FR3061203A1 - CHAMBER OF CULTURE AND IMAGING OF BIOLOGICAL SAMPLES - Google Patents

Abstract

The invention relates to a culture and imaging chamber (10) comprising a rigid support (1) and a cover (5), at least one microscope slide (2, 12) arranged facing an opening (11, 15). formed in the support (1) or in the cover (5), a seal (4, 14, 24) comprising at least one other opening (34) facing the microscope slide (2, 12) and a hydrogel layer (6) disposed within said other opening (34) of the seal (4, 14, 24), the hydrogel layer (6) comprising a well network, the well network comprising a plurality of open wells (26) of millimeter or sub-millimeter dimensions arranged at predetermined three-dimensional positions in an orthonormal frame linked to the support (1), each well (26) being adapted to receive a biological sample.

Description

© Agent (s): JACOBACCI CORALIS HARLE Simplified joint-stock company.

(54) CULTURE AND IMAGING CHAMBER OF BIOLOGICAL SAMPLES.

FR 3 061 203 - A1 (57) The invention relates to a culture and imaging chamber (10) comprising a rigid support (1) and a cover (5), at least one microscope slide (2, 12) arranged facing an opening (11, 15) formed in the support (1) or in the cover (5), a seal (4, 14, 24) comprising at least one other opening (34) disposed facing the strip microscope (2, 12) and a hydrogel layer (6) disposed inside said other opening (34) of the seal (4, 14, 24), the hydrogel layer (6) comprising a network of wells, the network of wells comprising a plurality of open wells (26) of millimeter or sub-millimeter dimensions arranged at predetermined three-dimensional positions in an orthonormal reference linked to the support (1), each well (26) being suitable for receiving a biological sample.

Technical field to which the invention relates

The present invention relates generally to the field of devices and methods for the culture and observation of biological samples.

In the present document, the term “biological sample” is understood to mean 3D biological cells such as spheroids, organoids or cellular aggregates (3D cells), embryos or even capsules containing biological cells.

It relates more particularly to a sample holder for an optical microscope suitable for visualizing for example the spatial and temporal evolution of living biological organisms or for imaging fixed samples, by way of example PFA type fixers and marked with any type of marker, such as antibodies against proteins.

TECHNOLOGICAL BACKGROUND

Light microscopy is commonly used in biology or biomedicine for the visualization of biological processes in biological organisms or cells. Different fields of biology including developmental biology, stem cells, tissue engineering or biophysics are interested in the study and analysis of processes at the cellular level. However, these analyzes require the acquisition of images of high spatial and temporal resolution, in a repetitive manner, in large quantity and with a sufficient yield to allow a reliable statistical analysis. Current optical microscopes offer different imaging techniques and configurations such as confocal, multi-photon or spinning disc microscopy that provide excellent spatial and temporal resolution for studying a particular sample. However, these microscopy devices do not allow a large number of biological samples to be assembled and processed automatically with high yield.

There are culture chambers, for example of the Petri dish type, which make it possible to grow living biological samples in a liquid culture medium.

Special devices for immobilizing living biological samples have been developed in certain laboratories. As summarized in a review article, J. Clarke ("Live imaging of development in fish embryos", Semin cell Dev Biol, 20 (8), 942-6, 2009) it has been demonstrated the visualization of zebrafish larvae included in an agarose gel. Although this approach is easy to implement, it is not suitable for long-term monitoring of the development of these larvae due to the mechanical stresses generated by the growth of the larvae in the agarose gel. In addition, this process is not automated, so it is difficult to obtain a large number of samples to obtain reliable statistics.

Also known is a culture chamber comprising a network of cylindrical wells formed by etching in a thick glass substrate. However, such a culture chamber is not compatible with viewing by an optical microscope in a straight configuration.

However, biological samples have a wide variety of shape, size and development conditions. For example, zebrafish larvae have an elongated shape and are very tolerant of growing conditions. Other embryos have a spherical shape but are more demanding in terms of culture conditions. Another category of three-dimensional biological samples includes multicellular aggregates, spheroids, organoids, embryos and capsules containing biological cells.

In biology or biomedicine experiments, for example, it is desirable to test drugs on in vivo models with high yield. It is therefore necessary to carry out automated image acquisitions on numerous targets with high spatial and temporal resolution.

There is no practical device for precisely positioning a large number of living biological samples on an optical microscope sample holder while reducing mechanical or biochemical interactions on these living biological samples.

Object of the invention

In order to remedy the aforementioned drawback of the state of the art, the present invention provides a culture and imaging chamber for biological samples by optical microscopy, said culture and imaging chamber comprising a support and a cover. , the support comprising at least one interior housing, at least one microscope slide disposed facing an opening formed in the support or in the cover facing said interior housing, a seal disposed between the support and the cover in contact with said at least one microscope slide, the seal delimiting at least one other opening arranged opposite the microscope slide, a layer of hydrogel disposed inside said other opening of the seal, the layer of hydrogel comprising a network of open wells formed at predetermined three-dimensional positions in an orthonormal reference frame linked to the support, each well being adapted to receive a biological sample, the support, the cover and the seal being configured so as to be made integral to form a culture and imaging chamber which is sealed against a liquid.

This culture and imaging chamber is compatible with imaging on many microscopes, is inexpensive to manufacture, easy to assemble, transport, while ensuring liquid tightness and gas permeability of the culture medium. It allows biological samples to be positioned individually with micrometric precision without applying mechanical stress to these biological samples.

Advantageously, the culture and imaging chamber comprises the mechanical holding means adapted to maintain the seal between the support and the cover.

Other non-limiting and advantageous characteristics of the culture and imaging chamber according to the invention, taken individually or in any technically possible combination, are the following:

- the wells are of millimeter or sub-millimeter dimensions or even up to micrometric dimensions;

- The support and / or the seal comprises a plurality of sealed compartments with respect to each other, at least one sealed compartment comprising said hydrogel layer and said network of wells;

- The support has a first opening, the cover has a second opening, said at least one microscope slide comprising a first microscope slide arranged opposite the first opening and a second microscope slide arranged opposite the second opening, the seal d the seal being in contact with the first microscope slide and the second microscope slide;

- The cover is opaque or the cover has a second opening, said at least one microscope slide has a second microscope slide disposed facing the second opening and a reflecting or dichroic optical component disposed in the interior housing of the support;

- The support is opaque or the support has a first opening, said at least one microscope slide comprising a first microscope slide facing the first opening and in which the cover is opaque or includes a reflecting or dichroic optical component;

the network of open wells is arranged in a grid having a predefined pitch in one direction or two transverse directions in the orthonormal reference frame;

- the well network comprises at least one open well having a parallelepiped shape, a cylindrical shape, a frustoconical shape, a flared shape or a closed shape at the level of a well opening;

the network of wells comprises at least one well having a wall inclined with respect to a horizontal surface of the hydrogel layer at an angle greater than 0 degrees and less than 135 degrees, or 110 degrees or preferably less than 100 degrees said horizontal surface of the hydrogel layer being that where the well openings are located;

- The network of wells comprises at least one well having a flat or concave bottom formed in the hydrogel layer and / or at least one well having a bottom formed by a microscope slide surface;

- The hydrogel layer has a plurality of registration marks, each registration mark being arranged near a well and being configured to identify this well;

- a bowl is formed in the hydrogel layer and is adapted to receive an immersion liquid;

- the support, the cover and the seal are formed by molding or by three-dimensional printing from polymer materials, preferably biocompatible;

- The rigid support has several openings and is adapted to receive a microscope slide facing each support opening;

- the support comprises a partition separating the interior housing into watertight compartments, each compartment comprising a seal and a microscope slide;

- the seal has a rectangular or square section;

- The support comprises at least one lateral fin adapted to receive a clamp for fixing the microscope slide on a sample holder for an optical microscope and for holding the support on the sample holder for an optical microscope;

- the microscope slide is made of glass, of polymer material and / or has a thin layer coating adapted to increase the adhesion of a biological sample;

The invention also provides a method of manufacturing a culture chamber and imaging biological samples by microscopy comprising the following steps:

- manufacture by stereo-lithography from a resin a pad comprising a plurality of projections having a base of transverse millimeter or submillimetric dimension and a height of millimeter or submillimetric dimension;

- have a first microscope slide in an interior housing of a rigid support,

- have a seal in the interior housing of the rigid support, the seal having an opening and the seal having a lower surface in contact with the microscope slide,

- pour a layer of liquid hydrogel into the opening of the seal so as to cover the surface of the microscope slide;

forming an imprint of the buffer in the layer of liquid hydrogel, so as to obtain a layer of gelled hydrogel comprising the imprint of the plurality of protrusions of the buffer and forming a network of wells of dimensions equal to the dimensions of the protrusions of the buffer ;

- take at least one biological sample and place the biological sample in a well of the network of wells; and

- attach a cover to the support to seal the culture and imaging chamber thus formed.

Detailed description of an exemplary embodiment

The description which follows with reference to the appended drawings, given by way of nonlimiting examples, will make it clear what the invention consists of and how it can be carried out.

In the accompanying drawings:

- Figure 1 shows an exploded view of the components of a culture and imaging chamber according to an embodiment of the invention;

- Figure 2 shows an exploded view of the elements of a mold to form a seal;

- Figure 3 shows views from above and in section along line AA and along line B-B of an example of a seal;

- Figure 4 shows a perspective view of an example of a pad provided with a network of projections to form a network of wells by imprint in a hydrogel layer;

- Figure 5 shows views from above and, respectively, in section along line A-A and line B-B, of an example of a pad for imprinting a network of wells in a layer of hydrogel;

- Figure 6 shows detailed views of an example of a projection for forming a flared well;

- Figure 7 shows detail views of another example of projection to form a well of rectangular parallelepiped shape;

- Figure 8 illustrates the stages of construction of a culture and imaging chamber according to an embodiment of the invention;

- Figure 9 shows examples of support and seals forming watertight compartments according to another embodiment of the invention;

- Figure 10A shows a detail view of an example of a tampon and fig. 10B is a detail view of a hydrogel layer in which the stamp of FIG. 10A network of wells with local identification marks associated with each well;

- Figure 11 illustrates for microscopy an example of a well;

- Figure 12 schematically illustrates an example of a process for transferring biological objects from a culture dish to the wells of a culture and imaging chamber;

- Figure 13 illustrates the positioning of a culture and imaging chamber on an optical microscope sample holder;

- Figure 14 shows an image reconstituted from 70 optical microscopy images according to a rectangular pitch grid, each well comprising a biological capsule;

- Figures 15A-15B represent images composed of 50 images of a network of wells comprising biological capsules taken respectively at an instant t = 0 and at another instant t = 15h;

- Figure 16 shows an example of a well for an experiment on a biological capsule and the trajectories of several cell nuclei within this capsule;

- Figure 17 shows another example of a network of wells for an experiment on zebrafish larvae.

Device and method

The present disclosure proposes a culture and imaging chamber which can be entirely manufactured and assembled by the user from common components such as microscope slides, inexpensive materials, such as a hydrogel (for example agarose , methylcellulose, phytagel) and an elastomer (for example silicone) and parts manufactured by 3D stereolithography using a 3D printer having a resolution of at least 50 μm in each direction and using a resin. Preferably, the resin used is a biocompatible resin.

In FIG. 1, the components of a culture and imaging chamber are shown according to an embodiment of the invention. The different components are here dismantled and placed next to each other in a reference (XYZ). The culture and imaging chamber includes a support 1, a first microscope slide 2, a hydrogel layer 6, a seal 4, a cover 5 and a second microscope slide 12.

For the remainder of the description, a vertical direction is a direction parallel to the axis OZ, the longitudinal plane is perpendicular to the vertical direction.

The support 1 has a length of 76 mm and a width of 26 mm which correspond to the dimensions of a conventional microscope slide. In this example, the support 1 has a flat bottom parallel to the longitudinal plane having an opening 11 and lower flanges 51 defining the opening 11. The support also has walls 41 extending in the vertical direction on four sides transversely to the bottom over a height of a few millimeters and upper edges 71. The bottom of the support is adapted to receive a microscope slide 2 having a length of 50 mm and a width of 24 mm. The opening 11 is here rectangular of length and width respectively less than the length and the width of the microscope slide 2. The edges 51 of the bottom and the walls 41 of the support 1 thus form an interior housing 61 adapted to receive and maintain the microscope slide 2 facing the opening 11. The opening 11 of the support 1 thus provides optical access to the interior housing 61. Advantageously, the support 1 here comprises two external fins 21 arranged in the extension of the length support to allow manipulation or fixing of the culture and imaging chamber on a microscope sample holder.

The cover 5 is generally rectangular in shape and has a flat upper face 35 and edges 45. The edges 45 extend transversely in the vertical direction to the upper face 35 of the cover on four sides to form a skirt. The edges 45 of the cover 5 are adapted to fit on the upper edges 71 of the support 1. The upper face 35 of the cover here has an opening 15 of rectangular shape, having a length of approximately 44 mm and respectively a width of about 18 mm, which are less than the length and respectively the width of a standard microscope slide. A slot 25 is provided in one of the edges of the cover to allow the insertion of the second microscope slide 12. Advantageously, the cover has guide rails to guide and hold the slide 12 in the cover 5. Thus, the cover 12 completely covers the opening 15 of the cover. The cover and the support are held together, in a reversible and sealed manner, by means of the seal 4. More specifically, the cover fits on the edges of the seal 4 which protrude outside the interior housing 61. The opening 15 of the cover 5 provides optical access to the interior housing 61. Advantageously, when the cover 5 is positioned on the support 1, the opening 15 of the cover 5 is aligned with the opening 11 support 1.

The cover 5 and the support 1 are preferably manufactured by 3D stereolithography, for example by means of a printer of the Micro-Plus type.

Hi-Re from Envision City with a resolution of at least 50 µm and preferably µm in each direction in each direction. A biocompatible resin is used such as HTM140 resin from Envision City. The resin has a low viscosity before photo-polymerization and a high rigidity after photo3061203 polymerization. After hardening, the support 1 and the cover 5 can be autoclaved a large number of times. Thus, there is a support 1 and a rigid cover 5. However, it is easy to fix the cover on the support. The cover is removable, for example to change the content of the culture and imaging chamber or to change the culture liquid.

The seal 4 is preferably a silicone seal, for example polydimethylsiloxane (PDMS). The seal here has the general form of a frame 14 of rectangular section surrounding a rectangular opening 34. The seal 4 is preferably formed by molding. The seal 4 has a thickness dimensioned so that it is in abutment along a continuous and closed line on the first microscope slide 2 and along another continuous and closed line on the second microscope slide 12, when the cover 5 is fixed to the support 1. The lower part of the frame 14 of the seal forms a flange 94. The flange is dimensioned so that when the seal 4 is placed in abutment on the first microscope slide in the internal housing 61, the upper part of the frame 14 exceeds the walls 41 of the support. When the cover 5 is positioned on the support 1, the edges 45 of the cover fit onto the upper part of the frame 14 to keep the cover fixed to the support in a leaktight manner. The seal 4, for example made of PDMS, can also be autoclaved a large number of times. In general, the different elements of the device, apart from the hydrogel layer, are autoclavable individually and / or assembled.

The manufacture of the hydrogel layer 6 will be described in more detail below. The hydrogel layer 6 comprises a network of open wells formed by stamping or by marking an imprint. The hydrogel layer 6 is preferably made of an agarose gel. Agarose has several advantages as a support for forming wells, including being easy to model, being transparent in the visible range, and being neutral to most living biological samples. In variants, the hydrogel layer consists of a phytogel, a methylcellulose gel, an alginate gel or a fluoropolymer. The hydrogel layer 6 is intended to be positioned on the first microscope slide 2 and inside the seal 4.

The seal 4 is a seal vis-à-vis an immersion liquid and gas permeable. Thus, the volume delimited by the two microscope slides 2, 12 and the seal 4 is a volume which is tight vis-à-vis an immersion liquid, but which allows gas exchange. The hydrogel layer is placed inside this volume. There are different types of microscopy imaging that may require one or two optical accesses. Thus, in fluorescence microscopy, the excitation and the emission are done by means of the same microscope objective, a single optical access is then sufficient. On the other hand, for bright field microscopy or for transmission microscopy (for example dark field, polarization imaging, by generation of second harmonic or SHG, or more generally any generation mode contrast for imaging requiring transmission detection), both optical accesses are required. The first microscope slide 2 allows for example to visualize the hydrogel layer 6 by means of an inverted microscope. The second microscope slide 12 makes it possible, for example, to visualize the hydrogel layer 6 by means of a straight microscope. This provides a culture and imaging chamber which is liquid tight and gas permeable.

The culture and imaging chamber is easily removable. The support 1, the cover 5 and the seal 4 can be sterilized for reuse. The microscope slides 2, 12 are inexpensive. The hydrogel layer is easy to make and also inexpensive. The hydrogel layer is one of the consumables.

FIG. 2 illustrates an example of a molding device for forming a seal. In Figure 2A, there is shown in exploded view the various elements of the mold and the seal obtained. The molding device comprises an outer frame 74, an inner frame 64 and a crosspiece 84 for positioning the inner frame 64 relative to the outer frame 74. In FIG. 2B, the mounted molding device is shown. To form the seal, a liquid PDMS resin (for example Sylgard 184, Corning mixed with 10% curing agent) is poured into the gap between the inner frame 64 and the outer frame 74 of the mold. After approximately 3 hours of hardening, the seal 4 is removed from the molding device.

Figure 3 shows views from above and in section along the lines

B-B and C-C of a seal 4 thus obtained. The seal 4 has a large rectangular opening 34 about 18 mm wide and 45 mm long.

The seal 4 has a total thickness of about 7 mm. The seal 4 advantageously comprises a planar lower surface 44 and a planar upper surface 54. The seal 4 is placed on the first microscope slide 2 in the housing 61 of the support 1, so that the lower surface 44 forms a continuous and closed contact line with the upper surface of the first slide 2 of microscope. The second microscope slide 12 being inserted into the cover 5, the cover 5 is placed on the support 1 so as to close the culture and imaging chamber. Thus, the upper surface 54 of the seal 4 forms another continuous and closed contact line with the lower surface of the second microscope slide 12. No glue is used between the various components of the culture chamber and of 'imagery. This gives a sealed culture and imaging chamber without glue, which avoids any risk of contamination of the biological samples inside the culture and imaging chamber.

We will now describe the fabrication of the network of open wells intended to receive biological samples.

Firstly, a tampon 13 is made comprising a plurality of raised projections. The pad is then used to form a network of wells by stamping in a layer of hydrogel. The pad 13 is preferably manufactured by 3D stereo lithography using a 3D printer having a resolution of at least 50 µm and preferably 25 µm in each direction. For example, the printer of the Micro-Plus Hi-Re type from Envision City and a biocompatible resin such as HTM140 resin from Envision City are used.

Figures 4-5 illustrate an example of a tampon 13 comprising a plurality of raised projections 23 arranged here in a regular two-dimensional network. For example, the buffer has 7 rows and 11 columns, or 77 projections, or else 9 rows and 17 columns, or 153 projections. The shape, the dimensions and the relative positions of the projections 23 determine the reverse shape, the dimensions and the positions of the wells that one seeks to manufacture. Preferably, the protrusions of the tampon all have the same shape and the same dimensions and are arranged in a regular mesh with one or two dimensions. The height of the projections is chosen to be preferably greater than or equal to 5 times the dimension of the biological samples considered. Advantageously, the buffer 13 has feet 33 which serve to determine the distance of the wells relative to the microscope slide. Preferably, the feet 33 of the pad 13 have a height greater than or equal to the height of the projections 23. The pad 13 has a total length and a width slightly less respectively than the length and the width of the opening 34 of the joint 4.

FIG. 6 represents an example of projection 23 for forming a well adapted to receive a biological object of spherical shape and of diameter between 100 μm and approximately 300 μm. The projection 23 has a height of about 1.2 mm. The projection here has a rectangular base of 1 mm by 0.5 mm and a disc-shaped top 53 of 0.3 mm in diameter. The corresponding well shape is a flared shape which allows precise 3D positioning and maintenance of a spherical biological object without applying mechanical stress. In a particularly advantageous manner, the buffer 13 also includes a mark 43 in relief or in hollow adjacent to the projection 23. This mark 43 is intended to generate a specific marking mark in the vicinity of each well in order to allow identification and easy marking. of each well on the microscopy images.

FIG. 7 represents another example of projection 23 for forming a well adapted to receive a biological object of elongated shape, for example a zebrafish larva. The projection 23 is here of rectangular parallelepiped shape. The projection 23 has a height of about 1 mm and a rectangular apex 53 of about 1.5 mm by 0.7 mm. The corresponding parallelepiped well shape prevents rotational movements of a larva on itself while reducing the mechanical stresses applied to the larva.

FIG. 8 illustrates an example of a method for manufacturing and assembling a culture and imaging chamber. In FIG. 8A, a first microscope slide 2 has been placed in the housing of a support 1 placed flat on a table. A seal 4 is placed on the microscope slide 2 and held in the interior housing 61 by a dimensional adjustment of the support 1 so as to form a continuous and closed seal line between the microscope slide 2 and the lower surface 44 of the seal 4. A hydrogel in liquid form is poured into the opening 34 of the seal 4 so as to form a layer of liquid hydrogel covering the surface of the microscope slide 2 inside the seal. sealing 4. By way of example, about 2 ml of agarose heated to a temperature suitable for making it liquid is used.

In FIG. 8B, before hardening of the hydrogel, the surface of the pad 13 comprising the network of projections 23 is applied to the layer of liquid hydrogel while preventing the formation of bubbles. The feet 33 of the buffer are advantageously in contact with the surface of the microscope slide 2. The assembly is cooled, for example at a temperature of 4 ° C for a few minutes until the hydrogel layer is gelled.

In the step of FIG. 8C, the buffer is removed after freezing of the agarose layer. The gelled agarose layer thus has the imprint of the projections 23 of the buffer 13 which form a network of wells in the agarose layer. The positions and dimensions of each well are thus determined precisely in three dimensions. Each well forms a cavity having a well opening formed on the surface of the gelled agarose layer, each well opening being of millimeter or sub-millimeter transverse dimension and each well having a millimeter depth. By construction, the wells are in fact arranged on the same grid as the projections of the buffer. The wells have the same dimensions as the corresponding protrusions of the buffer.

In a particularly advantageous manner, the bottom of each well is located at the same and unique distance from the microscope slide which facilitates the automatic acquisition of microscopy images on each well without requiring a development step between each image.

In FIG. 8D, a liquid medium is poured suitable for the culture and the imaging of the biological objects considered. This liquid medium is, for example, an aqueous saline solution of the PBS type, phosphate buffered saline, and derivatives or an aqueous culture medium suitable for 3D cell cultures, whether or not supplemented with pharmacological molecules or molecules intended to provide fluorescent exogenous labeling or chromogenic or other, or any other molecules of biological interest to be applied by balneation. In the step of FIG. 8E, the cover 5 provided with the second microscope slide is positioned on the support 1. The second microscope slide 12 comes into contact with the upper surface 54 of the seal 4. The chamber culture and imaging is then tight vis-à-vis liquids while remaining permeable to gases. The opening 11 of the support 1 is aligned with the opening 34 of the seal and with the opening 15 of the cover 5. According to the needs of the experiment, the culture and imaging chamber can be placed in a incubator to allow the liquid medium to diffuse in the agarose layer and / or to balance the gas exchanges.

After placing the biological samples in the hydrogel layer wells, the culture and imaging chamber is placed on a microscope sample holder as shown in Figure 8F. The objective and / or the condenser 100 of the microscope is here placed above the culture and imaging chamber.

The total height of the culture and imaging chamber is less than 8 mm and compatible with most optical microscopes. According to the modalities of imaging by reflection or by transmission, the lamella 2 and / or the lamella 12 allow the collection of a signal by reflection and / or by transmission.

The total time for assembling a culture and imaging chamber is less than 10 minutes. The loading of biological samples can be done quickly and very efficiently.

FIG. 9 illustrates a variant of seal 24 comprising here two openings 134. Each opening 134 of seal 24 here forms a compartment. Such a seal can be placed in a support as illustrated in FIG. 1 to form two watertight compartments relative to one another. This seal 24 makes it possible, for example, to form an identical network of wells in each compartment of the seal 24. The two openings 134 are intended to be arranged facing an opening in the support 1 or the cover 5.

According to another variant illustrated in FIG. 9, the support 1 comprises two compartments separated by a partition 31. A seal 94 adapted to the dimensions of each compartment makes it possible to have two networks of wells formed in two layers of agarose, the two compartments being sealed relative to each other while being formed in the same support 1. Each seal 94 has an opening 234 disposed opposite an opening 111 of a compartment of the support 1.

These variants are useful, for example, for easily comparing molecules dissolved in separate culture media while ensuring that the two compartments are subjected to the same external conditions. The dissolved molecules can be markers or drugs, which are administered to biological samples by bathing.

FIG. 10A illustrates a detailed view of a portion of buffer 13 for forming an array of wells by imprint in a layer of hydrogel. We observe the projections 23 and the marks 43 as well as a foot 33. In FIG. 10B, the layer of agarose comprises the imprint of the tampon of FIG. 10A. We observe the network of wells 26, and the marking marks 46 adjacent to each well. In this example, each registration mark 46 is made up of a letter indicating a line and a number indicating a column of the network. The footprint forms a bowl 36 which makes it possible to inject, collect and / or change the liquid medium of the culture and imaging chamber.

In FIG. 11, there is a microscopic view of a well 26 formed by the imprint of a projection 23 as shown in FIG. 6. The bottom 56 of the well 26 is flat and of circular shape with a diameter equal to about 300 micrometers. The walls of the well are flared towards the opening of the well. One of the walls is advantageously inclined at an angle of about 67 degrees relative to the horizontal surface of the agarose layer to allow the injection of a biological capsule via a pipette.

The transfer of the shape of the projections in the agarose is carried out in a very precise and faithful manner. In particular, we observe in FIG. 11 steps corresponding to the fabrication layer by layer in 3D of the projections of the pad. These steps allow for example to measure the depth of an image plane along the optical axis in a confocal microscopy image.

Advantageously, the ends of the projections are placed at a predetermined distance (for example by the height of the feet of the buffer) from the surface of the microscope slide so that the bottom of the wells consist of a thin layer of agarose and are all located at the same predetermined distance from the microscope slide surface.

FIG. 12 illustrates a method of transferring 27 spherical biological samples approximately 200 μm in diameter into a network of wells. In FIG. 12A, the biological samples 27 are placed in a liquid medium 17 in a Petri dish 9. In FIG. 12A, the biological samples are aspirated 27 one by one in an 8 Pasteur pipette in the direction of the arrow. The tip of the pipette is preferably chosen so that the internal diameter of the pipette is slightly greater than that of the biological samples 27. The suction is preferably configured so that the biological samples 27 are aligned and regularly spaced in the pipette 8. For example, up to 12 biological samples 27 are thus aspirated in a pipette. In FIG. 12B-12C, the end of the pipette is placed in a well 26 of the agarose layer in order to deposit a biological sample 27 in this well 26. The operation is repeated to individually deposit a biological sample 27 in each well of the agarose layer 6. The biological samples 27 are thus placed individually in the wells at predetermined positions in 3D. Generally, this step is carried out by hand, under visual control by observation with a stereomicroscope. Positioning accuracy depends on the size of the wells, the size of the biological samples, and the manual precision of an experimenter or the mechanical precision of a micromanipulator. The positioning accuracy is in a range from a few hundred micrometers to about ten micrometers.

The handling of biological samples 27 during aspiration and during expulsion can be monitored in real time using a stereomicroscope. Inclining a wall of the well at an angle of about 67 degrees from the vertical allows a biological sample 27 to be injected via the pipette without obscuring the image field in an upright microscope. On the other hand, tilting the opposite wall at an angle of about 80 degrees to the horizontal surface of the hydrogel layer helps to reduce optical aberrations in an upright microscope.

In this way, several dozen biological samples can each be placed in a well of the same culture and imaging chamber in a few minutes.

In a particularly advantageous manner, the dish 36 formed in the agarose layer 6 makes it possible to renew the liquid medium without risking touching one of the biological samples 27.

The culture and imaging chamber can then be sealed by the cover and the microscope slide 12. The seal 4 is gas permeable but is tight against liquids and prevents evaporation culture fluid. Thus the sterility of the biological samples is preserved in the chamber and the chamber can be subject to back and forth between the incubator where the biological samples are cultured under appropriate conditions and sometimes sterile and the microscope. Samples can be imaged for several days in a row, whether continuous or not, with or without return to the incubator. It is also possible to open the lid in a sterile environment or not to replace the culture medium by taking advantage of the cuvettes 36 formed in the corners of the agarose layer 6.

The shape and depth of the wells allow easy handling of a large number of biological samples which are individualized and easily identifiable by the registration marks. Round trips from the culture and imaging chamber between a preparation bench, an incubator and / or a microscope are thus facilitated. In addition, the risk of mixing individualized biological samples identified in the well network is extremely reduced.

Alternatively, the cover does not have an opening. The support has an opening and a microscope slide as described in the embodiment in connection with FIG. 1. This embodiment makes it possible to protect the biological samples from ambient light. The culture and imaging chamber can be viewed on an inverted optical microscope.

In another variant, the second microscope slide is replaced with an optical device or an optical system comprising for example a reflecting plate, such as a mirror, or a partially reflecting plate, a dichroic plate, a polarizer, a half-wave plate , a quarter wave plate and / or a lens. This variant can make it possible to carry out experiments requiring to selectively bring in or detect light in the culture and imaging chamber while observing the samples using an inverted microscope.

In another variant, the support does not have an opening, but nevertheless includes a microscope slide 2 or an optical device or an optical system comprising for example a reflecting plate, such as a mirror, or a partially reflecting plate, a dichroic plate, a polarizer, a half-wave plate, a quarter-wave plate and / or a lens. In this case, the hydrogel layer and the seal 4 are directly in contact with the reflective lamella or strip so as to seal the chamber. The cover 5 has an opening 15 and a microscope slide 12 as described in the embodiment in connection with FIG. 1. This embodiment can allow experiments requiring the presence of optical components on the light path to be carried out. either on the excitation (before the sample) or detection (after the sample) portion in the culture and imaging chamber while observing the samples using an upright microscope.

FIG. 13 illustrates the use of a culture and imaging chamber according to the present disclosure on a sample plate of a microscopy device. The motorized stage is programmed to allow the automatic acquisition of low or medium resolution image fields using a microscopy device depending on the pitch of the well network. The stage generally moves in the coordinates of the spatial reference frame of the microscope, conventionally along the X and Y axes in a horizontal plane. The displacement along the vertical Z axis, corresponding to the depth of the sample, is carried out either by moving the stage and / or, more often, the objective turret. The objective and / or the condenser 100 of the microscope is here placed above the culture and imaging chamber. It is no longer necessary to focus on each biological sample. The acquisition of a large number of microscopy images on many biological samples is thus greatly accelerated. The yield and the number of samples can thus be greatly increased. On the insert of FIG. 13, it can be seen that the opening 11 of the support is aligned with the opening 34 of the seal and with the opening 15 of the cover. These three openings 11, 34 and 15 can thus be aligned on the optical axis of a straight or inverted microscope to allow measurements in transmission.

FIG. 14 illustrates an example of 70 image fields corresponding to 70 wells of a culture and imaging chamber. The identification marks are identified adjacent to the wells despite the presence of bubbles formed on the surface of the agarose layer.

In FIG. 15, images are acquired using an epi-fluorescence and wide field microscope for taking pictures as a function of time. Advantageously, the microscope includes an enclosure under a temperature and gas controlled atmosphere surrounding the sample holder and the culture and imaging chamber. FIG. 15A represents the image acquisition of 50 biological capsules in wells at the start of the experiment (t = 0) and FIG. 15B represents the image of the same wells at the end of the experiment (t = 15h ). Intermediate images can be acquired automatically, for example every 30 min.

In another example, a confocal laser scanning microscope is used to acquire slices at different Z altitudes in biological cells, with a Z pitch of a few microns for example. We can then reconstruct the 3D image of the 50 biological capsules and follow the evolution of the cells with very high spatial and temporal resolution.

FIGS. 16A and 16B represent composite images comprising fluorescence channels superimposed on a bright field microscopy image. These images make it possible to follow the trace of the displacement of the cellular nuclei inside a capsule as a function of time, represented in FIG. 16C. Figure 16D illustrates the directional analysis of cell movements in Figure 16C, which can help determine the interaction between different populations of biological cells. It is observed that the biological capsule does not move inside a well and does not deteriorate. This culture and analysis chamber makes it possible to analyze a large number of biological samples with a high yield, while providing microscopy images of high spatial resolution, in 2D or 3D and over a long duration of experiment ranging up to several tens of hours or even several days.

In another example of application, wells having other shapes and / or dimensions adapted to other samples are used. By way of example, a network of wells of rectangular parallelepiped shape is used, as described in connection with FIG. 7, in order to have transgenic zebrafish larvae having a cell nucleus marker, for example of the NLS :: TagRFP type. On the one hand, a wide field microscope is used to acquire the images of the larvae fixed at 30 hpf (Fig. 17A-17C). On the other hand, a confocal microscope is used to acquire images of the same larvae incubated additionally in a Bodipy 505/515 lipid marker (Fig. 17D-17E). An image processing system reconstructs the image of the larvae in 3D. Given the size of the larvae, there are 77 larvae in a 7-line, 11-column culture and imaging chamber. A 4X microscope objective allows a complete scan of the chamber (Fig. 17A). A 10X microscope objective is used to scan a reduced area (5 lines by 5 columns, represented by a frame in fig. 17A) with better spatial resolution (Fig. 17B). Remarkably, the image in FIG. 17C makes it possible to distinguish the individual nuclei from the labeled cells. It is also observed that the identification mark of each well is legible, which makes it possible to trace the development history of each larva individually and to push the analyzes of a particular individual if necessary.

More generally, those skilled in the art will easily adapt the shape and dimensions of the wells according to the biological sample to be studied in vivo, in particular to all kinds of embryos and organoids.

The culture and imaging chamber of this disclosure is compatible with many light microscopes.

More specifically, this culture and imaging chamber is compatible with any type of microscopy comprising a microscope stand, a stage provided with a sample holder intended to receive a microscopy slide and whose objective and / or condenser of the microscope have a frontal distance compatible with the spatial congestion of the culture and imaging chamber.

In particular, it is compatible with the use of a multiphoton microscope, based on the use of an infrared excitation beam which allows greater penetration of light into a thick sample and a reduction in phototoxicity due out-of-focus excitation. A light sheet microscope can also be used for rapid scanning of a light sheet by real or digital scanning, which greatly increases the scanning speed. In this case, one can use a straight microscope for excitation and collection or a microscope having an optical axis inclined at 45 degrees.

The culture and imaging chamber is also compatible with a confocal microscope, or with rotating or super resolution discs.

Claims (9)

1. Culture and imaging chamber (10) of biological samples by optical microscopy, said culture and imaging chamber (10) comprising: - a support (1) and a cover (5), the support (1) comprising at least one interior housing (61); - at least one microscope slide (2, 12) disposed facing an opening (11, 15) formed in the support (1) or in the cover (5) facing said interior housing; - a seal (4, 24, 94) disposed between the support (1) and the cover (5) in contact with said at least one microscope slide (2, 12), the seal (4) delimiting at least one other opening (34) disposed opposite the microscope slide (2, 12); - a hydrogel layer (6) disposed inside said other opening (34) of the seal (4, 24, 94), the hydrogel layer (6) comprising a network of wells (26) openings formed at predetermined three-dimensional positions in an orthonormal reference linked to the support (1), each well (26) being adapted to receive a biological sample; - The support, the cover and the gasket being configured so as to be made integral to form a culture and imaging chamber (10) tight vis-à-vis a liquid. 2. Culture and imaging chamber according to claim 1 in which the support (1) and / or the seal (4, 94, 24) comprises a plurality of compartments which are sealed relative to one another, at least a sealed compartment comprising said hydrogel layer (6) and said well network (26). 3. Culture and imaging chamber according to claim 1 or 2 wherein the support (1) has a first opening (11), the cover (5) has a second opening (15), said at least one microscope slide (2, 12) comprising a first microscope slide (2) arranged opposite the first opening (11) and a second microscope slide (12) arranged opposite the second opening (15), the seal (4) being in contact with the first microscope slide (2) and the second microscope slide (12). 4. Culture and imaging chamber according to claim 1 or 2 wherein the cover (5) has a second opening (15), said at least one microscope slide comprises a second microscope slide (12) disposed facing the second opening (15) and a reflecting or dichroic optical component disposed in the interior housing of the support. 5. Culture and imaging chamber according to claim 1 or 2 in which the support (1) has a first opening (11), said at least one microscope slide (2, 12) comprising a first microscope slide (2 ) facing the first opening (11) and in which the cover (5) is opaque or comprises a reflecting or dichroic optical component. 6. Culture and imaging chamber according to one of claims 1 to 5 wherein at least one open well (26) has a parallelepiped shape, a cylindrical shape, a frustoconical shape, a flared shape or a shape closed at the level a well opening. 7. Culture and imaging chamber according to one of claims 1 to 6 in which the network of wells comprises at least one well having a wall inclined with respect to a horizontal surface of the hydrogel layer of a greater angle at 0 and less than or equal to 135 degrees. 8. Culture and imaging chamber according to one of claims 1 to 7 in which the network of wells comprises at least one well having a flat or concave bottom formed in the hydrogel layer and / or at least one well having a bottom formed by a microscope slide surface (2). 9. Culture and imaging chamber according to one of claims 1 to 7 in which the hydrogel layer comprises a plurality of registration marks (46), each registration mark (46) being disposed near a well (26) and being configured to identify this well (26). 1/4 3/4