imaged and/or screened”), a resin layer comprising a predetermined breaking point and a base. An exemplary screening sample is shown in Figure IE. The black tip represents the tissue sample of interest, the part below the tissue sample of interest is the resin layer comprising a predetermined breaking point, the larger rectangular body below is the base. The combination of said elements form the screening sample of the invention. A screening sample is advantageous since it can be loaded into the chaperone block thereby leading to the technical effects described in detail above.

As used herein “screening sample” refers to the entire entity described above comprising the tissue sample of interest, the resin layer comprising a predetermined breaking point and the base. After loading into the chaperone block, the portion above the predetermined breaking point is separated from the lower portion including the base. The portion that remains in the chaperone block may be referred herein as “screening sample”. Accordingly, the term “screening sample” refers to the entire entity in context of an unloaded screening sample and to the part remaining in the chaperone block in context of a loaded screening sample. When the screening sample is loaded into the chaperone block, the resin layer will break or be cut of, e.g. at the predetermined breaking point. The skilled person is aware, that the broken off portion of the screening sample may comprise the tissue sample of interest as well as the part of the resin layer up to the predetermined breaking point, or up to the point where the break/cut actually happened.

A screening sample in the sense of the present invention may comprise a tissue sample of interest, a resin layer, e.g. in form of a rod, comprising a predetermined breaking point, and a base. The base may be a resin base. According to the present invention, a screening sample is configured to be loaded into the chaperone block of the present invention. More specifically, a screening sample is configured to be loaded in a cavity comprised in the chaperone block. Thus, the shape and size of the tissue sample of interest and resin layer may correspond to the shape and size of the cavities comprised in the chaperone block. Otherwise, the shape and size of the screening sample is not particularly limited, but it is preferred that the tissue sample of interest and the resin layer have the same or substantially same dimensions as the cavity in the chaperone sample and the top resin layer of the chaperone block. As explained above, this is advantageous to ensure an overlap of tissue sample of interest and chaperone sample as well as an overlap of the resin layer of the screening sample with the top resin layer of the chaperone block. It is further preferred that the tissue sample of interest, the resin layer of the screening sample, the chaperone sample, and the top layer of resin of the chaperone block have the same relations to each other, e.g. are the same thickness. The pre-determined breaking point of the resin layer of the screening sample is configured to break after loading of the screening sample into the chaperone block, thereby separating the screening sample from its base. The pre-determined breaking point is comprised in a layer of resin arranged in between the tissue sample of interest and the base.

If there are reservations about cutting off the sample because the offset of the chaperone sample could be destroyed, a cutting technique can be used in which the screening sample is separated from the base at its pre-determined breaking point with a razor blade after being loaded into the chaperone block.

A screening sample in the sense of the present invention comprises a tissue sample of interest, e.g. for connectomic screening. A screening sample as defined herein can be configured to be inserted into a chaperone block, e.g. into a cavity of a chaperone block, wherein after insertion the tissue sample of interest overlaps with the chaperone sample. The screening sample can comprise a tissue sample of interest treated with heavy metal treatment, dehydrated, infiltrated with resin and embedded into a silicon mold. The screening sample may be of any shape that is feasible for loading into the chaperone block. The screening sample can be in a shape which allows insertion of the screening sample into the chaperone block. The screening sample can comprise a resin layer of resin comprising a predetermined breaking point, wherein the predetermined breaking point is configured to be broken after loading of the screening sample into a chaperone block preferably without breaking the tissue sample of interest. The stability and electrical conductivity of the tissue sample of interest is improved when loaded into the chaperone block.

Typically, the tissue sample of interest is a biological tissue sample. Herein, the tissue sample of interest can comprise any type of biological tissue sample collected from a living or dead subject. As used herein tissue sample of interest” in general refers to a sample of tissue or tissue sample comprised in the screening sample. Accordingly, the “tissue sample of interest” may be a tissue sample or comprise a tissue sample. The tissue sample of interests will be analyzed by cutting and imaging in contrast to the chaperone sample. Accordingly, the tissue sample of interest comprises the tissue of interest that is to be analyzed, e.g. for connectomic screening.

The tissue sample of interest may be arranged in the top part of the screening sample followed by a layer of resin comprising the predetermined breaking point further followed by a base. The tissue sample of interest may be embedded and/or infiltrated by a resin in the top portion of the screening sample. The screening sample may be arranged like a layer, e.g. similar to a resin layer, since it can be embedded and/or infiltrated by resin. An exemplary screening sample design is shown in Figure IE wherein the black top portion represents a tissue sample of interest.

Accordingly, the invention provides in one aspect a screening sample comprising a tissue sample of interest, a resin layer comprising a predetermined breaking point and a base, wherein the screening sample is configured to be loaded into a chaperone block, e.g. the chaperone block of the present invention. The tissue sample of interest may comprise any tissue. Tissue in the sense of the present invention is preferably biological tissue, e.g. from an organ. In general, the tissue sample of interest is a tissue that is to be imaged and/or analyzed such as for connectomic screening. The tissue sample of interest may comprise tissue of a similar type of that of the chaperone sample. The similarity of the chaperone tissue sample and the tissue sample of interest results in a homogenized surface and avoids any differences in electrical conductivity within the loaded chaperone block, e.g. when cutting the block and imaging/analyzing the tissue samples of interest. For example, choosing a tissue with similar structural properties allows for cutting the loaded chaperone sample without mayor differences in density and rigidity between the chaperone tissue sample and the tissue sample of interest. This allows for uniform cutting of the loaded chaperone block since differences in the density and rigidity can lead to breaking of the slice during cutting. Homogenizing the structural properties by choosing similar tissue for the chaperone tissue sample and the tissue sample of interest reduces mechanical stress when cutting the chaperone block, thereby allowing cutting of the chaperon block in slices as thin as 35 nm. This in turn enables high throughput connectomic screening of a multitude of samples in parallel via EM imaging. Furthermore, choosing a tissue with similar conductive properties, thereby homogenizing the electrical conductivity between the chaperone sample and the screening sample, allows for high-quality imaging of the slices of the loaded chaperone block without damaging the sample or introducing artifacts due to charge accumulation. Accordingly, the tissue sample of interest is not limited to a specific tissue type, the skilled person is rather capable of choosing a tissue sample of interest and optionally chose a chaperone tissue sample with similar tissue properties, thereby resulting in the above-described effects. In general, the chaperone tissue sample and the tissue sample of interest may be similar tissue, however, the tissue may originate from different species.

Typically, the tissue sample of interest to be used in the present invention is a biological tissue sample. In general, the tissue sample of interest used in the present invention may comprise any tissue from any organism. In a preferred embodiment of the screening sample of the invention the tissue sample of interest comprises a tissue sample comprising neurons. In some embodiment of the screening sample of the invention the tissue sample of interest comprises a tissue sample of a mammal, reptile, fish, or bird. In some embodiments, the tissue sample of interest comprises a tissue sample of a rodent or a primate. In some embodiments the tissue sample of interest comprises a tissue sample of a mouse, macaque, human, rat, ferret, rabbit, marmoset, Komodo dragon, turtle, salamander, zebrafish, zebra finch, or pigeon. In a preferred embodiment, the tissue sample of interest comprises a tissue sample of a mouse brain, macaque brain or a human brain. When a ventricle is present in the tissue sample of interest it can negatively affect the cuttability of the loaded chaperone block. Accordingly, in some embodiments, the tissue sample of interest does not comprise a ventricle.

In the context of the present invention, a tissue sample of interest comprising neurons is preferred. The neuronal tissue-of-interest may be a structure of several hundred micrometers in diameter, such as barrels in the mouse SI cortex, subnuclei in the thalamus, or directional preference columns in the visual cortex.

In the context of the present invention, a tissue sample of interest can comprise a tissue sample of the whole brain, hemispheres, cortical and/or subcortical volumes, spinal cords and peripheral tissues containing nerve endings and the like of any of the above-mentioned species. The tissue sample of interest may also comprise a tissue sample of the cortex such as the somatosensory cortex, parietal cortex and the like of any of the above-mentioned species. The tissue sample of interest may comprise an intact tissue sample, meaning a tissue sample that has maintained its native structural integrity, or may be a tissue sample that has been homogenized, e.g. has been milled/blended into a homogenous sample. Herein, the tissue sample of interest can comprise any type of biological tissue sample, e.g. a biological tissue sample of a living or dead subject/animal. Accordingly, the invention provides in one aspect a screening sample comprising a tissue sample of interest, a resin layer comprising a predetermined breaking point and a base, wherein the screening sample is configured to be loaded into a chaperone block, wherein the tissue sample of interest comprised in the screening sample is a tissue sample of a mammal, reptile, fish, or bird or a rodent, preferably a tissue sample of a mouse, macaque, human, rat, ferret, rabbit, marmoset, Komodo dragon, turtle, salamander, zebrafish, zebra finch, or pigeon brain, most preferably a mouse brain, a human brain or a macaque brain.

As discussed above, it is advantageous that the chaperone sample and the tissue sample of interest that may be loaded into the chaperone block have similar structural and conductive properties. Thus, the chaperone sample and/or the tissue sample of interest may comprise the same or similar tissue, e.g. from a same or similar type of organ having same or similar structural and conductive properties. As mentioned above, the chaperone sample may comprise a tissue-derived sample having same or similar structural and/or conductive properties as a tissue sample of interest even if the chaperone sample and the tissue sample of interest comprise a tissue from different species. For example, the chaperone sample may be a tissue-derived sample from a rodent brain while the loaded tissue sample of interests are samples from a macaque brain and vice versa, as long as the tissues have similar or same structural and/or conductive properties, thereby ensuring the homogenization of the chaperone sample surface when loaded with the tissue sample of interests and the electrical conductivity of both samples.

Accordingly, the invention provides in one aspect a screening sample comprising a tissue sample of interest, a resin layer comprising a predetermined breaking point and a base, wherein the screening sample is configured to be loaded into a chaperone block, wherein the tissue sample of interest comprises a tissue originating from the same or substantially same type of organ having similar or same or substantially same structural and conductive properties as a tissue of a chaperone sample in a chaperone block, e.g. the chaperone block of the present invention.

The size or shape of the screening sample is not particularly limited. In particular, size or shape of the screening sample may depend on the intended use of the sample. For example, screening sample comprising a thick tissue sample of interest may produce more sections after loading and processing into a chaperone block. In general, the dimensions of the tissue sample of interest are not particular limited and depend on the size of the tissue area of interest and on the chaperone block that is intended to be used for loading. To facilitate full insertion of a tissue sample of interest into a cavity penetrating the chaperone sample, the tissue sample of interest may be at least as thick as the chaperone sample. In general, the tissue sample of interest may correspond or substantially correspond to the size and shape of cavities of a chaperone block that is to be used for later loading of the tissue sample of interest.

The tissue sample of interest may be of any diameter, shape and size as long as they are suitable to be loaded with into a corresponding chaperone block. The tissue sample of interest may be polyhedric, rectangular (e.g., as shown in Fig. 6), cubic, pyramidic, cone shaped, prism shaped, spheric, rod shaped and the like. In particular, tissue sample of interests of a polyhedric shape, such as a cuboidal shape may be preferred over a cylindric shape since this allows for less movement of the tissue sample of interests in the corresponding cavities and can aid the separation of the tissue sample of interest from its base via breaking of the predetermined breaking point. Thus, in one embodiment, the tissue sample of interest is approximately polyhedric, rectangular, cubic, pyramidic, cone shaped, prism shaped, or spheric. In a preferred embodiment, the tissue sample of interest is approximately polyhedric, such as cuboidal. In a preferred embodiment, the tissue sample of interest is approximately cylindrical. In a preferred embodiment, the tissue sample of interest is polyhedric, such as cuboidal. In a preferred embodiment, the tissue sample of interest is cylindrical. As mentioned above, the screening sample of the present invention can comprise a tissue sample of interest of any shape. Accordingly, in some embodiments, the screening sample of the present invention comprises approximately polyhedron tissue sample of interest. In some embodiments, the screening sample of the present invention can comprise an approximately rectangular tissue sample of interest. In some embodiments, the screening sample of the present invention can comprise an approximately cylindrical tissue sample of interest. In some embodiments, the screening sample of the present invention can comprise an approximately cubic tissue sample of interest. In some embodiments, the screening sample of the present invention can comprise an approximately pyramidic tissue sample of interest. In some embodiments, the screening sample of the present invention can comprise an approximately cone shaped tissue sample of interest. In some embodiments, the screening sample of the present invention can comprise an approximately prism shaped tissue sample of interest. In some embodiments, the screening sample of the present invention can comprise an approximately spherical tissue sample of interest. In some embodiments, the screening sample of the present invention can comprise an approximately rod-shaped tissue sample of interest.

Accordingly, the invention provides in one aspect a screening sample comprising a tissue sample of interest, a resin layer comprising a predetermined breaking point and a base, wherein the screening sample is configured to be loaded into a chaperone block, wherein the cavities are approximately cylindrical, polyhedric, such as cuboidal, or rod-shaped.

As mentioned above, the screening sample of the present invention can comprise a tissue sample of interest of any diameter. The diameter of the tissue sample of interest may depend on the diameter of the cavities of the chaperone block for loading the tissue sample of interest. For example, it is advantageous, that the diameter of the tissue sample of interest is slightly smaller than that of a corresponding cavity for loading. This is to enable loading of the tissue sample of interest into the chaperone block. Accordingly, in some embodiments, the cavities in the chaperone block and the tissue sample of interest have approximately the same diameter. In some embodiments, the cavities in the chaperone block and the tissue sample of interest have substantially the same diameter, as long as the tissue sample of interest can still be loaded into the cavities of the chaperone block. In some embodiments, the diameter of the screening sample tissue is smaller than that of the cavities of the chaperone block. In some embodiments, the tissue sample of interest and the resin layer comprising the predetermined breaking point have the same or substantially same diameter. Accordingly, in some embodiments, the tissue sample of interest and/or the resin layer comprising the predetermined breaking point has a diameter of about 70- 2000 pm, such as about 70pm, about 80pm, about 90pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, about 300 pm, about 310 pm, about 320 pm, about 330 pm, about 340 pm, about 350 pm, about 360 pm, about 370 pm, about 380 pm, about 390 pm, about 400 pm, about 410 pm, about 420 pm, about 430 pm, about 440 pm, about 450 pm, about 460 pm, about 470 pm, about 480 pm, about 490 pm, about 500 pm, about 510 pm, about 520 pm, about 530 pm, about 540 pm, about 550 pm, about 560 pm, about 570 pm, about 580 pm, about 590 pm, about 600 pm, about 610 pm, about 620 pm, about 630 pm, about 640 pm, about 650 pm, about 660 pm, about 670 pm, about 680 pm, about 690 pm, about 700 pm, about 710 pm, about 720 pm, about 730 pm, about 740 pm, about 750 pm, about 760 pm, about 770 pm, about 780 pm, about 790 pm, about 800 pm, about 810 pm, about 820 pm, about 830 pm, about 840 pm, about 850 pm, about 860 pm, about 870 pm, about 880 pm, about 890 pm, about 900 pm, about 910 pm, about 920 pm, about 930 pm, about 940 pm, about 950 pm, about 960 pm, about 970 pm, about 980 pm, about 990 pm, about 1000 pm, about 1010 pm, about 1020 pm, about 1030 pm, about 1040 pm, about 1050 pm, about 1060 pm, about

1070 pm, about 1080 pm, about 1090 pm, about 1100 pm, about 1110 pm, about 1120 pm, about

1130 pm, about 1140 pm, about 1150 pm, about 1160 pm, about 1170 pm, about 1180 pm, about

1190 pm, about 1200 pm, about 1210 pm, about 1220 pm, about 1230 pm, about 1240 pm, about

1250 pm, about 1260 pm, about 1270 pm, about 1280 pm, about 1290 pm, about 1300 pm, about

1310 pm, about 1320 pm, about 1330 pm, about 1340 pm, about 1350 pm, about 1360 pm, about

1370 pm, about 1380 pm, about 1390 pm, about 1400 pm, about 1410 pm, about 1420 pm, about

1430 pm, about 1440 pm, about 1450 pm, about 1460 pm, about 1470 pm, about 1480 pm, about

1490 pm, about 1500 pm, about 1510 pm, about 1520 pm, about 1530 pm, about 1540 pm, about

1550 pm, about 1560 pm, about 1570 pm, about 1580 pm, about 1590 pm, about 1600 pm, about

1610 pm, about 1620 pm, about 1630 pm, about 1640 pm, about 1650 pm, about 1660 pm, about 1670 pm, about 1680 pm, about 1690 pm, about 1700 pm, about 1710 pm, about 1720 pm, about

1730 pm, about 1740 pm, about 1750 pm, about 1760 pm, about 1770 pm, about 1780 pm, about

1790 pm, about 1800 pm, about 1810 pm, about 1820 pm, about 1830 pm, about 1840 pm, about

1850 pm, about 1860 pm, about 1870 pm, about 1880 pm, about 1890 pm, about 1900 pm, about

1910 pm, about 1920 pm, about 1930 pm, about 1940 pm, about 1950 pm, about 1960 pm, about

1970 pm, about 1980 pm, about 1990 pm, or about 2000 pm.

In a preferred embodiment, the tissue sample of interest and/or the resin layer comprising the predetermined breaking point has a diameter of about 70-500 pm, such as about 70pm, about 80pm, about 90pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, about 300 pm, about 310 pm, about 320 pm, about 330 pm, about 340 pm, about 350 pm, about 360 pm, about 370 pm, about 380 pm, about 390 pm, about 400 pm, about 410 pm, about 420 pm, about 430 pm, about 440 pm, about 450 pm, about 460 pm, about 470 pm, about 480 pm, about 490 pm, or about 500 pm. In a preferred embodiment, the tissue sample of interest and/or the resin layer comprising the predetermined breaking point has a diameter of about 70-150 pm, such as about 70pm, about 80pm, about 90pm, about 100pm, about 110pm, about 120pm, about 130pm, about 140pm, or about 150pm. In a more preferred embodiment, the screening sample of the present invention can comprise a tissue sample of interest and/or a resin layer comprising the predetermined breaking point with a diameter of about 120 pm. In some embodiment, the screening sample of the present invention can comprise a tissue sample of interest and/or a resin layer comprising the predetermined breaking point with a diameter of about 1mm to 3mm.

Accordingly, the invention provides in one aspect a screening sample comprising a tissue sample of interest, a resin layer comprising a predetermined breaking point and a base, wherein the screening sample is configured to be loaded into a chaperone block, wherein the tissue sample of interest and/or the resin layer comprising the predetermined breaking point has a diameter of about 70-2000 pm.

The screening sample of the present invention may have any size or shape. It is preferred, that the resin layer comprising the predetermined breaking point has the same or substantially the same size or shape as the tissue sample of interest. This is advantageous since the resin layer can be seen as a continuation of the tissue sample of interest that will be partially inserted into the chaperone block and the overlaps with the top layer of resin of the chaperone block. Accordingly, features defined above in context of the shape and size of the tissue sample of interest apply mutatis mutandis to the resin layer comprising the predetermined breaking point.

Since the tissue sample of interest will ultimately be cut, the thickness of the tissue sample of interest, together with the thickness of the chaperone sample determines the number of sections that can be obtained from the chaperone block. At the same time, the thickness of the chaperone sample determines the maximum of thickness of a tissue sample of interest that may be loaded into the chaperone block. Accordingly, the thickness of the chaperone sample and the tissue sample of interest may be increased or decreased to modify the number of sections that can be obtained from a single chaperone block. In general, the chaperone sample and the tissue sample of interest may be the same or substantially same thickness. This is because only the part of tissue sample of interest that has been entirely loaded into the chaperone sample can be cut and subsequently analyzed. Accordingly, in one embodiment, the chaperone sample and the tissue sample of interest are the same thickness. The thickness of the chaperone and/or the tissue sample of interest may be scaled depending on the desired application. Accordingly, in one embodiment the thickness of the chaperone sample and/or the tissue sample of interest is at least about 100 pm to 5000 pm, such as about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, about 300 pm, about 310 pm, about 320 pm, about 330 pm, about 340 pm, about 350 pm, about 360 pm, about 370 pm, about 380 pm, about 390 pm, about 400 pm, about 410 pm, about 420 pm, about 430 pm, about 440 pm, about 450 pm, about 460 pm, about 470 pm, about 480 pm, about 490 pm, about 500 pm, about 510 pm, about 520 pm, about 530 pm, about 540 pm, about 550 pm, about 560 pm, about 570 pm, about 580 pm, about 590 pm, about 600 pm, about 610 pm, about 620 pm, about 630 pm, about 640 pm, about 650 pm, about 660 pm, about 670 pm, about 680 pm, about 690 pm, about 700 pm, about 710 pm, about 720 pm, about 730 pm, about 740 pm, about 750 pm, about 760 pm, about 770 pm, about 780 pm, about 790 pm, about 800 pm, about 810 pm, about 820 pm, about 830 pm, about 840 pm, about 850 pm, about 860 pm, about 870 pm, about 880 pm, about 890 pm, about 900 pm, about 910 pm, about 920 pm, about 930 pm, about 940 pm, about 950 pm, about 960 pm, about 970 pm, about 980 pm, about 990 pm, about 1000 pm, about 1010 pm, about 1020 pm, about 1030 pm, about 1040 pm, about 1050 pm, about 1060 pm, about 1070 pm, about 1080 pm, about 1090 pm, about 1100 pm, about 1110 pm, about 1120 pm, about 1130 pm, about 1140 pm, about 1150 pm, about 1160 pm, about 1170 pm, about 1180 pm, about 1190 pm, about 1200 pm, about 1210 pm, about 1220 pm, about 1230 pm, about 1240 pm, about 1250 pm, about 1260 pm, about 1270 pm, about 1280 pm, about 1290 pm, about 1300 pm, about 1310 pm, about 1320 pm, about 1330 pm, about 1340 pm, about 1350 pm, about 1360 pm, about 1370 pm, about 1380 pm, about 1390 pm, about 1400 pm, about 1410 pm, about 1420 pm, about 1430 pm, about 1440 pm, about 1450 pm, about 1460 pm, about 1470 pm, about 1480 pm, about 1490 pm, about 1500 pm, about 1510 pm, about 1520 pm, about 1530 pm, about 1540 pm, about 1550 pm, about 1560 pm, about 1570 pm, about 1580 pm, about 1590 pm, about 1600 pm, about 1610 pm, about 1620 pm, about 1630 pm, about 1640 pm, about 1650 pm, about 1660 pm, about 1670 pm, about 1680 pm, about 1690 pm, about 1700 pm, about 1710 pm, about 1720 pm, about 1730 pm, about 1740 pm, about 1750 pm, about 1760 pm, about 1770 pm, about 1780 pm, about 1790 pm, about 1800 pm, about 1810 pm, about 1820 pm, about 1830 pm, about 1840 pm, about 1850 pm, about 1860 pm, about 1870 pm, about 1880 pm, about 1890 pm, about 1900 pm, about 1910 pm, about 1920 pm, about 1930 pm, about 1940 pm, about 1950 pm, about 1960 pm, about 1970 pm, about 1980 pm, about 1990 pm, about 2000 pm, about 2100 pm, about 2200 pm, about 2300 pm, about 2400 pm, about 2500 pm, about 2600 pm, about 2700 pm, about 2800 pm, about 2900 pm, about 3000 pm, about 3100 pm, about 3200 pm, about 3300 pm, about 3400 pm, about 3500 pm, about 3600 pm, about 3700 pm, about 3800 pm, about 3900 pm, about 4000 pm, about 4100 pm, about 4200 pm, about 4300 pm, about 4400 pm, about 4500 pm, about 4600 pm, about 4700 pm, about 4800 pm, about 4900 pm, or about 5000 pm. In a preferred embodiment, the thickness of the chaperone sample and/or the tissue sample of interest is about 500 pm.

Accordingly, the invention provides in one preferred aspect a screening sample comprising a tissue sample of interest, a resin layer comprising a predetermined breaking point and a base, wherein the screening sample is configured to be loaded into a chaperone block, wherein the chaperone sample and/or the tissue sample of interest is at least about 100 pm to 5000 pm, preferably at least about 500 pm, more preferably about 500 pm thick.

In some embodiments, the chaperone block of the present invention comprises the tissue sample of interest, wherein the tissue sample of interest is between about 100 and 2000 pm thick. In some embodiments, the chaperone block of the present invention comprises the tissue sample of interest, wherein the screening sample is between about 100 and 1000 pm thick.

As mentioned above, the screening sample of the present invention can be of any suitable size and shape allowing loading of the tissue sample of interest and optionally part of the resin layer into the chaperone block. The dimensions of the screening sample are of variable size. The chaperon sample and tissue sample of interest may possess the same thickness. As defined herein, the screening sample comprises inter alia a tissue sample of interest and a resin layer comprising a predetermined breaking point. Accordingly, in some embodiments, the tissue sample of interest is about 100 to 2000 pm thick. Accordingly, in some embodiments, resin layer is about 100 to 2000 pm thick. Accordingly, in some embodiments, the tissue sample of interest is about 100 to 1000 pm thick. Accordingly, in some embodiments, the resin layer is about 100 to 1000 pm thick. In some embodiments, the tissue sample of interest is at least about 500 pm thick. In some embodiments, the resin layer is at least about 500 pm thick. In some embodiments, the tissue sample of interest and the resin layer are the same or substantially same thickness. In a preferred embodiment, the tissue sample of interest and the resin layer are about at least 500 pm thick. In a more preferred embodiment, the tissue sample of interest and the resin layer are about 500 pm thick.

A tissue sample of interest in the context of the present invention is configured to be loaded into a cavity comprised in a chaperone block of the present invention. Thus, the dimension and shape of the tissue sample of interest may approximately correspond to the dimension and shape of a cavity comprised in a chaperone block. Accordingly, in some embodiments, the tissue sample of interest is a of a polyhedric shape. In some embodiments, the tissue sample of interest is a of a rectangular shape. In some embodiments, the tissue sample of interest is a of a cylindrical shape. In some embodiments, tissue sample of interest is a of a cubic shape. In some embodiments, tissue sample of interest is a of a pyramidic shape. In some embodiments, the tissue sample of interest is of a conic shape. In some embodiments, the tissue sample of interest is of a prism shape. In some embodiments, the tissue sample of interest is of a spherical shape. In some embodiments, the tissue sample of interest of the same or substantially same shape as a cavity comprised in the chaperone block of the present invention. In some embodiments, the screening sample comprises a tissue sample of interest in a shape suitable to be loaded into a cavity comprised in the chaperone block of the present invention. Accordingly, in some embodiments, the screening sample comprises a resin layer, wherein the resin layer is of a polyhedric shape. In some embodiments, the resin layer is of a rectangular shape. In some embodiments the resin layer is of a cylindrical shape. In some embodiments, the resin layer is of a cubic shape. In some embodiments, the resin layer is of a pyramidic shape. In some embodiments, the resin layer is of a conic shape. In some embodiments the resin layer is of a prism shape. In some embodiments the resin layer is rod-shaped. In some embodiments, the resin layer is of a spherical shape. In some embodiments, the resin layer is of the same or substantially same shape as a cavity comprised in the top layer of resin of a chaperone block of the present invention. In some embodiments, the resin layer is suitable to be loaded into a cavity comprised in the chaperone block of the present invention, wherein the resin layer of the screening sample overlaps with the top layer of resin of the chaperone block when loaded.

Accordingly, the invention provides in one aspect a screening sample comprising a tissue sample of interest, a resin layer comprising a predetermined breaking point and a base, wherein the screening sample is configured to be loaded into a chaperone block comprising at least two layers of resin and, at least one layer of a chaperone sample and a multitude of cavities.

In the sense of the screening sample a “resin layer” is a layer of resin comprising a predetermined breaking point configured to break the tissue sample of interest off the base when loading the chaperone block. The resin layer is preferably arranged between the tissue sample of interest and the base. In general, the invention is not limited to any kind of resin, the skilled person is rather capable of choosing a feasible resin composition to securely embed the tissue sample of interest and form the resin layer with the predetermined breaking point. The resin provides structural integrity to the tissue sample of interest allowing for later cutting of the sample, e.g. when loaded with screening samples.

The resin used herein may be used to form one or more separate layers, e.g. a layer comprising the tissue sample of interest and the resin layer comprising the predetermined breaking point. In general, the size, shape, and/or thickness of the resin and/or the resin layers are not particularly limited. The skilled person is aware that size, shape and/or thickness may depend on the specific application of the screening sample, e.g. when a machine for processing requires certain dimensions. Resins used herein are preferably epoxy resins and include but are not limited to commercially available epoxy resins such as Epon 812 substitute resin, resin prepared according to Spurr, and the like. The resin according to Spurr (Spurr’ s resin, 10 g) consists of 3,4-epoxycyclohexylmethyl 3,4-epoxycyclo-hexaneca (ERL 4221, 4.1 g), diglycidyl ether of polypropylene glycol (DER 736, 0.95 g), nonenyl succinic anhydride (NSA, 5.9 g) and dimethylaminoethanol (DMAE, preferably 11 to 113 pl, more preferably 55 to 113 pl, even more preferably 113 pl). According to the present invention, the Epon 812 substitute resin (Epon 812, 10 g) consist of tri-glycidyl ether of glycerol (5.9 g), Methyl nadic anhydride (MNA, 3.7 g), Dodecenylsuccinic anhydride (DDSA, 2.25 g), 2,4,6-Tris(dimethylaminomethyl)phenol (DMP, 20 to 205 pl, more preferably 100 to 205 pl, even more preferably 205 pl).

In some embodiments, the resin may comprise metal. In a preferred embodiment, the resin of the screening sample is Epon 812.

Accordingly, the invention provides in one preferred aspect a screening sample comprising a tissue sample of interest embedded and/or infiltrated in/with a resin, a resin layer comprising a predetermined breaking point and a resin base, wherein the screening sample is configured to be loaded into a chaperone block, preferably wherein the resin is Epon 812.

The size or shape of the resin layer comprising a predetermined breaking point is not particularly limited. In particular, size or shape of the resin layer comprising a predetermined breaking point may depend on the intended use of the sample and in particular may depend on the top layer of resin of the chaperone block. In general, the dimension of the resin layer comprising a predetermined breaking point should at least be configured to accommodate the tissue sample of interest above, e.g. the resin layer should be sized so that the tissue sample of interest can be embedded. In general, the dimensions of the resin layer comprising a predetermined breaking point are not particular limited and may depend on the size of the cavities of a chaperone block and the top layer of resin. The skilled person is capable of choosing appropriate dimensions of the resin layer comprising a predetermined breaking point.

When loading the chaperone block, the tissue sample of interest will be introduced into the cavity of the chaperone sample, whereas the resin layer comprising a predetermined breaking point will be introduced into the cavity of the top layer of resin of the chaperone block. This is shown for example in Figure 3B. This ensures that the tissue sample of interest breaks of at the predetermined breaking point and allows loading of the tissue sample of interest into the chaperone sample. Thus, it is advantageous, that the chaperone sample and the tissue sample of interest are the same or substantially the same thickness. Furthermore, it is advantageous that the top layer of resin and the layer of resin comprising a predetermined breaking point of the screening sample are the same or substantially the same thickness. This way, the chaperone sample and the tissue sample of interest will overlap in the loaded chaperone block. Furthermore, the top layer of resin will overlap with the layer of resin comprising a predetermined breaking point of the screening sample. Accordingly, in some embodiments, the top layer of resin and the layer of resin of the screening sample are the same or substantially the same thickness. Accordingly, in some embodiments the thickness of the top layer of resin and/or the resin layer comprising a predetermined breaking point is at least about 200 pm to 2000 pm, such as about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, about 300 pm, about 310 pm, about 320 pm, about 330 pm, about 340 pm, about 350 pm, about 360 pm, about 370 pm, about 380 pm, about 390 pm, about 400 pm, about 400 pm, about 410 pm, about 420 pm, about 430 pm, about 440 pm, about 450 pm, about 460 pm, about 470 pm, about 480 pm, about 490 pm, about 500 pm, about 510 pm, about 520 pm, about 530 pm, about 540 pm, about 550 pm, about 560 pm, about 570 pm, about 580 pm, about 590 pm, about 600 pm, about 610 pm, about 620 pm, about 630 pm, about 640 pm, about 650 pm, about 660 pm, about 670 pm, about 680 pm, about 690 pm, about 700 pm, about 710 pm, about 720 pm, about 730 pm, about 740 pm, about 750 pm, about 760 pm, about 770 pm, about 780 pm, about 790 pm, about 800 pm, about 810 pm, about 820 pm, about 830 pm, about 840 pm, about 850 pm, about 860 pm, about 870 pm, about 880 pm, about 890 pm, about 900 pm, about 910 pm, about 920 pm, about 930 pm, about 940 pm, about 950 pm, about 960 pm, about 970 pm, about 980 pm, about 990 pm, about 1000 pm, about 1010 pm, about 1020 pm, about 1030 pm, about 1040 pm, about 1050 pm, about 1060 pm, about 1070 pm, about 1080 pm, about 1090 pm, about 1100 pm, about 1110 pm, about 1120 pm, about 1130 pm, about 1140 pm, about 1150 pm, about 1160 pm, about 1170 pm, about 1180 pm, about 1190 pm, about 1200 pm, about 1210 pm, about 1220 pm, about 1230 pm, about 1240 pm, about 1250 pm, about 1260 pm, about 1270 pm, about 1280 pm, about 1290 pm, about 1300 pm, about 1310 pm, about 1320 pm, about 1330 pm, about 1340 pm, about 1350 pm, about 1360 pm, about 1370 pm, about 1380 pm, about 1390 pm, about 1400 pm, about 1410 pm, about 1420 pm, about 1430 pm, about 1440 pm, about 1450 pm, about 1460 pm, about 1470 pm, about 1480 pm, about 1490 pm, about 1500 pm, about 1510 pm, about 1520 pm, about 1530 pm, about 1540 pm, about 1550 pm, about 1560 pm, about 1570 pm, about 1580 pm, about 1590 pm, about 1600 pm, about 1610 pm, about 1620 pm, about 1630 pm, about 1640 pm, about 1650 pm, about 1660 pm, about 1670 pm, about 1680 pm, about 1690 pm, about 1700 pm, about 1710 pm, about 1720 pm, about 1730 pm, about 1740 pm, about 1750 pm, about 1760 pm, about 1770 pm, about 1780 pm, about 1790 pm, about 1800 pm, about 1810 pm, about 1820 pm, about 1830 pm, about 1840 pm, about 1850 pm, about 1860 pm, about 1870 pm, about 1880 pm, about 1890 pm, about 1900 pm, about 1910 pm, about 1920 pm, about 1930 pm, about 1940 pm, about 1950 pm, about 1960 pm, about 1970 pm, about 1980 pm, about 1990 pm, or about 2000 pm.

In a preferred embodiment, the thickness of the top layer of resin and/or the resin layer comprising a predetermined breaking point is about 200 pm to 500pm, such as about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, about 300 pm, about 310 pm, about 320 pm, about 330 pm, about 340 pm, about 350 pm, about 360 pm, about 370 pm, about 380 pm, about 390 pm, about 400 pm, about 410 pm, about 420 pm, about 430 pm, about 440 pm, about 450 pm, about 460 pm, about 470 pm, about 480 pm, about 490 pm, or about 500 pm. In a further preferred embodiment, the thickness of the top layer of resin and/or the resin layer comprising a predetermined breaking point is about 500 pm.

Accordingly, the invention provides in one preferred aspect a screening sample comprising a tissue sample of interest, a resin layer comprising a predetermined breaking point and a base, wherein the screening sample is configured to be loaded into a chaperone block, wherein the chaperone sample of the chaperone block and/or the tissue sample of interest is at least about 100 pm to 5000 pm, preferably at least about 500 pm, more preferably about 500 pm thick, and wherein the at least two layers of resin are at least about 100 pm to 5000 pm, preferably about 500 pm thick.

The invention also provides a screening sample comprising a tissue sample of interest, a resin layer comprising a predetermined breaking point and a base, wherein the screening sample is configured to be loaded into a chaperone block, wherein the tissue sample of interest and/or the resin layer comprising a predetermined breaking point and/or the top layer of resin of the chaperone block, and/or the chaperone sample of the chaperone block is at least about 100 pm to 5000 pm, preferably at least about 500 pm, more preferably about 500 pm thick.

The invention also provides a screening sample comprising a tissue sample of interest, a resin layer comprising a predetermined breaking point and a base, wherein the screening sample is configured to be loaded into a chaperone block, wherein the tissue sample of interest and the resin layer comprising a predetermined breaking point are at least about 100 pm to 5000 pm, preferably at least about 500 pm, more preferably about 500 pm thick.

The invention also provides a screening sample comprising a tissue sample of interest, a resin layer comprising a predetermined breaking point and a base, wherein the screening sample is configured to be loaded into a chaperone block, wherein the tissue sample of interest, the chaperone sample of the chaperone block, the resin layer comprising a predetermined breaking point, and the top layer of resin of the chaperone block are at least about 100 pm to 5000 pm, preferably at least about 500 pm, more preferably about 500 pm thick.

In general, base of the screening sample may be of any shape or size. The base is configured to be picked up and released by a holder, e.g. a second holder of the apparatus of the present invention. This allows the reversible collection of a screening sample, which is then loaded into the chaperone block by breaking at the predetermined breaking point. Subsequently, the screening sample is discarded, and the loading process can be repeated with the next screening sample. The base can be a resin base. The base may be polyhedric, rectangular, cubic, pyramidic, cone shaped, prism shaped, spheric or the like. The base is preferably approximately polyhedric, such as cuboidal. The base may further comprise an alignment plane to allow for proper orientation of the screening sample, e.g. during the loading process.

In some embodiments, the screening sample of the present invention comprises a base, wherein the base is made of resin. In some embodiments, the screening sample of the present invention comprises a base, wherein the base is made of Epon 812. In some embodiments, the screening sample of the present invention comprises a resin layer comprising a predetermined breaking point, wherein the resin layer is made of Epon 812. In some embodiments, the screening sample of the present invention comprises a base, wherein the base has a width of about 500 pm to 3000 pm, such as a width of about 500 pm, about 600 pm, about 700 pm, about 800 pm, about 900 pm, about 1000 pm, about 1100 pm, about 1200 pm, about 1300 pm, about 1400 pm, about 1500 pm, about 1600 pm, about 1700 pm, about 1800 pm, about 1900 pm, about 2000 pm, about 2100 pm, about 2200 pm, about 2300 pm, about 2400 pm, about 2500 pm, about 2600 pm, about 2700 pm, about 2800 pm, about 2900 pm, or about 3000 pm, and wherein the resin base has a height of about 500 pm to 5000 pm, such as about 500 pm, about 750 pm, about 1000 pm, about 1250 pm, about 1500 pm, about 1750 pm, about 2000 pm, about 2250 pm, about 2500 pm, about 2750 pm, about 3000 pm, about 3250 pm, about 3500 pm, about 3750 pm, about 4000 pm, about 4250 pm, about 4500 pm, about 4750 pm, or about 5000 pm. In a preferred embodiment, screening sample of the present invention comprises a base, wherein the base has a width of 200pm and a height of 1mm.

The predetermined breaking point comprised in the resin layer of the screening sample allows the resin layer to break or fracture at a specific location under stress, rather than at a random point thereby releasing the tissue sample of interest into a chaperone block. In the present invention the stress is movement of the screening sample when the tissue sample of interest and part of the resin layer are loaded into the chaperone block against the chaperone block, thereby breaking of the tissue sample of interest. This is exemplary shown in Figure 3B. The invention is not particularly limited to any form of a predetermined breaking. The skilled person is capable of designing an appropriate breaking point. In some embodiments, the predetermined breaking point is a notch or groove in the resin layer. This creates a stress concentration point, which will be the weakest part of the resin layer and therefore the first to fail under stress. In some embodiments, the predetermined breaking point is an area in the resin layer having a reduced cross-sectional area. By reducing the cross-sectional area of the resin layer at the desired breaking point e.g., by tapering or necking, the material is weaker and more likely to break at that specific location. In some embodiments, the predetermined breaking point comprises material inclusions or inserts. By placing a different material than with lower strength or a different failure strain at the predetermined point can create a weak spot. This could be a softer resin, a brittle filler, or a perforated insert. In some embodiments, the predetermined breaking point comprises score lines or perforations. Similar to notches, but typically finer, score lines or perforations can be added to the resin layer to weaken it at a specific point. These can be created during the molding process or added afterward. In some embodiments, the predetermined breaking point comprises material property modification. Altering the material properties at a specific location can create a breaking point. This can be done by changing the composition, adding a plasticizer to make the resin layer more brittle, or by inducing residual stress through a localized heating and cooling process. In some embodiments, the predetermined breaking point comprises chemical weakening. Applying a chemical agent that reacts with the resin layer to weaken it at the predetermined point can also be an effective method. In some embodiments, the predetermined breaking point comprises internal structural features of the resin layer. Designing the resin layer with internal structural features that concentrate stress, such as holes, slots, or embedded fibers that stop at the predetermined point, can also create a controlled breaking point. The skilled person is aware that the individual application of the screening sample and the individual forces acting on the resin layer should be considered when designing the predetermined breaking point.

The tissue sample of interest may be an unprocessed or a processed tissue sample. As used herein, “processed tissue sample” refers to laboratory and preparatory methods known to a person skilled in the art, in particular to methods for preparing a tissue sample for EM imaging (Gour et al., 2021; Loomba et al., 2022; Motta et al., 2019; Shapson-Coe et al., 2024; Sievers et al., 2024). Such methods include for example tissue staining, fixation, embedding, infiltration, dehydration, washing, heavy metal treatment and the like. This is also explained in detail in the section regarding a method to produce a screening sample of the present invention below.

Method for producing a screening sample

The invention also provides a method for producing a screening sample, e.g. the screening sample of the invention as defined herein. An exemplary method for producing a screening sample of the present invention is described in detail in Example 1.

Accordingly, the invention provides in one aspect a method for producing a screening sample, e.g. the screening sample of the present invention, the method comprising:

(a) embedding a tissue sample of interest in a resin,

(b) milling the embedded sample into a shape comprising the tissue sample of interest, a resin layer and a base,

(c) introducing a predetermined breaking point into the resin layer between the tissue sample of interest and the base. In some embodiments, the base may be a resin base.

The invention also provides a method for producing a screening sample comprising a tissue sample of interest, a resin layer comprising a predetermined breaking point and a base, wherein the screening sample is configured to be loaded into a chaperone block, the method comprising:

(a) embedding a tissue sample of interest in a resin,

The screening sample produced by the method can comprise any of the features described herein in the context of the screening sample of the invention. Accordingly, the above-described advantages in context of the screening sample apply mutatis mutandis to the method for producing a screening sample. Furthermore, any of the features described in the context of the screening sample as a product herein, apply mutatis mutandis to the method for producing a screening sample with the provision that said features are formulated as method steps rather than product features. The method for producing a chaperone block can further comprises one or more of the following steps:

• Trimming an alignment plane into the basis e.g. for orientation of the screening sample during loading

• Putting the embedded sample in a 3D printed holder

• Imaging the embedded sample, e.g. with a binocular

• Defining the position of embedded sample and defining the shape of the screening sample to be milled, e.g. with CAD software

• Milling of the embedded sample to the shape defined in the previous step

• Milling of the embedded sample into a shape that a rod is formed comprising the tissue sample of interest on top and the layer or resin below the tissue sample of interest. The rod may preferably have a diameter of about 150 pm and a thickness of 1 mm, of which 500 pm is the tissue sample of interest followed by 500 pm of resin layer.

• Introducing a predetermined breaking point, e.g. by trimming the rod up to 2 mm on one side. This is exemplary shown in Figure IE.

A method for producing a screening sample of the present invention may further comprise one or more of the following steps, preferably in chronological order:

Provision of a biological tissue from a region of interest, e.g. from a brain of a mammal, reptile, fish, or bird. It is desirable for the tissue to comprise neurons. The mammal can be any mammal, such as a mouse, macaque, human, rat, ferret, rabbit, or marmoset. Suitable methods for isolating tissue are known to a person skilled in the art. The tissue can be isolated from a living or a dead subject. For example, the tissue may be isolated using biopsy metal punches with a diameter of 1 mm, 1.5 mm or 2 mm.

Fixing the biological tissue using an appropriate fixative according to methods known to the person skilled in the art to preserve the intracellular structures of the tissue. Biological tissue may be collected in tubes and storing it over night at 4°C. Each tube may comprise one tissue sample and a fixative, optionally wherein the fixative is cacodylate buffer. Appropriate fixatives include PFA 4%-40%, Osmium 2-4%, Ethanol 50-10%, Glutaraldehyde 50-2.5%, or 2-Propanol 50-10%. In general, any of the fixatives used in Sivers et al. 2024 and Loomba et al 2022, which are incorporated herein in their entirety, can be used in the sense of the sense of the present invention. A fixative in the sense of the present invention is preferably paraformaldehyde and/or glutaraldehyde. A fixative is preferably used comprised in a cacodylate buffer. An alternative fixative may be osmium.

Punching out an area of interest of about 1, 1.5 or 2 mm e.g. with a biopsy needle. The punched-out sample may be referred herein as tissue sample of interest.

Staining the tissue sample of interest, e.g. to facilitate microscopy-based image acquiring. The chaperone sample can be imaged using electron microscopy. To image tissue samples with an electron microscope, the tissue can be stained using heavy-metal staining e.g. to increase the contrast. Staining methods are known to a skilled person. The chaperone sample may be stained according to the protocol of Hua et al. 2015 or Song et al., 2023, which are incorporated herein in its entirety.

Dehydrating the stained tissue sample of interest.

Infiltrating the tissue sample of interest with a resin. The resin may be a resin that penetrates throughout the tissue to the molecular level, subsequently hardens and finally solidifies thereby rendering the sample rigid.

Putting the tissue sample of interest into a custom-made mold and embedding the same in a resin. The custom-made mold defines the shape and size of the later obtained screening sample. This shape however is not final and will late be milled to define the size and shape of the tissue sample of interest and the resin layer comprising the predetermined breaking point. The custom-made mold may be a silicon mold.

Curing the tissue sample of interest in an oven for about 3 days.

Removing the screening sample from the mold.

The screening sample is cured in a custom-made silicon mold for 3 days in an oven.

Trimming an alignment plane into the basis e.g. for orientation of the screening sample during loading. The alignment plane on the screening sample serves for orientation so that the screening sample can be milled into an appropriate shape. Thus, the alignment plane improves orientation. The screening sample is placed into an appropriate holder, for example into a custom-made 3D-printed holder or mold. By placing said sample into such an appropriate holder imaging is facilitated. Imaging may be performed with any suitable method, for example by at a magnification of 1.25x using a microscope which is connected to a camera.

Imaging the embedded sample, e.g. with a binocular. An image of the screening sample is acquired, optionally wherein the image is acquired from a top view. Based on this image the exact position of the tissue sample of interest and the shape of the screening sample are defined using a suitable software, for example a CAD software. This step allows defining the position of tissue sample of interest and defining the shape of the screening sample to be milled, e.g. with CAD software.

Milling the screening sample into a pre-defined shape, e.g. using a suitable software, such as a CAM software and a suitable milling cutter. The screening sample is placed into a milling machine and milled based on the collected data on position of the tissue sample of interest and the predefined shape via the imaging. The shape and size of the screening sample is not particularly limited and depends on the size and shape of the cavities and on the size and shape of the tissue that is to be analyzed. The structure obtained after the milling step is referred herein as screening sample. The screening sample comprises a tissue sample of interest, resin layer with a predetermined breaking point and a base. Introducing a predetermined breaking point, e.g. by trimming one side of the tissue sample of interest and the resin layer up to the base, e.g. up to 2 mm. This is exemplary shown in Figure IE.

Assessing the quality and/or shape of the screening sample after milling by acquiring an image. Such an image may, for example, be acquired at a magnification of 1.25x using a microscope connected to a camera.

Accordingly, in one aspect the invention provides a method for producing a screening sample, the method comprising:

(a) embedding a tissue sample of interest in a resin,

(b) milling the embedded tissue sample into a shape comprising the tissue sample, a resin layer and a base,

(c) introducing a predetermined breaking point into the resin layer between the tissue sample and the base, wherein the method further comprises staining the tissue sample.

The invention also provides a method for producing a screening sample, the method comprising:

(a) embedding a tissue sample of interest in resin,

(c) introducing a predetermined breaking point into the resin layer between the tissue sample and the base, further comprising treating the tissue sample with a heavy metal treatment.

(a) embedding a tissue sample of interest in a resin,

(c) introducing a predetermined breaking point into the resin layer between the tissue sample and the base, further comprising dehydrating and/or infiltrating the tissue sample with a resin.

(a) embedding a tissue sample of interest in a resin,

(b) milling the embedded tissue sample into a shape comprising the tissue sample of interest, a resin layer and a base,

(c) introducing a predetermined breaking point into the resin layer between the tissue sample and the base, further comprising milling an alignment plane into the base, preferably wherein the base is a resin base. In some embodiments, the method for producing the screening sample of the present invention further comprises milling the tissue sample of interest and/or the resin layer comprising a predetermined breaking point each to a thickness of about 200-500 pm, such as a thickness of 200 pm, 210 pm, 220 pm, 230 pm, 240 pm, 250 pm, 260 pm, 270 pm, 280 pm, 290 pm, 300 pm, 310 pm, 320 pm, 330 pm, 340 pm, 350 pm, 360 pm, 370 pm, 380 pm, 390 pm, 400 pm, 410 pm, 420 pm, 430 pm, 440 pm, 450 pm, 460 pm, 470 pm, 48 0pm, 490 pm, or 500 pm. In a preferred embodiment, the method for producing the screening sample of the present invention further comprises milling the tissue sample of interest and/or the resin layer comprising a predetermined breaking point each to a thickness of about 500 pm.

In any of the embodiments of the method for producing the screening sample provided herein, the screening sample may be the screening sample of the present invention.

In some embodiments the method for producing the screening sample, e.g. the screening sample of the present invention, does not comprise a step of obtaining a tissue sample of a subject/organism.

The collection of the tissue comprised in the tissue sample of interest may include biopsies or autopsies for example. According to the present invention, the tissue of interest sample is preferably collected from a mammal, reptile, fish, or bird. More preferably, tissue sample of interest is collected from mice, humans or macaques. In the context of the present invention, a tissue sample of interest containing neurons is preferred. The neuronal tissue-of-interest may be a structure of several hundred micrometers in diameter, such as barrels in the mouse SI cortex, subnuclei in the thalamus, or directional preference columns in the visual cortex.

In the context of the present invention, a tissue sample of interest may be collected from whole brain, hemispheres, cortical and/or subcortical volumes, spinal cords and peripheral tissues containing nerve endings and the like. Also included as defined herein is the cortex such as the somatosensory cortex, parietal cortex and the like. The tissue sample of interest can be an unprocessed or a processed biological tissue sample.

In some embodiments, the method for producing the screening sample of the present invention further comprises milling the tissue sample of interest and/or the resin layer into a pre-defined shape, such as a polyhedric, rectangular, cubic, pyramidic, cone, prism, spheric, or rod shape and the like. In a preferred embodiment, the method for producing the screening sample of the present invention further comprises milling the tissue sample of interest and/or the resin layer into a cylindric or polyhedric shape, such as a cuboidal shape.

The tissue sample of interest may be prepared for EM imaging by methods known in the art. This may include any of the following procedures:

Fixation of the tissue sample of interest to preserve the tissue in as close to its natural state as possible by using a fixative. Chemical fixatives such as glutaraldehyde or osmium tetroxide are commonly used because they cross-link proteins and stabilize the tissue's structure, preventing enzymatic degradation and autolysis. The tissue sample of interest may be immersed in the fixative for a specified period, which can vary depending on the tissue type and size. A fixative may also be delivered to the tissue sample of interest by transcranial perfusion.

After fixation, the tissue sample of interest may be washed with a buffer solution to remove excess fixative and any byproducts that may interfere with further processing of the tissue sample of interest.

The tissue sample of interest may undergo a second fixation step, e.g. with osmium tetroxide, which provides additional fixation and stains the tissue, enhancing contrast by reacting with lipids to create electron-dense areas.

The tissue sample of interest may be dehydrated through a graded series of e.g. ethanol or acetone solutions, starting with a lower concentration and gradually increasing to 100% to remove all water from the sample. This step can be advantageous because the embedding media, e.g. resin, may be hydrophobic and water would prevent proper infiltration.

After dehydration, the tissue sample of interest may be gradually infiltrated with a liquid embedding medium, such as any resin defined herein. The infiltration process may start with a mixture of the dehydrating agent and the resin and then transition to pure resin. The tissue sample can be left in the resin for it to completely permeate. In the sense of the present invention the tissue sample of interest may be infiltrated by a graded acetone-resin mixture, wherein the amount of resin increases throughout the grade. Finally, the tissue sample of interest can be infiltrated with pure resin, i.e. without acetone.

Once the tissue sample of interest is infiltrated, it can be placed in a mold, e.g. custom made mold adapted to the needs of the individual application, with fresh resin and polymerized (hardened) in an oven at a controlled temperature, e.g. 60°C. The result is a solid block with the tissue sample of interest embedded within it. This forms the tip of the screening sample of the invention.

Using an ultramicrotome, thin sections of the tissue are cut from the loaded chaperone block comprising the loaded tissue sample of interest. The thickness of these sections is typically around 50-100 nanometers for transmission electron microscopy (TEM) and can be thicker for scanning electron microscopy (SEM). As explained herein, the chaperone block loaded with the tissue sample of interest of the present invention is particularly advantageous since it allows for sections as thin as 35 nm. The sections may be collected on grids made of metal, such as copper or nickel or on carbon coated tape or silicon wafers.

The prepared and stained sections can be placed into the electron microscope for examination. The electron beam interacts with the sample, and the resulting images are captured, on a digital camera system. The images can then be analyzed e.g. for connectomics. Apparatus for loading a chaperone block

An apparatus for loading a screening sample into a chaperone block in the sense of the present invention comprises a first holder for holding the chaperone block, a second holder for holding the screening sample, wherein the apparatus is configured to move the tissue sample of interest by means of the second holder into a cavity of the chaperone block and to break of the tissue sample of interest from the base, thereby loading the screening sample into the chaperone block. The apparatus facilitates the insertion of the tissue samples, which are to be processed in a 3- dimensional electron microscopy-based connectomic screening, into cavities comprised in a chaperone block by providing stability through two holders and by providing means of precise movement of the screening sample into the cavities through a micromanipulator. To further reduce vibrations and instabilities all components of the apparatus are attached to an air table. The first holder, such as an 3D-printed holder, is fixed in an appropriate angle on a structure, such as a rod, wherein this structure may be fixed onto two additional structures, which are fixed on the air table, these structures optionally being rods fixed to the air table using screws. The second holder, such as an 3D-printed holder, can comprise a pneumatic gripper. The pneumatic gripper allows to collect a screening sample, e.g. collecting a from a magazine, hold the screening sample while moving, load the tissue sample of interest into a cavity of a chaperone block by moving the screening sample, break of the tissue sample of interest by a fast movement, while the screening tissue is loaded in the chaperone block and discard the screening sample after the tissue sample of interest has been loaded and has been broken off from its base. The screening sample may also manually be inserted into the second holder. In general, the pneumatic gripper can open the second holder for holding the screening sample. A screening sample may then be inserted into the second holder manually or automatically. Afterwards, the pneumatic gripper closes the second holder again, thereby fastening the screening sample in the second holder. For this the second holder may comprise two parts for holding a screening sample that can be opened by the pneumatic gripper. After loading of the chaperone block, the screening sample can be discarded by opening the second holder with the pneumatic gripper and discarding the screening sample. A new screening sample for loading can then be inserted into the second holder by repeating the above steps.

The pneumatic gripper is attached to a micromanipulator in an appropriate angle. The micromanipulator may be attached to a breadboard, such as an aluminum breadboard. The breadboard in turn is attached to the air table via a height adjuster. The micromanipulator allows for directed movement of the screening sample when in the holder in all three dimensions. The speed of the micromanipulator may be adjusted, e.g. to break off the tissue sample of interest at the predetermined breaking point. Preferably the first holder and the second holder are arranged at the same angle to the horizontal, e.g. 30° to the horizontal, wherein the holders face each other such that when holding a chaperone sample and a screening sample, both samples face each other. The holders are further characterized by their ability for securely hold and/or release the chaperone block and/or the screening sample. The apparatus may be placed under a microscope in such a way that the apparatus may be monitored during operation, e.g. during loading a chaperone block with a screening sample. An exemplary apparatus for loading a chaperone block of the present invention is shown in Figure 3 A.

Accordingly, the invention provides in one aspect an apparatus for loading a chaperone block, e.g. the chaperone block of the present invention with a screening sample, e.g. a screening sample of the present invention comprising:

(a) a first holder for holding the chaperone block,

(b) a second holder for holding the screening sample, wherein the apparatus is configured to move the tissue sample of interest by means of the second holder into a cavity of the chaperone block and to break of the tissue sample of interest from the base, thereby loading the screening sample into the chaperone block.

The invention also provides an apparatus for loading a chaperone block, e.g. the chaperone block of the present invention with a screening sample, e.g. a screening sample of the present invention comprising:

(a) a first holder for holding the chaperone block, wherein the first holder is attached to an air table,

(b) a second holder for holding the screening sample, wherein the second holder is attached to an air table, wherein the apparatus is configured to move the tissue sample of interest by means of the second holder into a cavity of the chaperone block and to break of the tissue sample of interest from the base, thereby loading the screening sample into the chaperone block.

(a) a first holder for holding the chaperone block,

(b) a second holder for holding the screening sample configured to move the screening sample in three dimensions, wherein the apparatus is configured to move the tissue sample of interest by means of the second holder into a cavity of the chaperone block and to break of the tissue sample of interest from the base, thereby loading the screening sample into the chaperone block.

(a) a first holder for holding the chaperone block,

(b) a second holder comprising a micromanipulator for holding the screening sample, wherein the apparatus is configured to move the tissue sample of interest by means of the second holder into a cavity of the chaperone block and to break of the tissue sample of interest from the base, thereby loading the screening sample into the chaperone block. The invention also provides an apparatus for loading a chaperone block, e.g. the chaperone block of the present invention with a screening sample, e.g. a screening sample of the present invention comprising:

(a) a first holder for holding the chaperone block,

(b) a second holder for holding the screening sample comprising a micromanipulator attached to a breadboard, e.g. a aluminum breadboard, wherein the apparatus is configured to move the tissue sample of interest by means of the second holder into a cavity of the chaperone block and to break of the tissue sample of interest from the base, thereby loading the screening sample into the chaperone block.

(a) a first holder for holding the chaperone block,

(b) a second holder for holding the screening sample comprising a micromanipulator attached to a breadboard, e.g. a aluminum breadboard, wherein the breadboard is attached to an air table via a height adjuster, wherein the apparatus is configured to move the tissue sample of interest by means of the second holder into a cavity of the chaperone block and to break of the tissue sample of interest from the base, thereby loading the screening sample into the chaperone block.

(a) a first holder for holding the chaperone block,

(b) a second holder for holding the screening sample, wherein the apparatus is configured to move the tissue sample of interest by means of the second holder into a cavity of the chaperone block and to break of the tissue sample of interest from the base, thereby loading the screening sample into the chaperone block, wherein the first and the second holder are arranged at an angle of about 0 to about 180°, such as about 0°, about 10°, about 20°, about 30°, about 40°, about 50°, about 60°, about 70°, about 80°, about 90°, about 100°, about 110°, about 120°, about 130°, about 140°, about 150°, about 160°, about 170°, or about 180° to the horizonal, with the holders facing each other such that when holding a chaperone sample and a screening sample, both samples face each other.

(a) a first holder for holding the chaperone block,

(b) a second holder for holding the screening sample, wherein the apparatus is configured to move the tissue sample of interest by means of the second holder into a cavity of the chaperone block and to break of the tissue sample of interest from the base, thereby loading the screening sample into the chaperone block, wherein the first and the second holder are arranged at an angle of about 30° to the horizonal, with the holders facing each other such that when holding a chaperone sample and a screening sample, both samples face each other.

(a) a first holder for holding the chaperone block,

(b) a second holder for holding the screening sample, wherein the apparatus is configured to move the tissue sample of interest by means of the second holder into a cavity of the chaperone block and to break of the tissue sample of interest from the base, thereby loading the screening sample into the chaperone block, wherein the first holder and the second holder further comprise fastening means to reversibly fasten the chaperone block and the screening sample. In one embodiment the first holder further comprises screws and screw sockets to reversibly fasten the chaperone block to the first holder. In a further embodiment the second holder further comprises a pneumatic gripper to reversibly collect, hold and/or discard the screening sample.

A method for preparing a sample for connectomic screening

The invention also provides A method for preparing a sample for parallel processing for 3- dimensional electron microscopy-based connectomic screening in the sense of the present invention refers to the provision and/or production of a screening sample and of a chaperone block and to the loading of said screening sample into the chaperone block with an apparatus. The features of the chaperone block, screening sample, apparatus as well as the features provided in the method for producing a chaperone block and the method for producing a screening sample of the present invention apply mutatis mutandis to the method for preparing a sample for connectomic screening, since said method combines all of the above.

Accordingly, the invention provides a method for preparing a sample for connectomic screening, the method comprising:

(a) providing one or more screening sample, e.g. a screening sample of the invention or producing a screening sample according to the method of the invention,

(b) providing a chaperone block, e.g. a chaperone block of the invention, or producing a producing a chaperone block according to the method invention,

(c) loading the one or more screening sample into the chaperone block, e.g. with the apparatus of the present invention.

The method for preparing a sample for connectomic screening according to the present invention may further comprise one or more of the following steps, preferably in chronological order:

Providing and/or producing a screening sample. The shape of the screening sample after milling is documented for later reference.

Providing and/or producing a chaperone block.

Providing an apparatus for loading a screening sample into a chaperone block.

The chaperone block is placed into a first holder comprised in the apparatus, wherein the first holder is fixed on a structure in an appropriate angle. The chaperone block is placed into the holder in such a way that the openings of the cavities are facing towards the second holder comprised in the apparatus.

The screening sample is placed into a second holder comprised in the apparatus. The second holder can comprise a pneumatic gripper and the second holder is attached in an appropriate angle, wherein the angle is the same angle as the angle in which the first holder is attached. The screening sample is placed into the second holder in such a way that the second holder holds the screening sample at the base.

The screening sample can be moved by operating a micromanipulator, wherein the screening sample can be moved in three directions and the movement of the screening sample may be monitored through binoculars, for example by monitoring the movement of the screening sample through the binoculars of a microscope.

One screening sample is loaded into one cavity of the chaperone block at a time. The tissue sample of interest and the resin layer each comprised in the screening sample are completely inserted into the cavity of the screening sample, wherein the screening sample is inserted in such a way that the tissue sample of interest is inserted into the cavity of the chaperone block first. The tissue sample of interest is inserted into the cavity of the chaperone block in such a way that it is fully surrounded by the chaperone sample. The resin layer of the screening sample is inserted into the cavity of the chaperone block is such a way that it is fully surrounded by the top layer of resin of the chaperone block. The base of the screening sample is not inserted into the cavity of the chaperone block. After insertion of the tissue sample of interest and the resin layer into the cavity, the resin layer is broken of the base at predetermined breaking point, by rapidly moving the screening sample away from the cavity sideways until the predetermined breaking point breaks. The predetermined breaking point breaks in such a way that the tissue sample of interest and the resin layer remain inserted in the cavity of the chaperone block and the resin base of the screening sample can be discarded. This process may be repeated as many times as desired, for example this process may be repeated until every cavity of the chaperone block has been loaded with one screening sample, wherein it is documented which screening sample is inserted in which cavity in such a way that the screening samples can be allocated for processing and imaging. This documentation of the position of each screening sample can be important for later reference. The loaded chaperone block is removed from the first holder.

An appropriate amount of resin is added on top of the chaperone block, more specifically on top of the top layer of the chaperone block, thereby filling the open spaces in the loaded cavities between the screening sample and the cavity walls. The appropriate amount of resin may be added all at once or in several steps. By adding an appropriate amount of resin the chaperone block is sealed and thus the tissue samples are preserved, furthermore the screening samples are secured in the chaperone block.

The chaperone block loaded with one or more screening samples is cured in the oven for 1 to 3 days. Spilled resin may be milled. Milling may, for example, be performed using a 6mm- diameter aluminum milling cutter on a milling machine.

The depth and position of each screening sample is determined by acquiring a control image, such as a pCT image. To fill visible gaps in the chaperone block identified by analyzing the pCT image, an appropriate amount of resin, such as a drop of resin, may be added and the chaperone block is placed into the oven for curing for 1 to 3 days.

Based on the information regarding the position from the pCT image resin is removed from the top of the chaperone block to expose a continuous layer of chaperone sample and tissue sample of interests. The continuous layer of chaperone sample and screening sample refers to a layer in one plane. The removal of resin may be performed using routine methods know to the person skilled in the art. These methods can comprise a diamond head milling system or a diamond head milling system, for example.

The chaperone sample loaded with the screening sample may further be sliced into slices of appropriate thickness, wherein an appropriate thickness is a thickness that allows processing, imaging and analyzing of the chaperone sample loaded with the screening sample, for example electron microscopy-based image acquisition.

Accordingly, the invention provides in one aspect a method for preparing a sample for connectomic screening, the method comprising:

(a) providing one or more screening sample, e.g. a screening sample of the invention or producing a screening sample according to the method of the invention, configured to be loaded into the cavities of the chaperone block,

The invention also provides a method for preparing a sample for connectomic screening, the method comprising:

(c) loading the one screening sample into each cavity of the chaperone block, e.g. with the apparatus of the present invention. The invention also provides a method for preparing a sample for connectomic screening, the method comprising:

(b) providing a chaperone block, e.g. a chaperone block of the invention, or producing a chaperone block according to the method invention,

(c) loading the one or more screening sample into the chaperone block, e.g. with the apparatus of the present invention, wherein the screening sample is loaded into the chaperone block by inserting the screening sample into a cavity of the chaperone block followed by breaking off the screening sample at a predetermined breaking point from its base hold by the second holder, thereby loading the chaperone block with the screening sample optionally, wherein the screening sample is broken off at a predetermined breaking point while being inserted into the cavity of the chaperone block by rapidly moving the second holder, further optionally by rapidly moving the second holder sideways along the surface of the chaperone block.

(c) loading the one or more screening sample into the chaperone block, e.g. with the apparatus of the present invention, wherein the screening sample is loaded into the chaperone block by inserting the screening sample into a cavity of the chaperone block followed by cutting off the screening sample at a predetermined breaking point from its base hold by the second holder, thereby loading the chaperone block with the screening sample. Cutting of the screening sample may be advantageous for large screening samples e.g. with a diameter of about 1mm.

In some embodiments, a screening sample is loaded into at least about 50 % to 100 %, such as about 50 %, about 55 %, about 60 %, about 65 %, about 70 %, about 75 %, about 80 %, about 85 %, about 90 %, about 90 %, about 91 %, about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 %, or about 100 % of cavities of the chaperone block.

In some embodiments, the method for preparing a sample for connectomic screening further comprises removing the loaded chaperone block from the first holder and adding resin on top of the chaperone block thereby filling open spaces in the loaded cavities between the screening samples and the cavity walls. In some embodiments, the method for preparing a sample for connectomic screening further comprises milling the top of the chaperone block to expose a continuous layer of chaperone sample and tissue sample of interests. This also ensures a homogenous surface for further processing of the loaded chaperone sample such as sectioning and/or imaging. In some embodiments, the method for preparing a sample for connectomic screening further comprises slicing or sectioning the chaperone sample loaded with the screening sample thereby producing sections of chaperone sample and tissue sample of interest for further analysis/imaging. As explained above, the invention surprisingly allows for sections as thin as 35 nm.

Batch sample

A batch sample in the sense of the present invention is a multitude of tissue samples of interest which are embedded in a resin. Herein the terms “batch sample” and “batch of rods” are used interchangeably. The terms relate to tissue sample rods placed in a holding sample (see, e.g., Figs. 7, 8 and 9A-C) which again could be a chaperone sample in the sense of the present disclosure (see, e.g., Fig. 9D), yet with a large cavity to hold the multitude of (e.g., more than 100) tissue samples. The rods may be subsequently dipped into the resin before placement on the batch in order to stick to the already placed rods. Alternatively, the resin may be applied to the growing batch repeatedly. The batch sample can, for example, be used for 3 -dimensional electron microscopy-based connectomic screening. The tissue samples in the batch sample are arranged to provide structural integrity to each other and thereby to the batch sample as a whole. The tissue samples comprised in the batch sample homogenize the electrical conductivity and the surface of the batch sample, e.g. when later used for sectioning. Homogenization of the surface of the batch sample is advantageous for the cuttability to avoid a disintegration of a slice when sectioning the batch sample, e.g. because of mechanical stress due to an inhomogeneous surface. In the sense of the present invention the tissue samples used in the batch sample may be tissue sample of interests as defined herein. Accordingly, all features provided herein in context of the screening sample (or the tissue sample of interest) apply mutatis mutandis to the tissue samples in the batch sample and the batch sample per se. The skilled person will understand that certain shapes of screening samples, e.g., rectangular shaped screening samples (shown in Fig. 6), may allow increased packing density within the chaperone cavity compared to other shapes.

A method for producing a batch sample may comprise one or more of the following steps:

Providing one or more tissue samples of interest from one or more mammal, reptile, fish, or bird, e.g. providing a screening sample of the present invention or producing the same according to the invention,

Cutting the tissue sample into an appropriate size and shape,

Arranging the one or more tissue samples in such a way that each sample provides structural integrity to one or more tissue samples, thereby stabilizing the batch sample, e.g. arranging the tissue samples in a pyramid, square, rectangle, triangle and the like such that the tissue samples are piled up like logs,

Embedding and/or infiltrating the one or more tissue samples in/with resin, Curing the tissue samples embedded in resin for 1 to 3 days, Slicing the tissue samples embedded in resin into slices of an appropriate size for connectomic screening.

A suitable technique for obtaining sample rods for batch positioning in the context of the present invention is shown in Fig. 7. The EM sample (tissue sample) described herein may be embedded in a holding resin structure being trimmed into a broad base and a thin sample-resin rod (Fig. 7A, left). This procedure is similar to the one used for hole-based sample batching described herein. The broad base can be gripped by a micromanipulator. A break-off template sample (Fig. 7A, right) may be prepared (of durable material, no requirement for conductivity etc). The break-off template sample may contain apertures of varying size to fit varying diameters of sample rods described herein. The base-sample unit (e.g. by the micromanipulator) and thereby the EM sample rod inserted into the break-off template may be moved (Fig. 7B). Then, the sample rod may be broken off the holding base by rapid movement of the micromanipulator. This procedure may be similar to the one used for hole-based sample batching described herein. From the other side, the sample rod is pushed back out of the break-off template using, e.g., high-pressure air puffs or a resin-rod, which may be held by another micromanipulator (Fig. 7C). Finally, the EM-sample rod may be pulled out of the break-off template using a micro-sample gripper that can non- destructively grab the EM sample rod (Fig. 7D).

Another suitable technique for obtaining sample rods for batch positioning in the context of the present invention is shown in Fig. 8. The EM sample (tissue sample) described herein may be embedded in a holding resin structure being trimmed into a broad base and thin sample-resin rod (Fig. 8A, left). This procedure is similar to the one used for hole-based sample batching described herein. The broad base may be gripped by a micromanipulator. (B) The EM sample rod may be grabbed (by another micromanipulator, e.g., ideally positioned to grab the resin part of the EM sample rod), and by rapid relative movement of the base versus the sample rod, the latter may be detached (broken off) from the resin base (Fig. 8B). This yields in a separated EM sample rod, held by a micromanipulator, ready for the final batching step (Fig. 8C).

In the context of the present invention, for positioning the sample of rods into a batch sample the EM sample rod obtained by, e.g., one of the two techniques described above, may be placed into the void of a properly shaped chaperone sample (Fig. 9A showing the side view of the positioning). The sample rod may be positioned to align to the chaperone sample posterior wall (Fig. 9B, showing the side view of the positioning). The micro-sample gripper may be removed, leaving the sample in position, with overhanging resin components, which may be trimmed off (Fig. 9C). The sample rods may be placed successively to yield efficiently area-covering sample rod arrangements. In the context of the present invention, the positioning may be performed in orthogonal alignment (Fig. 9B, left) or rotated 45° to improve rod placement accuracy (Fig. 9D, right). The fluid resin may be administered to the chaperone sample to improve stickiness of the first sample placements (Fig. 9B-D). In a final step, the remaining gaps may be filled by resin infiltration.

Typically, the batch sample to be used in the present invention is a biological tissue sample. Herein, the batch sample can include any type of biological tissue sample collected from a living or dead subject. The collection of the biological tissue sample may include biopsies or autopsies for example. According to the present invention, the batch sample is preferably collected from a mammal, reptile, fish, or bird. More preferably, the batch sample is collected from rodents or primates. Non-limiting examples of reptiles include dragons, turtles, or salamanders. More preferably, the batch sample is collected from mice, humans, macaques, rats, ferrets, rabbits, or marmosets. Most preferably, the batch sample is collected from humans. In the context of the present invention, a batch sample containing neurons is preferred. The neuronal tissue-of-interest may be a structure of several hundred micrometers in diameter, such as barrels in the mouse SI cortex, subnuclei in the thalamus, or directional preference columns in the visual cortex.

In the context of the present invention, a batch sample may be collected from whole brain, hemispheres, cortical and/or subcortical volumes, spinal cords and peripheral tissues containing nerve endings and the like. Also included as defined herein is the cortex such as the somatosensory cortex, parietal cortex and the like. The batch sample can be an unprocessed or a processed biological tissue sample. The batch sample can also be an organoid or a tissue culture, preferably comprising neurons and/or neuronal cells. As used herein, processing can refer to laboratory methods known to a person skilled in the art, such as tissue staining, fixation, embedding, infiltration, dehydration or the like. The size or shape of the batch sample is not particularly limited. In particular, the skilled person will understand that the size or shape of the batch sample may depend on the intended use of the sample. Furthermore, it is pointed out that using larger and/or faster microscopes (e.g., techniques commonly referred to as ultrafast electron microscopy (UEM)), the interface of the batch sample may get as large as, e.g., 1cm x 1cm). The skilled person will understand that no obvious physical limitation exists. To the contrary, when automated more than 1000 (tissue) samples can be placed in one batch. This in turn substantially decreases overhead times.

Accordingly, the invention provides in one aspect a batch sample comprising a multitude of tissue samples, such as tissue samples of interest, embedded in a resin, wherein the tissue samples, such as tissue sample of interest, are configured to provide structural integrity to each other and thereby to the entire batch sample, and wherein the tissue samples are configured to homogenize the electrical conductivity and surface of the batch sample.

The invention also provides a batch sample comprising a multitude of tissue samples, such as tissue sample of interest, embedded in a homogenate of stained tissue, wherein the tissue samples, such as tissue sample of interest, are configured to provide structural integrity to each other and thereby to the batch sample, and wherein the tissue samples are configured to homogenize the electrical conductivity and surface of the batch sample.

In the context of the present invention, a homogenate of stained tissue is a heterogeneous suspension obtained by disrupting (to a varying degree) cellular integrity of stained tissues. For instance, embedding the tissue sample of interest within (stained) intact cells (e.g., via floating in of blood cells) can similarly work.

The invention also provides a batch sample comprising a multitude of tissue samples, such as tissue sample of interest, embedded in a metal containing resin, wherein the tissue samples, such as tissue sample of interest, are configured to provide structural integrity to each other and thereby to the batch sample, and wherein the tissue samples are configured to homogenize the electrical conductivity and surface of the batch sample. A metal containing resin has the advantage, that it increases electrical conductivity which in turn is beneficial for EM imaging. The invention also provides a batch sample comprising a multitude of tissue samples, such as tissue sample of interest, embedded in a conductive material, such as a resin containing a metal and/or a conductive material (e.g., graphite), wherein the tissue samples, such as tissue sample of interest, are configured to provide structural integrity to each other and thereby to the batch sample, and wherein the tissue samples are configured to homogenize the electrical conductivity and surface of the batch sample. For example, the batch sample comprising a multitude of tissue samples, such as tissue sample of interest, can be embedded in carbon nanotubes. The skilled person will understand that in the context of the present invention any material enhancing electrical conductivity can be used to embed the (tissue) sample of interest.

Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted.

As used herein, the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise. The terms “include,” “such as,” and the like are intended to convey inclusion without limitation, unless otherwise specifically indicated.

As used herein, the term “or” is generally employed in its usual sense including “and/or” unless the content clearly dictates otherwise. The term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements.

As used herein, the term “comprising” also specifically includes embodiments “consisting of’ and “consisting essentially of’ the recited elements, unless specifically indicated otherwise.

As used herein, the term “about” indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” indicates the designated value ± 10%, ± 5%, or± 1%. In certain embodiments, where applicable, the term “about” indicates the designated value(s) ± one standard deviation of that value(s).

In some embodiments any one of the methods described herein can be in vitro and/or ex vivo. In some embodiments any of the method steps described herein can be in vitro and/or ex vivo.

The disclosures in context of the methods described herein are disclosed as corresponding use mutatis mutandis. The disclosures in context of the use described herein are disclosed as corresponding methods mutatis mutandis. Furthermore, product features described in the context of the chaperone block of the present invention are disclosed herein as corresponding method steps of the method for producing a chaperone block mutatis mutandis. Furthermore, product features described in the context of the screening sample of the present invention are disclosed herein as corresponding method steps of the method for producing a screening sample mutatis mutandis. Furthermore, product features described in the context of the chaperone block, the screening sample and the apparatus of the present invention are disclosed herein as corresponding method steps of the method for preparing a sample for connectomic screening mutatis mutandis.

In one aspect, the methods of the present invention are not methods for treatment of the human or animal body by therapy. In a further aspect, the methods of the present invention are not processes for modifying the germ line genetic identity of human beings. In one aspect, the methods of the present invention are in vitro or ex vivo methods. In a further aspect, the methods of the present invention are non-therapeutic or non-medical. In a further aspect, the methods of the present invention are not processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal.

The methods of the present invention relate to the use of fixed biological tissue samples and any means suitable for fixing biological tissue samples known in the art, such as perfusion, immersion and the like, can be used. The skilled person will understand that any fixative reagent, such as any fixative comprise in a cacodylate buffer, such as paraformaldehyde, glutaraldehyde and the like as well as mixtures of fixative reagents also in different buffers known in the art may be used in the context of the present invention. In the context of the present invention, in order to increase the density/electrical conductivity of the biological tissue sample for EM, heavy metal atoms may be incorporated from solution into the tissue-of-interest. These heavy metal atoms attach to subcellular structures within the tissue, increasing their electron density. As a result, the subcellular structures appear dark and stand out against a lighter background.

Typically, heavy metals/heavy metal compounds to be used according to the present invention include but are not limited to osmiumtetroxide, osmic acid, uranyl acetate (UA), uranyl formate, lanthanum nitrate, phosphotungstic acid, phosphomolybdic acid, ammonium molybdate, sodium urinate, lead perchlorate, erbium acetate, gadolinium acetate, methylamine tungstate, samarium acetate, thulium acetate, sodium silicotungstate, sodium phosphotungstate, lead hydroxide/citrate and the like. In case heavy metal solutions containing lead are used in the methods of the present invention, it is suggested to extend the incubation time to preferably 24 to 48 h and to renew the solution preferably 5 to 10 times.

After being stained, the biological tissue sample may be dehydrated by using a graded ethanol series. Furthermore, a biological tissue sample may be infiltrated by a graded acetone-resin mixture, wherein the amount of resin increases throughout the grade. Finally, the biological tissue sample is infiltrated with pure resin, i.e. without acetone. Any means suitable to dehydrate biological tissue samples are suitable in the methods of the present invention.

Typically, after being dehydrated, the biological tissue samples in the context of the present invention are embedded by using a low-viscosity epoxy resin embedding method.

In the context of the present invention, the term "resin infiltration" refers to treating the tissue with a resin that penetrates throughout the tissue to the molecular level, subsequently hardens and finally transforms into a solid thereby rendering the sample rigid.

According to the present invention, the term "embedding" relates to placing an infiltrated biological tissue sample in a mold in which the sample is typically surrounded with the same resin. The mold is then hardened to form an encasing block. The embedding reagent thus serves to provide rigid support and to facilitate the subsequent sectioning process.

Embedding mediums used herein are preferably epoxy resin and include but are not limited to commercially available epoxy resins such as Epon 812 substitute resin, resin prepared according to Spurr, and the like. Different resins provided herein may also be mixed with each other.

EM analysis is not limited to a certain method and preferably, volume EM, even more preferably high-throughput volume EM is used for connectomic analysis/dense neuronal circuit reconstruction, most preferably 3 -dimensional electron microscopy -based connectomic screening in the context of the present invention.

Typically, the image data needed for high-resolution connectomics may be acquired using transmission electron microscopy (TEM), preferably scanning electron microscopy (SEM), and/or section-based SEM (SBEM) and the like. Typically, the biological tissue sample of interest in the context of the present invention is cut into sections of preferably 20 to 200 nm, more preferably 30-40 nm, even more preferably 35 nm and/or 38 nm and further imaged by using high throughput SEM, preferably Multi-SEM.

As used herein “CAD software” preferably refers to the CAD software SolidWorks Professional 2024 developed by Dassault Systemes SolidWorks Corp.

As used herein “CAM software” preferably refers to the CAM software HyperMill developed by OPEN MIND Technologies AG.

Figure 1

Preparation of screening samples. Procedures shown for one screening sample; many such samples will be processed and placed into the chaperone sample (Fig. 2). (A) Extraction of screening sample from a brain section (or by biopsy), staining using established 3D EM en-bloc staining protocols (in particular Hua et al 2015, Song et al 2024). (B) Placement of the screening sample on resin. (C) Alignment of screening sample for inspection and preparation for milling of screening sample shape. (D) Top view of screening sample as seen through the inspection optics of (C), indicating milling planes. (E) Sample milling and resulting sample shape for each screening sample. Note dimensions are examples, ranges provided in application text.

Figure 2

Preparation of the chaperone sample. (A) Example extraction of chaperone sample from brain slices. (B) Placement of the chaperone sample in resin glued to sample holder, in relation to the sample trimming apparatus. (C) Arrangement of the sample alignment setup, indication of the alignment plane, and top view of chaperone sample before milling. (D) Overlay of chaperone sample top view with CAD-based definition of sample center for subsequent milling steps. (E) Arrangement of milling cutter and chaperone sample (not drawn to scale); sketch of cavity pattern and hexagonal sample shape (middle, not drawn to scale) and bright -field image of top view of chaperone sample with overlaid cavity pattern (right). (F) Arrangement of high-performance drill and chaperone sample, with sketch of drilling procedure and pCT images of a chaperone sample with 105 cavities after drilling.

Figure 3

Screening sample insertion setup. (A) Arrangement of screening sample holder and associated micromanipulator and pneumatic gripper (right) and chaperone sample holder placed at corresponding angles for friction-minimized insertion. A binocular microscope is placed at the top for observation of the insertion process, in particular the centered approach to avoid breaking off the screening sample before insertion. The arm of a light source is placed at an angle of about 30° along the screening sample holder (second holder) shining in the direction of the chaperone sample. (B) Detailed schematic of sample movement by remote controlled micromanipulator to detach screening sample from holding block after insertion into the chaperone sample. (C) Resulting arrangement of chaperone sample and multiple inserted screening samples after successful insertion and detachment, side view. Note that depth of insertion determines alignment of screening samples along insertion direction, critical for effective imaging in one plane. After insertion, resin is applied from the top to fill remaining cavities and space between screening sample and chaperone cavity. (D) Milling of chaperone sample containing screening samples into the final shape for 3D EM imaging.

Figure 4

3D EM Imaging of combined chaperone - screening sample block. (A) sketch and pCT imagery of chaperone block with 103 successfully inserted screening samples. Two cavities are empty. 93 tissue samples of interest have been completely inserted, 7 tissue samples of interest have been inserted half in diameter due to embedding/trimming, 3 tissue samples of interest have been inserted too deep in chaperone block and therefore have been cut after the first 100pm in the z- axis. (B) EM overview images of combined sample block imaged using multiSEM. (C-E) EM images at various resolutions reporting the integrity and sample quality of the combined chaperone sample - screening sample block; Dwell time: 50ns, Voxel size 4x4x35nm³.

Figure 5

106-screening sample experiment. (A) pCT image of a chaperone sample with 106 drilled encavings sized 150 pm in diameter, each, with about 50 pm chaperone tissue in between. Panels show xy, yz, xy, low-res xy views, respectively (clockwise from top left). (B) pCT image of the same chaperone sample as in (A), after placement of the 106 screening samples into the encavings. Same views as in (A). Note aligned placement of samples into encavings, and successful placement of 106/106 samples proving a high success rate of the methods described herein. (C) Snapshot from ATUM cutting of same sample.

Figure 6

Example of rectangular-shaped screening sample. pCT image of a screening sample trimmed in rectangular shape. This is beneficial for tighter sample placement in the batch-of-rod technique, and simplifies subsequent 3D data analysis.

Figure 7

Technique 1 for obtaining sample rods for batch positioning. (A) The EM sample is embedded in a holding resin structure that has been trimmed into a broad base (left) and thin sample-resin rod. This procedure is similar to the one used for hole-based sample batching. The broad base can be gripped by a micromanipulator. A break-off template sample (right) has been prepared (of durable material, no requirement for conductivity etc). The break-off template sample contains apertures of varying size to fit varying diameters of sample rods. (B) The base-sample unit is moved (e.g. by the micromanipulator) and thereby the EM sample rod inserted into the break-off template. Then, the sample rod is broken off the holding base by rapid movement of the micromanipulator (this procedure is similar to the one used for hole-based sample batching. (C) From the other side, the sample rod is pushed back out of the break-off template using high-pressure air puffs, or a resin-rod (potentially held by another micromanipulator, not shown). (D) Finally, the EM-sample rod is pulled out of the break-off template using a micro-sample gripper that can non-destructively grab the EM sample rod.

Figure 8

Technique 2 for obtaining sample rods for batch positioning. (A) The EM sample is embedded in a holding resin structure that has been trimmed into a broad base (left) and thin sample-resin rod. This procedure is similar to the one used for hole-based sample batching. The broad base can be gripped by a micromanipulator. (B) The EM sample rod is grabbed (by another micromanipulator, ideally positioned to grab the resin part of the EM sample rod), and by rapid relative movement of the base versus the sample rod, the latter is detached (broken off) from the resin base, (C) yielding a separated EM sample rod, held by a micromanipulator, ready for the final batching step.

Figure 9

Positioning of sample rods into sample batch. (A) The EM sample rod (obtained by either of the techniques shown in Figs. 7 and 8) is placed into the void of a properly shaped chaperone sample. Side view. (B) The sample rod is positioned to align to the chaperone sample posterior wall (side view shown). (C) The micro-sample gripper is removed, leaving the sample in position, with overhanging resin components (that can later be trimmed off). (D) Sample rods are placed successively to yield efficiently area-covering sample rod arrangements. Positioning can be performed in orthogonal alignment (left) or rotated 45° to improve rod placement accuracy (right). In Steps B-D, fluid resin can be administered to the chaperone sample to improve stickiness of the first sample placements. In a final step, the remaining gaps are filled by resin infiltration.

Figure 10

Batch-of-rod experiment. (A) Micro-CT image of a chaperone sample with an encaving, into which 13 screening samples were placed. The density of sample placement can be substantially further improved. (B) Example high-resolution EM image from the same sample as in (A), indicating high-quality ultrastructural tissue preservation at the border of one of the screening samples in (A). Note that using rectangular shaped screening samples (as shown in Fig. 6), higher packing density of the screening samples in the chaperone sample cavity can be achieved. In the following, the invention is further illustrated in a non-limiting example.

Example 1

Introduction

The connectomic analysis of brain samples has yielded surprising insights into the structure of neuronal circuits across various species and brain regions. However, these insights have so far been obtained from relatively small sets of experimental samples, ranging from n=l to about 12. For the systematic screening of connectomic variability across many relevant axes of variation, however, such as postnatal development, aging, pathological change, behavioral variability, disease states and pharmacological and other interventions, efficient data acquisition from ideally hundreds of volume EM samples is essential. Here, we developed an approach for the efficient sample preparation and imaging of order-of-magnitude 10² connectomic samples. By embedding a large number of samples into a chaperon tissue bloc, long-series ultrathin cuttability and artefact- free large-scale EM imaging could be obtained. We demonstrate the usability of this approach with 100 samples from mouse cortex, imaged in the multiplexed sample setting. With this, connectomic screening at a scale of 10² to the 10³ samples is made possible, enabling connectomic analyses of unprecedented magnitude.

The effort to obtain connectomic volume EM samples at a scale required for neuronal circuit analysis has so far impeded large-scale connectomic screening efforts. Most connectomic studies have so far been conducted on N=1 samples (Bock et al., 2011; Briggman et al., 2011; Helmstaedter et al., 2013; Hildebrand et al., 2017; Kornfeld et al., 2017; Lee et al., 2016; Shapson- Coe et al., 2024; Svara et al., 2022; Winding et al., 2023; Zheng et al., 2018) or n=2..3 samples (Motta et al., 2019; Schmidt et al., 2017), or in some rare cases of 8 to 13 samples (Gour et al., 2021; Loomba et al., 2022; Witvliet et al., 2021).

While some of these studies have provided fundamental insights about neuronal circuit structure, the relationship of connectomic properties to important parameters of variation or relevant events of brain formation cannot be systematically screened. For such investigations, screening at a scale of n=10² to 10³ samples is required.

The effort to sample and process a single 3D EM sample of the size 100 pm³ is currently about 4 weeks of combined imaging and cutting on a single-beam EM setup (or alternatively about 36 hours of imaging time or a total time of 77 hours including overhead time for a 100pm³ sample using ATUM+multiSEM). Evidently, scaling this to 100 samples would yield on the order of 10 years of imaging time for single beam setups (and 150 days for imaging time or a total time of 322 days including overhead time for a 100pm³ sample in multi -beam setups). Thus, the full scalability into this range of connectomic screening is not plausible.

At the same time the imaging of large tissue blocks at sized one to two millimeters in-plane has become realistic with the multiSEM imaging approach (Loomba et al., 2022; Shapson-Coe et al., 2024; Sievers et al., 2024). Here, within about 6 weeks a volume of 2mm by 2mm by about 100pm can realistically be imaged/acquired.

Our method utilizes the ability to image very large planes combined with the multiplexing of sample configuration to leverage the imaging speed into the ability to screen hundreds of EM samples, a throughput increase by a factor of 16 up to 26.

Results

The key impediment of single sample repeated data acquisition is both the difficulty of cutting and handling very small samples, collecting sections reliably at small size and the overhead of sample preparation and movement during imaging.

The goal of obtaining a multiplex sample preparation procedure was to make the preparation of about 100 smaller samples as similar as possible to the corresponding volume of one large sample. With this approach, the already obtained solutions to issues of sample homogeneity for ultrathin cutting, large scale, high throughput, electron microscopic imaging, avoidance of electrical charging during the imaging process, and subsequent steps of data analysis could be assumed to operate successfully on the multiplexed sample setting.

The procedure consists of three parts: 1) preparation of (many) individual samples for connectomic analysis (screening sample preparation); 2) preparation of the chaperone sample for hosting the about 100 screening samples; 3) Reliable insertion of screening samples into the chaperone sample. This is followed by ultrathin slicing and EM imaging as currently applied to large-volume samples.

These procedures are described in the following and Figures 1-4.

Generation of screening samples

Brain samples were generated as described previously (Hua et al., 2015; Loomba et al., 2022; Motta et al., 2019). Briefly, the brain was sliced into 500 pm thin sections using a Leica VT 1200S Vibratome and kept in fixative. The brain regions of interest were extracted using biopsy metal punches (KAI medicals, USA) with a diameter of 1 mm or 1.5 mm (Fig. 1A). The punches were collected separately in 2 ml Eppendorf tubes (round bottom) filled with 0.15M cacodylate buffer and were stored overnight at 4°C.

Samples were then stained using the protocol described in Hua et al. 2015 with slight modifications as described in (Loomba et al., 2022) (Fig. 1A). All staining steps were performed manually, staining solutions were freshly prepared prior to the respective staining steps. To avoid tissue damage, samples were handled carefully using pipettes.

After heavy metal treatment, the samples were dehydrated and infiltrated with resin. (EPON, as in (Loomba et al., 2022)). The samples were then embedded in a custom-made silicon mold (Fig. IB) (mb3DEngineering; Germany). Care was taken to place the sample in the middle of the mold to ensure that the center of the sample would be aligned for the milling step. The samples were placed in an oven at 60°C for 3d.

Then, the screening samples were trimmed to achieve the required shape. For this, an alignment plane was milled into the resin using a 6 mm -diameter aluminum milling cutter (Fig. 1C) (DATRON neo plus; DATRON AG; Germany). The sample was then fixated using a clamp in a custom-made mold (Markforged; Mark Two), and images of the sample were acquired before milling using a Leica MZ1 OF (camera DFC450C; Leica) at magnification 1.25x (Fig. 1C).

Finally, the sample was then milled to a pre-defined shape: a pointed rod of diameter 120pm and length 1mm, sitting on a basis of 500 pm width and 1 mm height, followed by the remaining original sample (Fig. IE). The sample shape was pre-defined using CAD software (Fig. ID) (SolidWorks Professional 2024) and milled using CAM (Open Mind; hyper MILL Inventor) on a milling machine (DATRO neo plus; DATRON AG; Germany) with a special milling cutter (Garant; Fa. Hoffman; Germany). The sample shape was designed to allow a controlled breaking- off of the sample rod after insertion into the chaperone sample, see below. After sample milling, another image was taken at 1.25x magnification using a Leica MZ1 OF (Fig. IE).

Generation of the chaperone sample

A 500 pm thick slice of adult mouse brain was prepared in fixative using a vibratome (Leica VT 1200S Vibratome). Then, using razor blades, a sample of size 3mm x 2.5mm was cut out from this slice, ensuring that no ventricle would be contained.

Then, the sample was stained using the protocol in Hua et al 2015 with modifications as in Loomba et al 2022 (Fig. 2A). The chaperone sample were handled with a brush. Infiltration was performed as described above for the screening samples.

Then, the chaperone sample was embedded on an aluminium pin (Fig. 2B), which had been prepared beforehand by adding a resin layer on top of the aluminium pin. The chaperone sample could then be placed onto this resin layer such that no direct contact between the chaperone sample and the aluminium pin occurred. With this, it was possible in subsequent steps to trim the chaperone sample very precisely without the risk of cutting into the aluminium pin.

Then, an alignment plane was milled onto the aluminum pin using a 6mm-diameter aluminum milling cutter (DATRON AG; Germany) on a milling machine (DATRON neo plus; DATRON AG; Germany) (Fig. 2C). Then, the resin was trimmed away from the top using a diamond head milling system (EM TRIM2, Leica Microsystems), so that a 500 pm-thick layer of resin remained on top of the sample (Fig. 2C).

The sample was then fixated in a custom-made mold (Markforged; Mark Two) using a clamp, such that it could be imaged (Leica MZ10F at lx magnification). Then, a CAD model (using SolidWorks Professional 2024) of a hexagonal shape was centered onto the sample (Fig. 2D), and the sample was milled accordingly using the CAM software (Open Mind; hyper MILL Inventor) on the milling machine (DATRON neo plus; DATRON AG; Germany) using a 6mm - diameter aluminum milling cutter (DATRON AG; Germany).

Finally, patterns of 80-100 holes of diameter 100-150pm each, separated by 50pm thick walls, arranged with offset in order to improve identification of the samples afterwards, and arranged within rectangular surrounds were pre-defined (see Fig. 2E). One of these designs was then used to guide a high-performance small drill (VHM; GUHRING KG). To avoid accumulation of fine dust in the holes, drilling was repeated once for each hole, and the chaperone sample was put in an ultra-sonic bath for a few seconds after drilling. A pCT image (Fig. 2F) was acquired afterwards to assess the quality of the milling and drilling.

Insertion of screening samples into the chaperone sample

For this step, a dedicated setup was built (Fig. 3 A) and placed on an air table (Newport S-2000) to absorb vibrations. First, a holder for the chaperone sample was built using a 3D printer (Markforged; Mark Two) and attached to a rod (Optical Post TR300/M; Thorlabs). This rod was clamped (right-angle post clamp; Thorlabs) onto two additional adjacent rods (Pedestal Pillar Post, M6 Taps, Thorlabs) that were in turn attached to the air table (Clamping Fork; CF125CM; Thorlabs) at an angle of 30° (Fig. 3 A). The chaperone sample was fixated to the holder via screws. Then, a holder for the respective screening sample was built using a 3D printer (Markforged; Mark Two). This holder was also positioned at an angle of 30°. The holder was then equipped with a pneumatic gripper (DHPS-10-A-NC-P5; parallel gripper; Festo). By operation via a button press (VHES-P-M52-M-G18; Festo), the pneumatic gripper closes the holder parts and clamps the screening sample in place. The pneumatic gripper was attached to a micromanipulator (Sensapex 3; npi electronic; Germany), which in turn was screwed first on an aluminum breadboard (MB1530/M;Thorlabs) and then to a height adjuster (LabJack; L490/M; Thorlabs).

With this, the screening sample could be moved in three directions, and placed into the respective hole of the chaperone sample (Fig. 3B). Once the sample was completely inserted into the respective hole, the micromanipulator speed was set to ‘very high’, such that the screening sample would break off at the intended location (transition between sample and resin base, see above). All these steps were monitored via a microscope placed on top of the setup (Fig. 3 A).

During the screening sample insertion process, the shape of each screening sample after milling, and the insertion position of each screening sample within the chaperone sample were carefully documented for later reference.

This process was repeated for 70-100 times, until the desired number of screening samples had been inserted into the chaperone sample.

When all screening samples had been inserted, the chaperone sample was removed from the holder and placed under the microscope in a petri dish on double-sided tape, preventing the sample from tripping over. A drop of freshly prepared resin (EPON) was added to the top of the sample using a Ipl pipette (Research plus; Eppendorf); this was repeated after about 10 minutes (Fig. 3C). Then, the chaperone sample was placed in an oven at 60°C for 3d (range 1 -3d, minimum is 24h). If after that time resin had spilled at the edges of the sample, the sample was re-milled on the milling machine using a 6mm-diameter aluminum milling cutter (DATRON AG; Germany). Then, the sample was imaged in a pCT (XRadia 520 Versa; Zeiss) to determine the depth position of the brain samples within the holes of the chaperone sample (Fig. 4A). Then, the top of the chaperone sample was trimmed off accordingly using either a diamond head milling system (EM TRIM2, Leica Microsystems) or an ultramicrotome (EM UC7; Leica with a Diamond knife; DiATOME 45°) to expose all screening samples in one plane (Fig. 3D).

In case during pCT imaging, empty spaces were detected around any screening sample, an additional drop of resin was added to the surface of the chaperone sample for complete immersion of all screening samples (Fig. 3D). The chaperone sample was then placed in the oven (range 1- 3d, minimum is 24h).

Imaging and cutting of the combined screening/chaperone samples

The final embedded chaperone sample containing all required screening samples was then prepared for ATUM slicing (Fig. 4B) and 3D EM imaging as described in Loomba et al 2022 and Sievers et al 2024 (Fig. 4C, 4B).

Proof of concept for 106 screening samples

Fig. 5 exemplarily shows that the procedures described herein, including drilling of the chaperone sample (Fig. 5A), embedding of the tissue samples (Fig. 5B) and ATUM slicing (Fig. 5C), can be applied to a chaperone sample for hosting 106 screening samples.

Batch-of-rod experiment

Generation of the chaperone sample for Batch-of-rod experiment

Then, the sample was stained using the protocol in Hua et al 2015 with modifications as in Loomba et al 2022. The chaperone sample were handled with a brush. Infiltration was performed as described above for the screening samples.

Then, the chaperone sample was embedded on an aluminium pin (Fig. 2B), which had been prepared beforehand by adding a resin layer on top of the aluminium pin. The chaperone sample could then be placed onto this resin layer such that no direct contact between the chaperone sample and the aluminium pin occurred. With this, it was possible in subsequent steps to trim the chaperone sample very precisely without the risk of cutting into the aluminium pin. Then, an alignment plane was milled onto the aluminum pin using a 6mm-diameter aluminum milling cutter (DATRON AG; Germany) on a milling machine (DATRON neo plus; DATRON AG; Germany). Then, the resin was trimmed away from the top using a diamond head milling system (EM TRIM2, Leica Microsystems), so that a 500 pm-thick layer of resin remained on top of the sample (Fig. 2C).

The sample was then fixated in a custom-made mold (Markforged; Mark Two) using a clamp, such that it could be imaged (Leica MZ10F at lx magnification). Then, a CAD model (using SolidWorks Professional 2024) of a hexagonal shape was centered onto the sample, and the sample was milled accordingly using the CAM software (Open Mind; hyper MILL Inventor) on the milling machine (DATRON neo plus; DATRON AG; Germany) using a 6mm - diameter aluminum milling cutter (DATRON AG; Germany).

Finally, a rectangular pattern with a length of 1 mm and a width of 0.7 mm was defined. This shape was then used to guide a high-performance small drill (solid carbide; GUHRING KG). A pCT image is then taken to assess the quality of the milling and drilling.

Insertion of screening-rod-samples into the chaperone-batch sample

For this step, a dedicated setup was built (Fig. 3 A) and placed on an air table (Newport S2000) to absorb vibrations. First, a holder for the chaperone sample was built using a 3D printer (Markforged; Mark Two) and attached to a rod (Optical Post TR300/M; Thorlabs). This rod was clamped (right-angle post clamp; Thorlabs) onto two additional adjacent rods (Pedestal Pillar Post, M6 Taps, Thorlabs) that were in turn attached to the air table (Clamping Fork; CF125CM; Thorlabs) at an angle of 30° (Fig. 3 A). The chaperone sample was fixated to the holder via screws.

Then, a holder for the respective screening sample was built using a 3D printer (Markforged; Mark Two). This holder was also positioned at an angle of 30°. The holder was then equipped with a pneumatic gripper (DHPS-10-A-NC-P5; parallel gripper; Festo). By operation via a button press (VHES-P-M52-M-G18; Festo), the pneumatic gripper closes the holder parts and clamps the screening sample in place. The pneumatic gripper was attached to a micromanipulator (Sensapex 3; npi electronic; Germany), which in turn was screwed first on an aluminum breadboard (MB1530/M;Thorlabs) and then to a height adjuster (LabJack; L490/M; Thorlabs). Fine forceps attached to an additional micromanipulator can now grip the screening sample on the resin part and break it off at the predetermined breaking point.

With this, the screening sample could be moved in three directions, and placed into the respective chaperone sample. This can be done in two ways: (a) either the sample is first dipped into a small container to coat it with resin and then place it in the chaperone sample or (b) the sample is placed in the chaperone block and then coated with resin. All these steps were monitored via a microscope placed on top of the setup. After each layer of stacked samples, the chaperone was placed in a 60° C oven overnight before starting the next layer.

During the screening sample insertion process, the shape of each screening sample after milling, and the insertion position of each screening sample within the chaperone sample were carefully documented for later reference. This process was repeated until the chaperone sample is completely filled. Then, the chaperone sample was placed in an oven at 60°C for Id and subsequently imaged in a pCT (XRadia 520 Versa; Zeiss). The top of the chaperone sample was trimmed off accordingly using either a diamond head milling system (EM TRIM2, Leica Microsystems) or an ultramicrotome (EM UC7; Leica with a Diamond knife; DiATOME 45°) to expose all screening samples in one plane (Fig. 10A)

In case during pCT imaging, empty spaces were detected around any screening sample, an additional drop of resin was added to the surface of the chaperone sample for complete immersion of all screening samples. The chaperone sample was then placed in the oven (range 1 -3 d, minimum is 24h).

Imaging and cutting of the combined batch- rod screening/chaperone samples

The final embedded chaperone sample containing all required screening samples was then prepared for ATUM slicing and 3D EM imaging (Fig. 10B) as described in Loomba et al 2022 and Sievers et al 2024. Fig. 10 shows a pCT image of a chaperone sample with an encaving into which 13 screening samples were placed (Fig. 10A). A high-resolution EM image from the sample was taken (Fig. 10B) which shows high-quality ultrastructural tissue preservation at the border of one of the screening samples. List of References

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Claims

1. A chaperone block for holding one or more biological tissue sample(s) of interest, particularly for electron microscopy (EM), wherein the chaperone block comprises at least two layers of resin, at least one layer of a chaperone sample and a multitude of cavities.

2. The chaperone block of claim 1, wherein the chaperone block is configured to provide structural integrity to one or more screening sample(s) which comprise said tissue sample(s) of interest, and wherein the chaperone block is further configured to homogenize the electrical conductivity and surface of the one or more screening sample(s).

3. The chaperone block of claim 1 or 2, wherein the chaperone block comprises a lower layer of resin, a middle layer of chaperone sample, and a top layer of resin and wherein the cavities are configured to be loaded with screening samples.

4. The chaperone block of any one of claims 1 to 3, wherein the cavities have a diameter of about 70-2000pm, preferably of about 100-150pm and/or wherein the chaperone block comprises about 2-2500 cavities, preferably comprising about 80-120 cavities, and/or wherein each of the cavity is arranged about 20-1000pm, preferably at least 20pm, apart from each other cavity.

5. The chaperone block of any one of claims 1 to 4, wherein the cavities at least partially penetrate the chaperone sample, preferably, wherein the cavities penetrate the top layer of resin and at least partially penetrate the chaperone sample, more preferably wherein the cavities penetrate the chaperone sample.

6. The chaperone block of any one of claims 1 to 5, comprising a screening sample in one or more of the multitude of cavities, preferably comprising a screening sample in each cavity.

7. The chaperone block of any one of claims 1 to 6, wherein the chaperone sample is a tissue sample.

8. A method for producing the chaperone block of any one of claims 1 to 7, the method comprising:

(a) embedding a chaperone sample onto a lower layer of resin,

(b) adding a top layer of resin on top of the chaperone sample,

9. A screening sample comprising a tissue sample of interest, a resin layer comprising a predetermined breaking point and a base, wherein the screening sample is configured to be loaded into a chaperone block of any one of claims 1 to 7.

10. The chaperone sample of any one of claims 1 to 7, or the screening sample of claim 9, wherein the chaperone sample and/or the tissue sample of interest comprises a sample of a mammalian, reptile, fish, or bird tissue, preferably a tissue of a mouse brain or a human brain, and/or wherein the tissue sample of interest comprises a tissue originating from the same type of organ having similar or same structural and conductive properties as a tissue of the chaperone sample.

11. A method for producing the screening sample of claim 9 or 10, the method comprising:

(a) embedding a tissue sample of interest in a resin,

(c) introducing a predetermined breaking point into the resin layer between the tissue sample of interest and the base.

12. An apparatus for loading a chaperone block of any one of claims 1 to 7 or 10 with a screening sample of claim 9 or 10 comprising:

(a) a first holder for holding the chaperone block,

(b) a second holder for holding the screening sample, wherein the apparatus is configured to move the screening sample by means of the second holder into a cavity of the chaperone block and to break of the tissue sample of interest from the base, thereby loading the tissue sample of interest into the chaperone block.

13. The apparatus of claim 12, wherein the second holder is configured to move the screening sample in three dimensions, and/or wherein the first holder and the second holder are arranged at an angle of 30° to the horizontal, with the chaperone block and the screening sample facing each other.

14. A method for preparing a sample for connectomic screening, the method comprising:

(a) providing one or more screening sample according to claim 9 or 10, or producing a screening sample according to claim 11,

(b) providing a chaperone block according to any one of claims 1 to 7 or 10, or producing a chaperone block according to claim 8,

(c) loading the one or more screening sample into the chaperone block with the apparatus according to claim 12 or 13.

15. The method of claim 14, wherein the screening sample is loaded into the chaperone block by inserting the screening sample into a cavity of the chaperone block followed by breaking off the screening sample at a predetermined breaking point from its base hold by the second holder, thereby loading the chaperone block with the screening sample, wherein the screening sample is broken off at a predetermined breaking point while being inserted into the cavity of the chaperone block by rapidly moving the second holder.

16. A batch sample comprising a multitude of tissue samples embedded in a resin, wherein the tissue samples are configured to provide structural integrity to each other and thereby to the batch sample, and wherein the tissue samples are configured to homogenize the electrical conductivity and surface of the batch sample.