WO2020011735A2

WO2020011735A2 - Systems, apparatuses, and methods for preparation of tissue samples

Info

Publication number: WO2020011735A2
Application number: PCT/EP2019/068307
Authority: WO
Inventors: Daniel KIRSCHENBAUM; Adriano Aguzzi
Original assignee: Universität Zürich
Priority date: 2018-07-09
Filing date: 2019-07-08
Publication date: 2020-01-16
Also published as: WO2020011735A3

Abstract

The invention relates to a method for preparing a tissue sample (101, 201) for microscopy, comprising the steps of submitting a tissue sample (101, 201) to a chemical fixation treatment, mounting the fixed tissue sample (101, 201) inside of an electrically insulating sample holder (102, 202, 902), said sample holder (102, 202, 902) defining a sample volume (121, 221) configured to receive, particularly to hold, the tissue sample (101, 201) within the sample holder (102, 202, 902), the sample holder (102, 202, 902) further having a first opening (117A, 217A) and a second opening (117B, 217B), mounting said sample holder (102, 202, 902) between a first electrolyte chamber (103A) comprising a first electrode (104 A, 904A) and a second electrolyte chamber (103C) comprising a second electrode (104B, 904B), in a clearing step, applying an electric potential difference (voltage) to said first electrode (104A, 904A) and said second electrode (104B, 904B). The invention further relates to a device for electrophoretic clearing, washing and/or staining of a tissue sample (101, 201).

Description

SYSTEMS, APPARATUSES, AND METHODS FOR PREPARATION OF TISSUE

SAMPLES

The present application relates to systems, apparatuses, and methods for use in the preparation of biological samples, and particularly thick tissue samples, for histological analysis.

The present application claims the benefit under 35 U.S.C. 1 19(e) of U.S. Provisional Application 62/695,477 filed on July 9, 2018, the disclosure of which is incorporated herein by reference.

Background

To study complex organs and tissues, such as a mammalian brain and a tumor mass, it is often necessary to understand both its 3D structure and molecular makeup. Current methods, exemplified by array tomography or serial block-face scanning electron microscopy, can provide sub-cellular details, but involve prohibitively inefficient and damaging mechanical sectioning and reconstruction. Optical sectioning techniques combined with tissue clearing methods have been developed, in which light-scattering is reduced to increase the depth at which tissue can be imaged. While these methods can bypass laborious mechanical sectioning and reconstruction processes, they are applicable to immunostaining and molecular phenotyping to a limited extent. A technology capable of preparation of biological tissue for microscopic analysis, using tissue clarification and/or or tissue staining, while maintaining the 3-D integrity of the tissue and of the sub-cellular structures therein, while also making biomolecules within the tissue accessible for labeling with molecular probes at deeper regions in the tissue, is highly desirable.

Tissue clarification methods currently available are based on detergent based extraction of lipids from tissue, while successful staining of tissue is based on the homogeneous distribution of dyes in the tissue volume. Without intervention, the kinetics of both processes are limited by passive diffusion and tissue porosity. Tissue clarification methods utilizing electrophoresis apply electric current to the bulk volume of buffer in which the tissue resides. This leads to a unidirectional stream of ions along the path of lowest electrical resistance, which in this case is mostly the ionic detergent buffer, whereas the tissue acts more like an insulator. Thus, there is a need for improved throughput of tissue clarification and tissue staining by molecular dyes.

The problems outlined in the preceding paragraphs are overcome by the methods and devices laid out in the independent claims, with further advantageous embodiments specified in the dependent claims, the specification and examples.

Summary

Systems, apparatuses, and methods for preparing tissue samples for processing and analysis are described herein.

An aspect of the invention relates to an apparatus that includes a chamber that includes a first chamber portion, a second chamber portion, and a third chamber portion. The second chamber portion is configured to hold a tissue sample. The first chamber portion is configured to hold a first buffer in contact with the tissue sample, and the third chamber portion is configured to hold a second buffer in contact with the tissue sample. The apparatus further includes a set of electrodes coupled to the chamber, the set of electrodes being configured to apply an electrical signal to the sample to induce electrophoresis in the sample. A first electrode of the set of electrodes is disposed in the first chamber portion and a second electrode of the set of electrodes is disposed in the third chamber portion of the chamber. The first electrode and the second electrode of the set of electrodes are disposed such that during application of an electric field between the first electrode and the second electrode, the electric field lines of the electric field pass only through or predominantly through the sample in the second chamber portion.

A alternative of this aspect relates to a device for electrophoretic clearing, washing and/or staining of a tissue sample, said device comprising a sample holder made of an electrically insulating material. The sample holder defines a sample volume configured to receive a tissue sample within the sample holder. The sample holder further has a first opening and a second opening, with the sample volume disposed between the first and the second opening. The first opening is configured to be brought into fluid communication with a first electrolyte chamber and the second opening is configured to be brought into fluid communication with a second electrolyte chamber or second electrolyte volume.

The device further has a first electrode and a second electrode. The first electrode is configured to be brought in electrically conductive contact with a first electrolyte contained in the first electrolyte chamber, and the second electrode is configured to be brought in electrically conductive contact with a second electrolyte contained in the second electrolyte chamber, and the first and second electrode are configured to be connected to a voltage source.

Another aspect of the invention relates to a method that comprises removably disposing a sample holder comprising a tissue sample in a second chamber portion of a chamber of an apparatus, the chamber including a first chamber portion, the second chamber portion, and a third chamber portion; flowing a first buffer in the first chamber portion, the first buffer in contact with the tissue sample; flowing a second buffer in the third chamber portion, the second buffer in contact with the tissue sample; applying an electrical signal to the sample to induce electrophoresis in the sample via a first electrode and a second electrode of a set of electrodes of the apparatus, the first electrode of the set of electrodes disposed in the first chamber portion and the second electrode of the set of electrodes disposed in the third chamber portion, wherein during application of an electric field between the first electrode and the second electrode, the electric field lines of the electric field pass only through or predominantly through the sample in the second chamber portion.

The method can further include applying a first pressure wave to the sample via a transducer of the apparatus such that applying the first electrical signal and applying the first pressure wave collectively results in at least partial lipid extraction from the tissue sample. Further, the method may include, at a second time different than the first time, flowing a third buffer in the first chamber portion and flowing a fourth buffer in the third chamber portion. The method may then include disposing a staining agent in the chamber, applying a second electrical signal to the sample to induce electrophoresis in the sample, and applying a second pressure wave to the sample via the transducer of the apparatus, such that applying the second electrical signal and applying the second pressure wave collectively results in the staining of the tissue sample with the staining agent.

A alternative of this aspect relates to a method for preparing a tissue sample for microscopy that comprises the steps of

• providing a tissue sample;

• submitting the tissue sample to a chemical fixation treatment, yielding a fixed tissue sample;

• mounting the fixed tissue sample inside of an electrically insulating sample holder, the

sample holder defining a sample volume configured to receive, particularly to hold, the sample within the sample holder, the sample holder further having a first opening and a second opening, with the sample volume disposed between the first and the second opening;

• mounting the sample holder between a first electrolyte chamber comprising a first electrode and a second electrolyte chamber comprising a second electrode, and wherein

i. said first electrolyte chamber is configured to comprise an electrolyte solution in contact with said tissue sample;

ii. said second electrolyte chamber is configured to comprise an electrolyte solution in contact with said tissue sample;

• in a clearing step, applying an electric potential difference (voltage) to said first and said second electrode wherein

said first electrolyte chamber is filled with a first electrolyte solution comprising an ionic detergent having an amphiphilic ion and a counterion, and

said second electrolyte chamber is filled with a second electrolyte solution, and wherein said second electrode is an anode if said amphiphilic ion has a negative charge, and wherein said second electrode is a cathode if said amphiphilic ion has a positive charge.

In certain embodiments, the method includes disposing a tissue sample in a sample holder, and removably disposing the sample holder in a second chamber portion of a chamber of an apparatus, the chamber of the apparatus including a first chamber portion, the second chamber portion, and a third chamber portion. The method further includes flowing a first buffer in the first chamber portion such that the first buffer is in contact with the tissue sample and flowing a second buffer in the third chamber portion such that the second buffer is in contact with the tissue sample. The method further includes applying an electrical signal to the sample to induce electrophoresis in the sample via a first electrode and a second electrode of a set of electrodes of the apparatus, where the first electrode of the set of electrodes is disposed in the first chamber portion and a second electrode of the set of electrodes disposed in the third chamber portion. The electrodes are configured such that during application of an electric field between the first electrode and the second electrode, the electric field lines of the electric field pass only through or predominantly through the sample in the second chamber portion.

In certain embodiments, a method, as described herein, includes disposing a tissue sample in a sample holder, and removably disposing the sample holder in a second chamber portion of a chamber of an apparatus, the chamber including a first chamber portion, the second chamber portion, and a third chamber portion. The method includes, at a first time, flowing a first buffer in the first chamber portion and flowing a second buffer in the third chamber portion, the first buffer and the second buffer including ionic detergents and optionally in addition, non-ionic detergents, and applying a first electrical signal to the sample to induce electrophoresis in the sample via a first electrode and a second electrode of a set of electrodes of the apparatus. The first electrode of the set of electrodes is disposed in the first chamber portion and the second electrode of the set of electrodes is disposed in the third chamber portion such that during application of an electric field between the first electrode and the second electrode, the electric field lines of the electric field pass only through or predominantly through the sample in the second chamber portion. The method further includes applying a first pressure wave to the sample via a transducer of the apparatus such that applying the first electrical signal and applying the first pressure wave collectively results in at least partial lipid extraction from the tissue sample. Further, the method includes, at a second time different than the first time, flowing a third buffer in the first chamber portion and flowing a fourth buffer in the third chamber portion. The method then includes disposing a staining agent in the chamber, applying a second electrical signal to the sample to induce electrophoresis in the sample, and applying a second pressure wave to the sample via the transducer of the apparatus, such that applying the second electrical signal and applying the second pressure wave collectively results in the staining of the tissue sample with the staining agent.

Brief Description of the Figures

FIG. 1 is an illustration of components of a system or device for focused sono-electro-osmosis, according to an embodiment of the invention.

FIG. 2 is an illustration of an exploded view of the system or device of FIG.1 .

FIG. 3A is an illustration of an example sample holding chamber or sample holder, particularly to be used in a system for focused sono-electro-osmosis, according to an embodiment. FIGS. 3B-3D illustrate the example sample holding chamber (sample holder) in FIG. 3A, including a sample (tissue sample) encapsulated in paraffin, and including a dye (staining preparation) embedded in hydrogel, respectively.

FIG. 4 is a cross sectional view of the assembled example sample holding chamber (sample holder) illustrated in FIGS. 3A-3D.

FIG. 5 illustrates a set of components that can be used to assemble example sample holding chambers (sample holders) to accommodate tissue samples of varying sizes, according to an embodiment.

FIG. 6 is an illustration of example components of a system or device for sono-electro-osmosis, according to an embodiment.

FIG. 7 is an illustration of the components of FIG. 6 as assembled.

FIG. 8 is a cross-sectional illustration of the assembly illustrated in FIG. 7, in use during sono- electro-osmosis, according to an embodiment.

FIGS. 9A-9C show images illustrating the difference in the extent of staining of sample tissue, with the use of sonication (US) and without (CTRL), using a system for sono-electro-osmosis, according to an embodiment. FIG. 9A is an image of a first imaging plane of the sample tissue. FIG. 9B is an image of a last imaging plane of the sample tissue. FIG. 9C is an orthogonal projection.

FIGS. 10A-10D are a set of plots quantifying the quality of imaging as a function of signal intensity and depth of tissue, when the tissue samples have been stained without sonication (CTRL, FIGS. 10A, C) and with sonication (US, FIGS. 10B, D), using a system for sono-electro- osmosis, according to certain embodiments. FIGS. 10A-B show results from labelling with To- Pro3 stain and FIGS. 10C-D show results from labelling with the DAPI stain.

FIGS. 1 1A-1 1 D shows images of tissue phantoms held in a gel and stained, with fluorescent dyes from the gel surface (indicated by the orange lines), with or without the treatment of the tissue phantoms with electrophoresis and/or ultrasound. The depth of stain is indicated by the arrows. FIGS. 1 1 A-D illustrate the best extent of staining as evidenced by the length of arrows is achieved in the case of combined treatment (FIG. 1 1 D) with both electrophoresis and ultrasound.

FIG. 12 shows a flowchart describing a method of processing a tissue sample, according to an embodiment.

FIG. 13 shows a flowchart describing a method of processing a tissue sample, according to another embodiment.

FIG. 14 and 15 show an embodiment of the device for electrophoretic clearing, washing and/or staining of a tissue sample according to the invention which is specifically adapted to perform the staining step of the method according to the invention. FIG. 16-19 show an embodiment of a sample holder for holding a tissue sample according to the invention configured for performing electrophoresis in a vertical direction comprising two membranes for holding the tissue sample.

FIG. 20 shows an array of hollow microneedles used to stain the tissue sample using electrophoresis according to the invention.

Detailed Description

Embodiments described herein relate to systems, apparatuses, and methods for use in the preparation of three-dimensional biological samples for analyses, such as for microscopic analysis and/or other imaging techniques. The systems, apparatuses and methods described herein can, for example be useful in medicine (including but not limited to pathological anatomy and histopathology) and research to, for example, diagnose or monitor disease (such as cancer, metastases, autoimmune and inflammatory diseases, neuropathies, or others) or graft transplantation, to monitor the outcome and impact of therapy (such as, for example, stimulation of lymphocytes to fight cancer), to study healthy or diseased tissue, to screen candidate agents for toxicity and efficacy in disease modification, and/or the like. Embodiments described herein can also allow for improved inspection of thick biological specimens (e.g. fixed samples of 3D tissue blocks) without requiring any thin or micro thin sectioning of the tissue that may cause damage to a specimen or result in loss of structural information of the specimen. Embodiments described herein can be further useful to allow significantly improved throughput of tissue clarification and/or staining of intact tissue blocks, permitting significantly better penetration of probing signals during analyses and significantly better results from such analyses. For example, thick, intact, three-dimensional blocks of biological samples can be significantly better probed through light microscopy to obtain better imaging results from techniques such as fluorescence imaging as described in greater detail herein. These and other objects, advantages, and features of the described embodiments will become apparent to those persons skilled in the art upon reading the details of the compositions and methods as more fully described below.

As described herein, analysis of three-dimensional tissue samples requires optical sectioning, which is possible in samples that have undergone tissue clarification and sometimes also tissue staining. Currently available methods of tissue clarification are limited by passive diffusion and tissue porosity. In some instances, when tissue clarification and/or staining is aided by electrophoresis, the clarification and staining of the sample is limited by the presence of an ionic buffer solution bathing the sample. The ionic buffer offers a path of lower electrical resistance for the applied current to pass through relative to the sample itself. Embodiments described herein are configured to dispose and/or otherwise distribute the buffer relative to the sample such that the current path necessarily passes through the sample rather than mostly through a more conductive buffer solution, aiding in more efficient and faster tissue clarification and/or staining at lower current and/or voltage levels. In some embodiments, electro-osmosis is used to achieve increased efficiency and throughput in tissue clarification and/or staining. In some embodiments, sono-electro-osmosis is used to achieve tissue clarification and/or staining.

Systems and methods described herein, in some instances, include focused electrophoresis combined with sonication (also referred to as sono-electro-osmosis) to achieve faster and more homogeneous tissue clarification and/or tissue staining in three-dimensional tissue samples (e.g. in large blocks of tissue samples). By applying pulsed pressure waves and/or standing acoustic waves to a tissue sample, transient changes in porosity of the sample can be induced. Without wishing to be bound by any particular theory, these changes in porosity are believed to lead to improved extraction of lipid-laden detergent micelles and may also lead to improved distribution or localization of stain in the tissue sample. In other words, by applying pressure waves and/or acoustic waves, the local porosity of tissue is transiently modified, while certain acoustic waves (e.g., standing waves) can create local dye-concentration maxima and microstreaming effects. Local concentration maxima and microstreaming effects can improve reaction kinetics, while modulation of porosity improves the diffusion of molecules through tissue.

For example, staining biological samples often involves immunohistochemical interactions between protein macromolecules including an antigen and an antibody. The antigen is immobilized on a portion of fixed tissue sample and the antibody is bound to the stain or dye and is mobile. An epitope is a portion of the immobilized antigen and the paratope in a portion of the antibody that recognizes and binds to the epitope of the antigen. Thus paratope-epitope interactions are central to tissue staining methods and dependent on reaction kinetics to enable access and binding between the mobile paratope and the immobilized epitope. Transient modulation of porosity offers channels of movement for the dye molecules carrying the paratope. Focused electrophoresis combined with sonication aids in the formation of localized regions of high dye concentrations aiding in faster, stronger interactions between paratope and epitopes proximal to that region.

Some embodiments described herein include a transducer coupled to the chamber, the transducer configured to apply a standing wave, such as a standing acoustic wave, or a continuous wave, such as a continuous acoustic wave, to the tissue sample held in the chamber, the set of electrodes and the transducer collectively configured to induce staining of the sample upon exposure to a staining agent. In some instances, the set of electrodes and the transducer can be collectively configured to induce molecular modifications to the sample or to some previously induced stain upon exposure to biologically active molecules, enzymes.

In some embodiments, tissue clarification as achieved by embodiments disclosed herein can be achieved via methods and compositions for preparing biological specimens as generally disclosed in International Application No. PCT/US2013/031066 (“the Ό66 application”) titled “METHODS AND COMPOSITIONS FOR PREPARING BIOLOGICAL SPECIMENS FOR MICROSCOPIC ANALYSIS” filed March 13, 2013, the entire disclosure of which is hereby incorporated by reference. A first aspect of the invention relates to a method for preparing a tissue sample for microscopy (particularly for electrophoretic clearing, washing and/or staining of a tissue sample), particularly by means of a device according to the second aspect of the invention, comprising the steps of

• providing a tissue sample;

• submitting said tissue sample to a chemical fixation treatment, yielding a fixed tissue sample;

• mounting said fixed tissue sample inside of an electrically insulating sample holder made (particularly according to the third aspect of the invention) of an electrically insulating material [elsewhere herein referred to as the second portion of the chamber], said sample holder defining a sample volume configured to receive, particularly to hold, the sample within the sample holder, the sample holder further having or comprising a first opening and a second opening opposite the first opening, with the sample volume disposed between (and connected to) the first and the second opening;

• mounting said sample holder between a first electrolyte chamber comprising a first electrode and a second electrolyte chamber comprising a second electrode, wherein said first and second electrodes are connectable to a voltage source, and wherein

i. said first electrolyte chamber is configured to comprise an electrolyte solution in electrically conductive contact with the first electrode and said tissue sample;

ii. said second electrolyte chamber is configured to comprise an electrolyte solution in electrically conductive contact with the second electrode and said tissue sample;

said second electrolyte chamber is filled with a second electrolyte solution, and wherein said second electrode is an anode if said amphiphilic ion has a negative charge so that the amphiphilic ion is drawn towards the second electrode through the sample, and wherein said second electrode is a cathode if said amphiphilic ion has a positive charge.

In certain embodiments, the ionic detergent is or comprises an alkylsulfate, particularly a C8 to C20 alkylsulfate. In a particular embodiment the ionic detergent is or comprises sodium dodecylsulfate (SDS). In certain embodiments, the ionic detergent is or comprises a bile acid. In a particular embodiment the ionic detergent is or comprises deoxycholic acid or a salt thereof, particularly the sodium salt of deoxycholate. In certain embodiments, the electric potential difference is in the range of 10V to 100V, particularly in the range of 20V to 60V. In certain embodiments, the electric potential difference is adjusted, particularly throughout the clearing step, to yield an electric current of 5 mA to 200 mA, particularly of 100-150 mA.

One advantage of the invention as embodied by the method and device as described herein, is that the clearing, washing and staining of the tissue sample can be effected at relatively low currents, leading to less heat development than prior art methods and devices. Less heat development in turn allows for better preservation of biological structures within the sample.

During the clearing step, particular embodiments provide for a presetting of the current at 5 mA to 200 mA, particularly of 100-150 mA. In certain embodiments, the current and/or voltage applied to the sample is adjusted in relation to the sample size and the setup (particularly the distance between the electrodes) to allow a sample temperature between 37°C to 42°C, particularly from 38,5°C to 40,5°C. This temperature range has proven to be of particular advantage as it allows to both rapidly clear the sample while preserving structural integrity, including the integrity of protein structure of proteins remaining in the sample after clarification.

In certain embodiments, the sample holder comprises a temperature sensor connected to the voltage source or controller in order to allow adjustment of the voltage to keep the temperature between 37°C to 42°C, particularly from 38,5°C to 40,5°C.

In certain embodiments, the electric potential difference is kept at constant voltage between 15 and 35 V, particularly at around 20 V during the washing and staining step.

In certain embodiments, the diameter or a diameter of the sample volume is between 3mm and 30mm. In certain embodiments, the distance between the first electrode and the second electrode is 10 mm to 150 mm, particularly between 20 mm and 100 mm. The diameter and electrode distance correspond to current routine pathology sample taking practices and tissue samples being prepared for microscopy in research settings.

In the context of the present specification, in case of a non-spherical shape of the sample volume or tissue sample, the term diameter refers to a maximum extension of the sample volume or tissue sample, particularly along the first axis or along a direction of the electric field applied between the electrodes during electrophoresis.

In certain embodiments, the fixation step is conducted by keeping the tissue sample in a solution comprising 2 % to 6% (particularly 4%) (w/v) acrylamide comprising, in relation to the acrylamide content, 0,5% to 2% (particularly 1 ,2%) (w/w) bisacrylamide, and/or 0,5% to 2% (w/v) formaldehyde or glutaraldehyde. The inventors have found that both aldehyde fixation and acrylamide fixation alone can provide samples amenable to the method of the invention. Combining the two fixation methods, however, has rendered the best results in the inventors’ practice. In certain embodiments, the concentration of the ionic detergent is 2% to 8% (w/v) (20 to 80g/L).

In certain embodiments, the concentration of ions inside of the sample volume, the first and the second electrolyte solution without counting the concentration of the ionic detergent, is 150 to 250 mOsm.

In certain embodiments, the clearing step is conducted for <24h to achieve full migration of the ionic detergent from the first electrolyte chamber to the second electrolyte chamber. One major advantage of the invention is that it allows for an order-of-magnitude acceleration of sample preparation.

In certain embodiments, the clearing step is followed by a washing step in which the first electrolyte chamber is emptied of the first electrolyte and filled with a different (third) electrolyte solution that is devoid of detergent. Similarly, the second electrolyte chamber is emptied of the second electrolyte and filled with a different (fourth) electrolyte solution devoid of detergent. Subsequently, an electric potential difference or the electric potential difference (voltage) is applied to said first and said second electrode. This exchange can of course also be performed in continuous mode by linking a recycling cycle connected to the first and second electrolyte chambers to a different source of electrolyte buffer.

As the clearing step employs agents detrimental to the preservation of protein tertiary structure, these agents need to be removed from the sample prior to staining of the sample.

In certain embodiments, subsequent to the clearing step (and the washing step, if applicable) a staining preparation comprising a dye specific for a biomolecule comprised in the tissue sample is introduced into the first electrolyte chamber and/or into the tissue sample, and an electrical potential difference is applied effecting the dye being drawn towards the second electrode. This step is referred to herein as the staining step.

Many staining agents can be employed. Ionic dyes and/or fluorescently labelled antibodies, which carry an electric charge due to the side chains comprised in the protein backbone, are of advantage as their entry into the sample is accelerated by the electric field. In certain embodiments, at least one further electrolyte, particularly a fifth and sixth electrolyte, is employed in the first and second electrolyte chamber, respectively. These electrolytes are adapted as regards the pH of the electrolyte to impart an electrical charge to the dye molecules comprised in the staining preparation in order to facilitate their migration into the sample. For example, a staining preparation can include antibody molecules covalently modified to comprise lysin residues to impart a positive charge to the antibody backbone, and the electrolyte will tend to a slightly acidic pH (5.0 to 6.5) to increase the positive charge of the antibody.

In certain embodiments, the staining preparation is applied comprised in, or in the form of, an electrolyte solution. This facilitates easy transport of a dye molecule into the sample by directing the flow of electrolyte to the sample from an electrolyte reservoir comprising an electrolyte that contains the dye. The dye, however, will be diluted in the electrolyte, and only a small fraction of the dye molecules will actually enter the sample, which results in unfavourable economics particularly with regard to dyes or staining molecules that are expensive or difficult to obtain (antibodies, advanced organic dye molecules).

In certain embodiments, the staining preparation is applied comprised in, or in the form of, a hydrogel preparation, particularly comprising or consisting of (low melting) agarose, positioned between the first electrode and the sample. This allows to apply a concentrated form of dye, all of which will enter the sample.

Another alternative for applying the staining preparation, which can be combined with either of the preceding methods of applying a staining preparation, is to insert one or several hollow microneedles into the tissue sample and injecting the staining preparation into the tissue sample by action of a micropump, or by applying an electric potential difference between the tip on the inside of the hollow microneedle and the sample (ionotophoresis). The microneedles typically have a diameter less than (<) 1/100 of the diameter of the sample volume (particularly a diameter of 20 pm to 150 pm). In certain embodiments, an array of microneedles is employed to apply the staining preparation. Non limiting examples of such arrays comprise 9 microneedles, 16 microneedles, 25 microneedles, 36 microneedles, 49 microneedles, 64 microneedles or 81 microneedles. The arrangement of the needle-array can be flexibly adjusted to follow the geometry of the sample. A hollow microneedle as used herein is a needle having a diameter between 1 pm and 999 pm and comprising a hollow inner volume. A microneedle electrode as used herein is an electrode that is placeable within the hollow inner volume of a microneedle.

The action of such microneedles facilitates the deposition of staining preparation depots within the sample, which can significantly accelerate the staining step.

In certain embodiments, pressure waves are applied to the sample during the clearing step, the washing step and/or the staining step. Particularly advantageous embodiments employ acoustic (sound) waves in the range of 200kHz to 5MHz, more particularly in the range of 1 to 2 MHz. As is demonstrated in the examples contained herein, the application of ultrasound greatly accelerates the process, and improves the results of the clearing and staining preparation of samples for microscopy.

In certain embodiments, the clearing step, the washing step and/or the staining step are conducted maintaining a temperature of 37°C to 42°C, particularly a temperature of 38,5°C to 40,5°C. The inventors observed significantly less satisfactory results at 30-35°C; at 45°C the sample started to suffer disintegration or denaturation. The clearing step must be conducted at >15°C to avoid precipitation of SDS.

In certain embodiments, the electrolyte solution comprised in the first and/or second electrolyte chamber is circulated or exchanged during application of an electric potential difference. The electrolyte can be exchanged or recycled, and in addition, may be treated to remove sample components cleared from the sample during the clearing step. The inventors have found that circulating the electrolyte at moderate rates improves the results of the method.

In certain embodiments, the sample holder is a simple plastic tube or a tube from an electrically insulating material.

In certain embodiments, the sample holder is separable into two parts, a first part comprising the first opening and a second part comprising the second opening, particularly with the sample volume being confined or defined by the first and second part. Particular embodiments of such sample holders are described below and depicted in the figures.

In certain embodiments, prior to the staining step a cross-linking hydrogel, particularly polyacrylamide, is provided in a first section of the first electrolyte chamber and/or the second electrolyte chamber, wherein a second section of the first electrolyte chamber is filled with the first electrolyte solution and/or wherein a second section of the second electrolyte chamber is filled with the second electrolyte solution, particularly wherein a hydrogel preparation, more particularly from a non-crosslinking hydrogel (such as low-melt agarose) comprising a staining preparation (comprising a dye) is embedded in the cross-linking hydrogel, more particularly adjacent to the sample volume and/or adjacent to the tissue sample.

In particular, a monomer solution is provided, particularly poured into, the first section of the first and/or second electrolyte chamber, wherein the monomer solution is subsequently allowed to polymerize into the cross-linking hydrogel or cross-linked hydrogel.

In certain embodiments, a recess is provided in the cross-linking hydrogel or cross-linked hydrogel in the first section of the first and/or second electrolyte chamber, wherein the noncrosslinking hydrogel comprising the staining preparation is embedded into the recess.

In certain embodiments, an opening (more particularly an axial opening) of the first and/or second electrolyte chamber leading to the first section or respective first section of the first or second electrolyte chamber) is closed by a liquid-tight seal prior to providing the cross-linking hydrogel in the first section, particularly prior to providing the monomer solution in the first section.

In certain embodiments, the liquid-tight seal comprises a protrusion facing the first section of the first and/or second electrolyte chamber, such that the recess is formed in the cross-linking or cross-linked hydrogel.

In certain embodiments, prior to said clearing step, washing step and/or staining step, the tissue sample is arranged between a first membrane and a second membrane, wherein particularly the first membrane, the tissue sample and the second membrane are arranged, particularly stacked, in a vertical direction, wherein the electric potential difference (in said clearing step, washing step and/or staining step) is applied in the vertical direction, more particularly wherein a positive electric potential is applied above the tissue sample (in other words the electrode above the tissue sample is the anode).

In certain embodiments, the first membrane and/or the second membrane is a semi-permeable membrane. Therein, the term‘semi-permeable’ designates a membrane allowing a first class of compounds to pass through the membrane, wherein passage of a second class of compounds through the membrane is blocked. In particular, the first class and the second class are distinguished by a physical property, more particular molecular size and/or charge and/or polarity. For example, the first and the second membrane may be permeable for compounds below a certain molecular size, such as small molecules (e.g. below 3 kDa), e.g. water, ions, buffer components, detergents and/or staining preparations, but not for larger compounds such as macromolecules (e.g. proteins). The advantage of such membranes is that the tissue sample is held in a fixed position, but remains accessible e.g. for detergent molecules during the clearing step and/or staining preparations (dyes) during the staining step. For example, the first and/or the second membrane may comprise or consist of cellulose, particularly have a plurality of pores with a defined pore size.

According to a particular embodiment, the first membrane and/or the second membrane is non- permeable for at least one dye molecule comprised in a staining preparation used in the staining step (in other words the membrane is capable of blocking the passage of this dye molecule, e.g. due to its higher molecular weight compared to other components of the used electrolyte solution(s)). Thus, the dye molecules are retained between the two membranes and the dilution of the staining preparation by the electrolyte solution is prevented. This significantly improves the efficiency of the staining step. In addition, the staining preparation can also be used efficiently in liquid phase, eliminating the need for embedding the staining preparation in a hydrogel.

In a second aspect, the invention relates to a device for electrophoretic clearing, washing and/or staining of a tissue sample, particularly a device usable in a method according to the first aspect of the invention, said device comprising a sample holder made of an electrically insulating material [elsewhere herein referred to as the second portion of the chamber], said sample holder defining a sample volume configured to receive, particularly to hold, a tissue sample within the sample holder, the sample holder further having or comprising, along a first axis or second axis, a first opening and a second opening opposite the first opening, with the sample volume disposed between (and connected to) the first opening and the second opening. The first opening is configured to be brought into fluid communication or fluid contact with a first electrolyte chamber or first electrolyte volume and said second opening is configured to be brought into fluid communication or fluid contact with a second electrolyte chamber or second electrolyte volume. The device further comprises a first electrode and a second electrode, wherein the first electrode is configured to be brought in electrically conductive contact with a first electrolyte contained in said first electrolyte chamber or first electrolyte volume, and wherein said second electrode is configured to be brought in electrically conductive contact with a second electrolyte contained in the second electrolyte chamber or second electrolyte volume, said first and second electrode being configured to be electrically connected to opposite poles of a voltage source.

In certain embodiments, the first electrode and the second electrode are configured to generate an electric field along a first axis (in other words the field vectors of the electric field are parallel to the first axis). In particular, the device and/or a chamber of the device extends along the first axis, more particularly between the first electrolyte chamber and the second electrolyte chamber.

In certain embodiments, the tissue sample is embedded in a hydrogel.

In certain embodiments, the first electrolyte chamber and/or the second electrolyte chamber is/are comprised in the device.

In certain embodiments, the device comprises a voltage source, wherein said first electrode and said second electrode are electrically connected to opposite poles of the voltage source.

In certain embodiments, the device further comprises a generator for generating pressure waves, particularly acoustic waves, more particularly ultrasound waves, wherein said generator is configured to transmit said pressure waves to said sample holder, particularly to said sample volume, such that said pressure waves are transmittable to a tissue sample arranged in said sample volume of the sample holder. Pressure waves, particularly ultrasound waves, advantageously further improve clearing of the tissue sample and its accessibility to staining preparations as described above in relation to the first aspect.

In particular, the generator is configured to generate pressure waves, particularly ultrasound waves, parallel to the first axis or perpendicular to the first axis (in other words, the generated pressure waves propagate along the first axis or perpendicular to the first axis).

In certain embodiments, said generator is configured to generate waves in the range of 200kHz to 5MHz, particularly in the range of 1 to 2 MHz. This frequency range was determined by the inventors to render particularly advantageous results. The indicated frequency range however is not to be construed to limit the scope of the invention.

In certain embodiments, the sample holder comprises a third opening and a fourth opening configured to be brought in fluid communication with said sample volume, particularly wherein said third opening and said fourth opening are arranged along a first axis and said first opening and said second opening are arranged along a second axis which is non-parallel to the first axis, more particularly wherein said first axis is perpendicular to said second axis. In particular, the device further comprises a third electrode and a fourth electrode, wherein the third electrode is configured to be brought in electrically conductive contact with a first electrolyte, contained in said first electrolyte chamber or a third electrolyte chamber, and wherein said fourth electrode is configured to be brought in electrically conductive contact with a second electrolyte, particularly contained in the second electrolyte chamber or a fourth electrolyte chamber, said third electrode and said fourth electrode being configured to be connected to a voltage source. In particular, the third electrode and the fourth electrode are configured to generate an electric field perpendicular to the first axis (in other words, the field vectors of the electric field are oriented perpendicular to the first axis) and/or perpendicular to the direction of the electric field generated by the first and the second electrode. For example, it is possible to perform electrophoresis in two directions (in the direction of the first and the second opening and in the direction of the third and fourth opening) using such a sample holder.

In certain embodiments, the sample volume is configured to be brought in fluid communication with a third and/or fourth electrolyte chamber, particularly wherein the third electrode chamber is comprised in the device, by means of the third opening and the fourth opening.

In certain embodiments, the sample holder defines a first compartment, a second compartment, a third compartment and/or a fourth compartment adjacent to the sample volume, wherein said first opening leads to said first compartment, said second opening leads to said second compartment, said third opening leads to said third compartment, and said fourth opening leads to said fourth compartment, particularly wherein said first compartment, said second compartment, said third compartment and/or said fourth compartment is delimited by a respective conical inner wall tapering towards said sample volume.

That is, the conical inner wall of the first compartment tapers from the first opening towards the sample volume, the conical inner wall of the second compartment tapers from the second opening towards the sample volume, the conical inner wall of the third compartment tapers from the third opening towards the sample volume, and/or the conical inner wall of the fourth compartment tapers from the fourth opening towards the sample volume. This can allow the inserting of staining preparation in the form of a pre-cast hydrogel conus that fits into the opening.

In certain embodiments, the first opening, the second opening, the third opening and/or the fourth opening comprised in the sample holder are conical. This can allow the inserting of staining preparation in the form of a pre-cast hydrogel conus that fits into the opening.

In certain embodiments, the first compartment, said second compartment, said third compartment and/or said fourth compartment is configured to receive, particularly hold, a hydrogel preparation, particularly from a non-crosslinking hydrogel, comprising a staining preparation.

In certain embodiments the sample holder is separable into two parts, a first part comprising the first opening and a second part comprising the second opening, with the sample volume being confined or defined by the first and second part, wherein particularly the first part comprises the third opening and the second part comprises the fourth opening or the first part comprises the fourth opening and the second part comprises the third opening. .

In certain embodiments, the first part and the second part are configured to be connected at a connection surface, wherein a surface normal of the connection surface (being perpendicular to the connection surface) is non-parallel to the first axis and the second axis (along which the sample holder extends), particularly wherein the surface normal is arranged at an angle of 45° with respect to the first axis and the second axis.

In certain embodiments,

- said first electrolyte chamber comprises an axial opening configured to be brought in fluid communication with said first opening of said sample holder and said second electrolyte chamber comprises an axial opening configured to be brought in fluid communication with said second opening of said sample holder, and wherein

- said first electrolyte chamber comprises a first section extending along a first axis between said axial opening of the first electrolyte chamber and a lateral wall of the first electrolyte chamber and a second section adjacent to said first section, wherein said second section extends along a second axis perpendicular to said first axis from said first section to a top opening of said first electrolyte chamber, particularly wherein said first electrolyte chamber is L-shaped when viewed in a cross-sectional plane defined (or held) by the first axis and the second axis, and/or

- wherein said second electrolyte chamber comprises a first section extending along said first axis between said axial opening of the second electrolyte chamber and a lateral wall of the second electrolyte chamber and a second section adjacent to said first section, wherein said second section extends along a third axis perpendicular to said first axis from said first section to a top opening of said second electrolyte chamber, particularly wherein said second electrolyte chamber is L-shaped when viewed in a cross-sectional plane defined (or held) by the first axis and the third axis. Advantageously, such a device may be used to generate a plug or barrier from a cross-linking hydrogel in the first section of the respective electrolyte chamber, and dispose electrophoresis buffer in the second section. In particular, it is possible to embed a noncrosslinking hydrogel comprising a staining preparation within such a cross-linking hydrogel in an easy manner using the described device.

In certain embodiments, the first electrolyte chamber is comprised in a first block comprising a first half shell and a second half shell, wherein said first half shell and said second half shell jointly form said first electrolyte chamber when the first half shell and the second half shell are assembled, and/or wherein said second electrolyte chamber is comprised in a second block comprising a first half shell and a second half shell, wherein said first half shell and said second half shell jointly form said second electrolyte chamber when the first half shell and the second half shell are assembled Such a device is easy to disassemble after use, particularly to remove a cross-linking hydrogel and clean the components.

In certain embodiments, the sample holder comprises a first membrane and a second membrane, wherein said tissue sample is arrangeable between said first membrane and said second membrane.

In certain embodiments, the sample holder comprises a bottom part configured to be placed in a liquid reservoir, particularly a second electrolyte chamber, such that said first membrane, said tissue sample and said second membrane are arranged, particularly stacked, in a vertical direction, particularly wherein said sample holder extends along a first axis (extending in the vertical direction during the preferred operational mode of the device).

In certain embodiments, the bottom part comprises a ring-shaped base extending in the circumferential direction in respect of the first axis, wherein said base defines a space.

In certain embodiments, said base comprises at least one recess configured to allow entry of said second electrolyte from said liquid reservoir into said space via said recess. In certain embodiments, the bottom part comprises an end face limiting the space defined by the base along the first axis, wherein the end face has a conical shape, particularly with the end of the conus pointing towards the base. This advantageously prevents accumulation of gas bubbles generated during electrophoresis (e.g. by electrolysis of buffer components) below the first membrane.

In certain embodiments, the sample holder is a hollow tube.

In certain embodiments, the sample holder is separable into two parts, a first part comprising the first opening and a second part comprising the second opening, particularly wherein the sample volume is confined or defined by the first part and the second part. In particular, the first part further comprises the third opening and the second part further comprises the fourth opening, or the first part further comprises the fourth opening and the second part further comprises the third opening.

In certain embodiments, the device further comprises at least one hollow microneedle, particularly a microneedle having a diameter < 1/100 of a diameter of the sample volume (more particularly a diameter of 20 pm to 150 pm), said microneedle being configured to be inserted into said sample volume such that a tissue sample arranged in the sample volume is penetrable by said hollow microneedle, particularly such that a staining preparation is injectable into said tissue sample by said microneedle. The injection can be effected by a micropump delivering a preset amount of staining preparation volume into the sample at a particular position of the sample. Alternatively, or in addition to the action of the micropump, the hollow microneedle(s) comprise a microneedle electrode capable of applying an electrical potential difference between a tip of said microneedle and said sample volume of the sample holder or relative to said first and/or second electrode.

In certain embodiments, the hollow microneedle comprises a microneedle electrode capable of applying an electrical potential difference between a tip of said microneedle and said sample volume of the sample holder or relative to said first electrode and/or said second electrode. In particular, the microneedle electrode is electrically connectable to a voltage source, such that an electric potential can be applied to the microneedle electrode. A hollow microneedle as used herein is a needle having a diameter between 1 pm and 999 pm (particularly 20 pm to 150 pm) and comprising a hollow inner volume. A microneedle electrode as used herein is an electrode that is placeable within the hollow inner volume of a microneedle.

In certain embodiments, the device further comprises a third electrode and a fourth electrode, wherein the third electrode is configured to be brought in electrically conductive contact with a first electrolyte, particularly contained in said first electrolyte chamber or a third electrolyte chamber, and wherein said fourth electrode is configured to be brought in electrically conductive contact with a second electrolyte, particularly contained in the second electrolyte chamber or a fourth electrolyte chamber, said third electrode and said fourth electrode being configured to be connected to a voltage source. In particular, the third electrode and the fourth electrode are configured to generate an electric field perpendicular to the first axis (in other words, the field vectors of the electric field are oriented perpendicular to the first axis) and/or perpendicular to the electric field generated by the first and second electrodes.

In certain embodiments, the device comprises a temperature sensor connected to a voltage source or controller in order to allow adjustment of the voltage between the first and the second electrode to keep the temperature between 37°C to 42°C, particularly from 38,5°C to 40,5°C.

In certain embodiments, the device comprises a recirculatory apparatus configured to circulate and/or exchange the electrolyte solution comprised in the first and/or second electrolyte chamber (particularly the first and/or second electrolyte solution) during application of an electric potential difference between the first and the second electrode.

A third aspect of the invention relates to a sample holder for receiving, particularly holding, a tissue sample, particularly for a device according to the second aspect of the invention.

The sample holder is made of an electrically insulating material and defines a sample volume configured to receive, particularly to hold, a tissue sample, particularly a tissue sample embedded in a hydrogel, within the sample holder, the sample holder further having a first opening and a second opening with the sample volume disposed between the first opening and the second opening, wherein the first opening is configured to be brought into fluid communication with a first electrolyte chamber and the second opening is configured to be brought into fluid communication with a second electrolyte chamber.

In certain embodiments, the sample holder comprises a third opening and a fourth opening configured to be brought in fluid communication with said sample volume, particularly wherein said third opening and said fourth opening are arranged along a first axis and said first opening and said second opening are arranged along a second axis which is non-parallel to the first axis, more particularly wherein said first axis is perpendicular to said second axis. In particularly, the third opening is configured to be brought in fluid communication with the first electrolyte chamber or a third electrolyte chamber, and the fourth opening is configured to be brought in fluid communication with the second electrolyte chamber or a fourth electrolyte chamber.

In certain embodiments, the sample volume is configured to be brought in fluid communication with a third electrolyte chamber, particularly wherein the third electrode chamber is comprised in the device, by means of the third opening and the fourth opening.

In certain embodiments the sample holder is separable into two parts, a first part comprising the first opening and a second part comprising the second opening, with the sample volume being confined or defined by the first and second part, wherein particularly the first part comprises the third opening and the second part comprises the fourth opening or the first part comprises the fourth opening and the second part comprises the third opening.

In certain embodiments, said base comprises at least one recess configured to allow entry of said second electrolyte from said liquid reservoir into said space via said recess.

In certain embodiments, the bottom part comprises an end face limiting the space defined by the base along the first axis, wherein the end face has a conical shape, particularly with the end of the conus pointing towards the base.

In certain embodiments, the sample holder comprises a temperature sensor connected to a voltage source or controller in order to allow adjustment of the voltage between the first and the second electrode to keep the temperature between 37°C to 42°C, particularly from 38,5°C to 40,5°C.

Specific examples of the invention are described below with reference to the figures, which are meant to illustrate the invention, but not to limit its scope.

FIGS.1 and 2 show example embodiments of a system (elsewhere in this specification also referred to as‘device’) 100 that can be used for tissue clarification via sono-electro-osmosis, as described herein. Embodiments of the system 100 can be configured to perform focused electrophoresis on a tissue sample, for application of acoustic or pressure waves on the tissue sample, or a combination of both in the form of sono-electro-osmosis. Some embodiments, of the system 100 described above can be configured for tissue clarification, for tissue-lipid extraction, and/or for molecular interrogation. Some embodiments of the system 100 can be configured to perform enzyme application on the tissue sample, to treat the tissue sample, for example to digest certain molecules present in the sample, to wash the tissue sample or to label certain molecules or structures (such as by staining). In some instances, certain molecules can be introduced into the tissue sample using some embodiments of the system 100 and the introduced molecules can then be treated using some embodiments of the system 100.

The system 100 includes a chamber 103 that includes a first chamber portion 103A (elsewhere in this specification referred to as‘first electrolyte chamber’), a second chamber portion 103B (elsewhere in this specification referred to as‘sample volume’ 121 , 221 , 321 , 421 , 521 , 621 , 721 , 821 ) and a third chamber portion 103C (elsewhere in this specification referred to as‘second electrolyte chamber’). The different portions of the chamber 103 can be defined by one or more fixed or removable structures, such as partitioning or fixture plates 107A, 107B. The second chamber portion 103B is configured to removably hold a sample holder 102 that includes a tissue sample101 , or the second chamber portion 103B may be a sample volume 121 comprised in or arranged within the sample holder 102. The first chamber portion 103A is configured to hold a first buffer 1 10A (elsewhere in this specification referred to as‘first electrolyte solution’ in contact with the tissue sample 101 and the third chamber portion 103C is configured to hold a second buffer 1 10B (elsewhere in this specification referred to as‘second electrolyte solution’ in contact with the tissue sample 101 . The system 100 further includes a set of electrodes that can include groups of electrodes 104A, 104B, each within the first 103A and third 103C chamber portions of the chamber 103 respectively. The group of electrodes 104A and 104B are coupled to the chamber 103, positioned with respect to the sample holder 102, and configured to apply an electric field to interact with the sample 101 , to induce electrophoresis in a focused manner. The electrodes 104A, 104B and the sample holder 102 are configured such that during application of an electric field E, between the first electrode 104A and the second electrode 104B, the electric field lines of field E, and the electric current, pass only through the sample 101 in the second chamber portion 103B or sample volume 121.

In some embodiments, the system 100 can include a controller coupled to the set of electrodes 104A, 104B. The controller (not shown in FIG.1 and FIG. 2) can be configured to activate one or more electrodes of the set of electrodes 104A, 104B. For example, in some instances the set of electrodes 104A and 104B can further include groups of electrodes. The controller can be configured to selectively activate each electrode of the group of electrodes of the set of electrodes, such that the activated electrode can function as an anode. In other instances, the controller can activate each electrode in the group of electrodes of the set of electrodes such that the electrode can function as a cathode. In some instances, the controller can be configured to activate a group of electrodes concurrently to function as anodes or cathodes. The controller can also be configured to transiently change the polarity of individual electrodes or groups of electrodes such that each electrode or each group of electrodes can alternatively or sequentially function as an anode and then as a cathode, such that the direction of the applied electric field vector can be changed. In other words, the controller can activate a first electrode or a first group of electrodes to be anodes, and a second electrode or a second group of electrodes to be cathodes at a first time. The controller can be configured to activate the first electrode or first group of electrodes to be cathodes and the second electrode or second group of electrodes to be anodes at a second time, reversing the polarity.

Changing the direction or spatial distribution of the electrical field applied to the sample can help to overcome less than ideal homogenicity in the effect of clearing, washing and staining of the sample.

The system 100 further includes a transducer 106 (elsewhere in this specification referred to as ‘generator for generating pressure waves’), coupled to the second portion 103B of the chamber 103 and configured to apply a pressure wave signal (e.g. a pulsed wave or a continuous wave) to the tissue sample 101 held by the sample holder 102. In some instances, the pressure wave can be an ultrasonic wave of a predetermined frequency of set of frequencies, with the transducer 106 being an ultrasonic transducer. In some instances, the pressure wave can be an acoustic wave with the transducer 106 being an acoustic transducer. In some instances, the acoustic pressure wave can be a standing wave.

The chamber 103 is configured to hold a conductive buffer or a system of buffers 1 10A, 1 10B, surrounding the group of electrodes 104A, 104B in the first portion 103A and the third portion 103C of the chamber 103. In some embodiments, different buffers can be used in the different portions 103A-103C of the chamber 103. For example, some embodiments of the system 100 can use a first buffer in the portion of the chamber 103 housing the anode (e.g., the portion 103A) and a second buffer, different from the first buffer, in the portion of the chamber 103 housing the cathode (e.g., the portion 103C). In some embodiments, any of the buffers described herein, and generally referred to as the buffer 1 10 (elsewhere in this specification referred to as ‘electrolyte solution’) for simplicity, can be a detergent solution containing ionic and non-ionic detergents (e.g., an SDS-containing lipid-removal buffer) that can be recirculated through two independent circulation routes using suitable recirculatory apparatus 1 12, as indicated by the dashed arrows in FIG. 1. In some embodiments, any of the buffers disclosed herein can be useful for preparing biological specimen as generally disclosed in the Ό66 application. The system 100, in some embodiments, can include additional support and/or positioning features to position and/or hold the exchangeable capsule 105, such as, for example, positioning posts 1 13 indicated in FIG. 1 .

The sample holder 102 in the second portion 103B of the chamber 103 of the system 100 can be configured to be a portion of a removable, or standard exchangeable capsule 105. The sample holder 102 includes a first opening 1 17A (not visible in FIG. 2), to interface with the first buffer 1 10A in the first chamber portion 103A of the chamber 103, and a second opening 1 17B to interface with the second buffer 1 10B in the second chamber portion 103C. In some embodiments, the sample holder 102 or the system 100 (device 100) can include positioning rings 108A (not visible in FIG. 2) and108B, at or near the openings 1 17A, 1 17B defined on the sample holder 102. The positioning rings 108A, 108B can be configured to engage with the openings 1 17A, 1 17B of the sample holder 102. In some embodiments, the sample holder 102 can be configured and/or structured such that it limits the amount of conductive buffer 1 10A, 1 10B surrounding the tissue sample 101. In such a configuration the detergent ions carrying the electric current, applied through the set of electrodes 104A, 104B, are forced to flow through the tissue sample 101 , significantly enhancing the clarification process. Such a configuration also reduces the amount of buffer 1 10A, 1 10B consumed by electrophoresis due to relatively lower electric field, voltage, or current values. In such a configuration, with the tissue sample 101 being the only conducting medium in the second portion 103B of the chamber 103, tissue clarification and/or staining can be carried out with the use of (up to) ten-fold lower current compared to other available electrophoretic methods, avoiding potential tissue deformation and damage.

The sample holder 102 can be configured to hold biological or engineered tissue sample 101 which may be in any suitable form, such as thin-slice format, whole-mount format, in medical biopsy or punch format, and/or the like. In some embodiments, the sample 101 may be tissue prepared in a hydrogel or hydrogel matrix to preserve tissue structure. In some instances, the tissue sample 101 can be prepared using one or more procedures such as formaldehyde fixation, hydrogel embedding of formaldehyde fixed tissue, and combined formaldehyde and hydrogel fixation. During preparation of the sample 101 , such procedures may be applied on fresh tissue or on tissue already processed such as, for example, formalin-fixed-paraffin- embedded (FFPE) tissue. In some instances, the above described method(s) can be used on frozen tissue to prepare the sample 101. In some instances, the sample 101 can be associated with a unique identifier that can be generated and stored in a storage medium in a suitable form, such as, for example a look up table. The unique identifier can be associated with the sample 101 throughout the procedure involved in processing the sample 101. For example, the unique identifier can be a unique bar-code or a unique Quick Response (QR) code that can be read by a corresponding reader. In some instances, the sample 101 can be acquired (e.g. in an operating theater or at a clinic) and the container holding the sample 101 can be affixed with the unique identifier. In some embodiments, the sample is disposed in a sample holding chamber as described herein, and the unique identifier is associated with the sample holding chamber. In this manner, each sample can be uniquely tracked before, during, and/or after processing with the system 100.

The electrodes 104A, 104B can be platinum electrodes used to form an anode and a cathode, each housed within the first portion 103A and the third portion 103C of the chamber 103, respectively. The sample holder 102 and the electrodes 104A, 104B can be collectively configured such that each of the electrodes 104A, 104B is disposed about 5 - 50 mm from the nearest surface of the sample 101 . In some embodiments, the electrodes 104A, 104B can be separated from the openings 1 17A, 1 17B of the sample holder 102 by the positioning of fixture plates 107A, 107B that can include a grid patterned cross-section. In some embodiments, each electrode of the set of electrodes 104A and 104B can be configured to have the same surface area or a substantially matching surface area as the portion and/or surface of the tissue sample 101 facing the electrode.

The electrodes 104A, 104B can be connected to a power source or voltage source 1 1 1 , for example a DC power supply (not shown in FIGS. 1 , 2). In some embodiments, the system 100 and/or the power source can be configured to energize one or more pairs of electrodes such that two or more systems that are structurally and/or functionally similar to the system 100 may be run in parallel. In some instances, the two or more systems running in parallel may operate on the same sample. In some other instances, the systems running in parallel may operate of separate samples. In such embodiments, the group of electrodes of the two or more systems (similar to group of electrodes 104A and 104B of the system 100) may be switched, interleaved or run in parallel in any suitable manner, such as by a processor such as a microcontroller (not shown). While the system 100 in FIGS. 1 and 2 is illustrated to have one chamber 103, a system used for high throughput tissue clarification via sono-electro-osmosis, as described herein, can include several chambers that are parallelized with each other and configured to be run in parallel. In some embodiments, the several chambers that are in parallel can be configured to be individualized in terms of isolation of samples and one or more buffers. For example, samples isolated in parallel but individualized chambers can be efficiently run in parallel while reducing or eliminating cross-contamination between the isolated samples, or the buffers, staining agents, enzymes or other components used in association with each of the isolated or individualized chambers.

The power source may be a bipolar source whose polarity may be reversed. In some embodiments, for example, the power source can be operated at 30-1000 mA in the constant current (CC) mode. In the CC mode, the system 100 can be configured such that the applied voltage is between about 20-200 V, including all values and subranges in between. In some other embodiments, the power source may be operated at 5-500 V in a constant voltage (CV) mode. In some embodiments, the power source may be operated for a predefined time set, for example, by a timer and/or clock. As an example, the power source may be programmed to operate for a predefined time period of 10-1000 minutes, for more than 1000 minutes, and/or the like.

The buffer (or electrolyte solution) 1 10 can be one or more buffers, which can be detergent solutions containing ionic and/or non-ionic detergents that can be recirculated through two independent circulation routes using suitable recirculatory apparatus 1 12, as described above. The buffer(s) 1 10 (e.g. buffer 1 10A, and/or buffer 1 1 OB) can include ionic and non-ionic detergents. In some instances, the buffer(s) 1 10A, 1 10B can include a buffer system, a lead ion and a terminating ion, such as (for example) tris (e.g. trisphosphate, EDTA, etc.,), chloride-ion and tricine (or glycine) respectively. In some embodiments, the buffer 1 10A, 1 10B can be configured to allow isotachophoresis, or the selective separation or concentration of ionic analytes such as during tissue staining with molecular dyes.

The first portion 103A and the third portion 103C of the chamber 103 can include inflow or inlet ports 1 15A, 1 15B and outflow or outlet ports 1 16A, 1 16B that can be suitably configured to engage with the recirculatory apparatus 1 12 though suitable devices such as tubing or other connections. For example, the inlet port 1 15A and the outlet port 1 16A in the first chamber portion 103A can be used to recirculate a first buffer 1 10A housed in the first chamber portion 103A, and the inlet port 1 15B and the outlet port 1 16B included in the third chamber portion 103C can be used to recirculate a second buffer 1 10B housed in the third chamber portion 103C. In some embodiments, the inflow port can be limited to one central port directly flushing or irrigating the sample in the second chamber portion 103B, while the outflow ports are two separate ports disposed in 103A and 103C.

The system 100 can be configured such that the buffer 1 10A, 1 10B flows at a rate of about 10- 100 ml/min, including all values and sub ranges in between. In some embodiments, the outflow ports 1 16A, 1 16B can be configured to be positioned adjacent or proximal to the group of electrodes 104A, 104B respectively. For example, as shown in, FIG. 1 , the outflow port 1 16B can be configured to have its opening adjacent to the position of the electrode 104B. In some instances, the outflow ports 1 16A, 1 16B can be configured to remove buffer from near the electrodes with consideration of external forces such as gravity. Each pair of inflow and outflow ports in the first and third portions of the chamber 103 can be operated independent of the other using any suitable pumping mechanism. For example, the pair of ports 1 15A, 1 16A in the first portion 103A can be operated by a pump (not shown) independent of the pump operating the pair of ports 1 15B, 1 16B in the third portion 103C. In some embodiments, the system can have fluid communication between the chamber portions. For example, in some embodiments, the system can include one inlet port to direct flow of a buffer into one chamber portion and a pair of outflow ports to direct flow of the buffer or vice versa i.e., the system can include two inlet ports in two respective chamber portions and one outlet port within one chamber portion. For example, in some embodiments, the system can include one inlet port in the second chamber portion and two outlet ports in the first and second chamber portions such that a buffer can flow into the second chamber portion and flow out of the first and second chamber portions.

The system 100 can be further configured to maintain the recirculated buffer 1 10 at a temperature between about 4-80°C, including all values and sub ranges in between. In some embodiments, the recirculated buffer 1 10 can be maintained at temperature range between about 4-80°C, including all values and sub ranges in between. In some embodiments, such as embodiments configured to allow isotachophoresis, the system 100 can be configured to maintain the buffers 1 10A and/or 1 10B at a pH between about 4-10, including all values and sub ranges in between. In some embodiments, the system 100 can be configured such that portions of the chamber 103 and/or the buffer 1 10A, 1 10B can be maintained with a pH gradient and/or an osmotic gradient.

The transducer 106 of the system 100 can include one or more transducers that are configured to be in contact with the buffer 1 10A, 1 10B in one or more portions (e.g. the second portion 103B) of the chamber 103. In some instances, the transducer 106 can be positioned such that the operational end of the transducer 106 faces the sample holder 102 housing the tissue sample 101 . In some embodiments, the transducer 106 can be configured with respect to other components in the system 100 such that the electrodes 104A, 104B, and the sample holder 102, such that the pressure waves applied by the transducer 106 travel and act upon the sample 101 in the sample holder 102 in a direction parallel to the direction of flow of current due to an electric field generated by the electrodes 104A, 104B. In some other embodiments, the transducer 106, the sample holder 102 and/or the electrodes 104A, 104B can be configured such that the pressure waves are applied in a specific direction with respect to the direction of the electric field (e.g. orthogonal direction to the electric field vector of the applied electric field). For example, as best illustrated in FIGS. 4, 8, the pressure waves may be applied in a direction orthogonal to the electric field vector. In some embodiments, the system 100 can be configured such that a rotary component may be employed to hold and or manipulate the transducer 106 such that acoustic pressure waves can be generated to bombard the sample 101 along different axes. In some embodiments, acoustic pressure waves along various axes can be used to bombard the sample serially. In some other embodiments, rotary systems can be used to hold and manipulate two or more transducers such that acoustic pressure waves can be used to bombard the sample along various axes, substantially simultaneously. The transducer 106 can be configured such that it can be operated in a standalone manner, or in parallel with the electrodes 104A, 104B configured to perform electrophoresis. In some instances, the acoustic or pressure waves generated by the transducer 106 may be applied on the sample 101 simultaneously with the electric field applied on the sample by the electrodes 104A, 104B. In some other instances, the acoustic or pressure waves from the transducer 106 may be applied in an interleaved manner with the electric field from the electrodes 104A, 104B. In some instances, the pressure waves from the transducer 106 can be applied on the sample 101 in an overlapping manner in time, with the electric field from the electrodes 104A, 104B.

The transducer (or generator) 106 can be configured to produce standing waves such as acoustic pressure waves directed at the sample 101 with suitable values of intensity, frequency, and/or the like. The pressure waves may be applied in a pulsed wave form, or in a continuous wave form, or a combination thereof. For example, the transducer 106 can in some embodiments be configured to produce continuous pressure waves in the frequency range of about 40-4000 KHz, including all values and sub ranges in between. In some instances, the pressure waves can be generated as standing waves.

Particularly, in the example shown in FIG. 1 , the system 100 or device 100 comprises a part 1 18 comprising a bore 1 19 extending along a first axis A1 and defining the chamber 103. For better visibility of the components, only half of the first part 1 18A (cut along the first axis A1 ) is depicted in FIG. 1 . The first electrode 104A and the second electrode 104B are shaped as spirals extending parallel to each other on each side of the sample holder 102 in a plane perpendicular to the first axis A1. Thus, the electric field resulting from an electric potential difference between the first electrode 104A and the second electrode 104B is oriented parallel to the first axis A1.

In particular, the exchangeable capsule 105 comprising the sample holder 102 (and the tissue sample 101 ) can be inserted into the second chamber portion 103B along a second axis A2 perpendicular to the first axis A1 (in a radial direction in respect of the first axis A1 ), from above in the examples shown in FIG. 1 and FIG. 2.

The transducer or generator 106 is particularly oriented perpendicular to the second axis A2 below the second chamber portion 103B, such that pressure waves, particularly ultrasound waves, generated by the transducer (generator) 106 travel along the second axis A2 towards the sample holder 102 (and the tissue sample 101 ).

Furthermore, the inlet ports 1 15A, 1 15B described above are particularly oriented in an axial direction in respect of the first axis A1 , and the outlet ports 1 16A, 1 16B described above are particularly oriented in a radial direction in respect of the first axis A1. Of course, the inlet ports 1 15A, 1 15B and outlet ports 1 16A, 1 16B may also be arranged in any other suitable orientation.

In particular, the system or device 100 shown in Fig. 2 comprises a cross-shaped part 18 comprising a first section 1 18A extending along the first axis A1 and comprising a bore 1 19A forming the chamber 103, a second section 1 18B and a third section 1 18C, wherein the second section 1 18B and the third section 1 18C are arranged along a third axis A3 perpendicular to the first axis A1 . Particularly, the second section 1 18B and the third section 1 18C each comprise a respective groove 1 19B, 1 19C extending along the second axis A2. Particularly, the positioning posts 1 13 each comprise a respective (upper) clamp portion 1 13A configured to engage a plate portion 105A of the exchangeable capsule 105 and a respective (lower) bolt portion 1 13B configured to be inserted into a respective groove 1 19B, 1 19C of the second or third section 1 18B, 1 18C of the part 1 18. Therein, in particular, the plate portion 105A, to (the bottom of) which the cylindrical sample holder 102 is attached is configured to close the chamber 103. Particularly, the exchangeable capsule 105 further comprises a handle 105B for manual manipulation of the capsule 105.

In embodiments of the system 100 that are configured for isotachophoresis of molecular dyes, the system 100 can be configured to apply one or more molecular stains or dyes that have an electric charge. The dyes or stains can include, but are not limited to, protein, nucleic acid, luminescent conjugated oligothiophenes and polythiophenes, amyloidotropic dyes such as Thioflavin T and its derivatives, protein-fluorochrome and nucleic-acid-fluorochrome conjugates, Forster-Resonance-Energy-Transfer (FRET)-compatible fluorochromes, or small molecule dyes targeting one or more nucleic acids or proteins. In some embodiments, as disclosed previously, the system 100 can be configured to apply one of more enzymes to the sample or treat the sample with one or more enzymatic agents (e.g. BOLORAMIS, protein digestion). The system 100 is configured such that the electric field applied by the electrodes 104A, 104B or the pressure or acoustic waves applied by the transducer 106, or both can interact with the dyes or stains or the enzymes. For example, when used together (e.g. concurrently or in an interleaved manner) the acoustic waves can be configured to transiently or temporarily increase porosity of the sample tissue 101 allowing better or enhanced access of tissue structures by the charged stains or dyes controlled by a directional electric field.

In some instances, the charged dyes and/or the enzymes (as applicable to specific embodiments) can be in dissolved in the buffer 1 10 contained in one or more portions of the chamber 103, such as the first portion 103A or portion 103C. In some instances, the charged dyes and/or enzymes can be entrapped in a non-crosslinking gel positioned suitably with respect to the sample 101 in the sample holder 102. In some instances, the non-crosslinking gel entrapping the dyes can be cast on a portion of the surface of the sample 101 , for example the portion of the surface of sample 101 facing one of the portions of the chamber 103. In some instances, the non-crosslinking gel entrapping the dye and/or enzyme can be encapsulated and the capsule containing the gel entrapping the dye and/or enzyme can be included in at least one portion of the chamber 103.

In some embodiments, apparatuses and methods described herein include application of molecular dyes that are embedded and concentrated in a non-crosslinking hydrogel medium at one side of the sample. The embedding of concentrated dyes allows for focused electrophoretic mobilization of the dyes, and for a significant reduction in the amount of dye- used. The positioning of the dyes can be modified based on the properties of the dye and the properties of the system 100 such as the polarity of the electrodes 104A and 104B. For example, if a dye carries a net negative charge, the dye may be placed in the portion of the chamber 103 which also includes the cathode of the electrodes 104A, 104B. As another example, if a dye carries a net positive charge the dye may be placed (in the buffer or in an encapsulated form) in the portion of the chamber 103 that includes the anode of the electrodes 104A, 104B.

Embodiments of the system 100 described above can be configured to perform tissue clarification, treatment of tissue samples with enzymatic agents, or incorporation of labels or dyes targeting specific molecules or structure in the tissue sample, in any order, to transform the tissue sample into a transparent or semi-transparent or translucent tissue sample carrying molecular labels and modifications of interest. Such transformed tissue samples may be then used for high quality high throughput volumetric microscopic imaging as needed for applications like medical diagnostics, scientific research, education and entertainment, etc. The use of the disclosed system for the above mention or other uses can significantly improve efficiency and throughput in operation. For example, systems can be configured to advance process times for clearing three-dimensional biological tissue from a matter of weeks to a few hours.

In some embodiments, a set of systems, with each system being similar to the system 100, can be run and independently operated in parallel for processing multiple tissue samples as described herein. In such embodiments, at least two systems of the set of systems can be operated with an independent set of reagents for the clarification and/or staining of its tissue sample. In this manner, cross-contamination between different tissue samples can be avoided. Additionally, in some embodiments, at least two systems of the set of systems can carry out the clarification and/or staining processes under different operating conditions (e.g., different duration of treatment with enzymatic reagent). When combined with the tissue sample and/or the sample holding chamber having a unique identifier as described herein, aspects of these embodiments are useful for continued sample tracking and independent treatment using multiple versions of the system 100 operating in parallel.

FIG. 3A shows a perspective view of an example sample holder 202 that can be used with the system 100 and/or the system 900. The sample holder 202 can be structurally and/or functionally similar to the sample holder 102. In some embodiments, the sample holder 202 can constructed of any suitable inert material and assembled from two or more components. FIGS. 3B-3D illustrate various embodiments of the sample holder 202 being assembled, receiving a sample 201 cast in paraffin, and receiving one or more dyes embedded in a hydrogel medium 223, respectively. As best illustrated in FIG. 3B, in some embodiments, the sample holder 202 can be assembled from sample holder portions 202A and 202B having one or more mating features. For example, the portions 202A and 202B can each have cones 222 and 224 that form a pair of cones. The cones 222 and 224 of the cone pair can be configured such that a longitudinal axis of each cone and/or of the assembled cone pair can be orthogonal to either the axis of application of one or more electric fields or to the axis of application of pressure waves. The system 100 can be also be configured in some embodiments such that a longitudinal axis of one set of cones (e.g. cones 222 of the parts 202A and 202B) can be orthogonal to an applied directional electric field while being parallel or in-axis with pressure waves applied from the transducer 106.

The sample holder 202 can define openings 217A, 217B, 217C, 217D (openings 217A, 217C and 217D being visible in FIGS 3A-3D), and a designated space 221 defined by the assembled parts 202A, 202B such that the space 221 can hold the tissue sample 201 upon assembly. The sample holder 202 can be made from any of a variety of parts to form the sample holder 201 , for example to suit samples of different sizes or shapes. The sample holder 202 isolates the region occupied by the tissue sample 201 from the buffer-containing portions such that, during operation, the tissue sample 201 provides the only electrically conductive path for the flow of electric current due to the application of electric field by electrodes in the sono-electro-osmosis system housing the sample holder 202.

During preparation, in some instances, the tissue sample 201 can be embedded in a conductive medium. In some instances, the tissue sample 201 can be embedded in a suitable non- conductive, castable medium (e.g. the tissue sample 201 can be embedded in paraffin) using casting molds of various sizes and inserted into the space 221 of the sample holder 202, before, during or after assembly of the sample holder 202. FIG. 3C shows the sample holder 202 including a tissue sample 201 cast in paraffin and held in the space 221. In embodiments configured for staining of the sample tissue 201 , the sample holder 202 can further include the dye/stain embedded in a hydrogel matrix 223 (e.g. dye cast in low melting agarose) and placed at or near one or more of the openings defined on the sample holder 202, for example the openings 217A and 217D as shown in FIG. 3D. In some embodiments, prior to casting the dye, some tissue surface is exposed towards each cone in order to allow the stains and buffers to contact the tissue The embedded dye can be cast in one or both of the parts 202A, 202B forming the sample holder 202. The top image in FIG. 3D shows a fully assembled sample holder 202 with dyes and hydrogel cast in cones 217A and 217 D and the bottom image shows the same structure rotated by 180 degrees.

In particular, FIG. 3-5 show sample holders 202 comprising a sample volume 221 and a first, a second, a third and a fourth opening 217A, 217B, 217C, 217D configured to be brought in fluid communication with the sample volume 221 , wherein the third opening 217C and the fourth opening 217D are arranged along a first axis A1 , and the first and the second opening 217A, 217B are arranged along a second axis A2 perpendicular to the first axis.

Therein, the sample holder 202 defines a first compartment 225A, a second compartment 225B, a third compartment 225C and a fourth compartment 225D, wherein the first opening 217A leads to the first compartment 225A, the second opening 217B leads to the second compartment 225B, the third opening 217C leads to the third compartment 225C, and the fourth opening 217D leads to the fourth compartment 225D, wherein the first compartment 225A, the second compartment 225B, the third compartment 225C and/or the fourth compartment 225D is delimited by a respective conical inner wall tapering towards said sample volume 221 .

In particular, the first compartment 225A, the second compartment 225B, the third compartment 225C and/or the fourth compartment 225D (the first compartment in the embodiment shown in Fig. 3D) is configured to receive and hold a hydrogel preparation 223, particularly from a noncrosslinking hydrogel, comprising a staining preparation, in close proximity to the tissue sample 201 .

In use, the system 100 can be used to perform focused sonication, focused electrophoresis, or focused sono-electro-osmosis on a tissue sample, as described herein. In an example instance, a sono-electro-osmosis system (such as the system 100 described here) can be used by disposing a tissue sample in the removable sample holder, disposing the removable sample holder in the second chamber portion of the system, and flowing buffers in the first and third chamber portions of the system. For example, the first chamber portion of the chamber can be flowed with a first buffer, and the third chamber portion of the chamber of the system can be flowed with a second buffer. In some instances, the first and second buffers can be different and in some other instances the first and second buffers can be identical of the same buffer. The buffers can be flowed such that they bathe the electrodes positioned the first and third chamber portions of the system, and can be recirculated by the independent recirculatory systems that include separate inlet ports, outlet ports and /or pumps associated with the first buffer and the second buffer. The system can be powered suitably and used for sonication by applying pressure waves focused on the tissue sample in the sample holder. In some embodiments, the system can perform focused electrophoresis by applying one or more directional electric signals, on the sample in the sample holder, via the first electrode (or group of electrodes) disposed in the first chamber portion and the second electrode (or group of electrodes) disposed in the third chamber portion of the system. The electric field can be applied such that the electric field lines pass only or predominantly or a greater extent through the sample in the second chamber portion. In some instances, sonication and focused electrophoresis can be performed in an overlapping manner, that is the application of one or more directional pressure waves and the application of one or more directional electric fields (E1 , E2, etc.) can be substantially overlapped to be concurrently acting on the sample. In some other instances the pressure waves and the electric field(s) can be applied in an interleaved manner or in any suitable manner such that the pressure wave(s) and the electric field vectors collectively impact the sample for applications like tissue clarification, or lipid extraction, or tissue staining, or tissue interaction with enzymatic agents or the like. The pressure waves and the one or more electric fields can be configured to have specific direction vectors with respect to each other. For example, one of the electric fields (e.g. E1 ) can be co axial with a pressure wave (US) while another electric field (e.g. E2) can be orthogonal to the pressure wave US). In some instances, one or more buffers may be used to perform tissue clarification or lipid extraction from a tissue sample followed by a third or fourth buffer used to carry out tissue staining on the clarified tissue sample. The process of clarification and staining with separate buffer systems can also be alternated repeatedly in some instances.

In certain embodiments, the clearing step will be conducted with a buffer comprising significant amounts of chaotropic compounds (the ionic detergent) for clearing the sample. In order to effect staining with antibodies or other staining agents sensitive to the presence of such agents capable of disturbing or dissolving a tertiary peptide structure, thereby destroying for example the specificity of a staining antibody, the sample must be rinsed/washed to remove the chaotropic agent. This can be achieved in the washing step using a third and fourth buffer different from the first and second buffer, where the third and fourth buffer do not contain detergents or chaotropic agents. The properties of the pressure wave(s) and electric field(s) can be suitably configured for the process of tissue clarification and changed appropriately for the process of tissue staining. For example, a first set of pressure wave(s) of a particular intensity, frequency and/or direction, and a first set of electric field(s) of a suitable amplitude, and/or direction can be used for performing the lipid extraction. A second set of pressure wave(s) of suitable intensity, frequency and/or direction, and a second set of electric field(s) of a suitable amplitude, and/or direction can be used for performing the staining of the sample with suitable dyes or staining agents.

FIG. 4 shows the sample holder 202 during use in a system for sono-electro-osmosis, and specifically during the application of both an acoustic ultrasound wave US (e.g., generated by the transducer 106) as well as a directional electric fields E1 and E2, E1 being applied in parallel, E2 orthogonally relative to acoustic waves FIG.5 illustrates a variety of sample holders 302-802 (302, 402, 502, 602 702, 802) assembled from parts as described above in reference to the sample holder 202 shown in FIGS.3A-D, to incorporate samples of varying sizes and/or shapes, with respective spaces 321-821 (321 , 421 , 521 , 621 , 721 , 821 ) for holding the samples of varying sizes and/or shapes. FIGS. 3A-D illustrate various sample holder designs, along with a corresponding mold (303, 403, 503, 603, 703, 803) allowing for casting the samples in any suitable material such as, for example, paraffin.

FIG. 6 illustrates a system or device 900 for sono-electro-osmosis that can be structurally and/or functionally similar to the system or device 100. The system 900 includes a chamber 903 with a stationary chamber block 901 A and a mobile chamber block 901 B as indicated in FIG. 6. The stationary chamber block 901 A includes a sample holder 902, orthogonal electrodes and an electrode 904A that, in this example instance, is configured for operation as an axial cathode. The mobile chamber block also includes an electrode 904B that, in this example instance, is configured for operation as an axial anode. Furthermore, the mobile chamber block 901 B comprises a reflection plate 905 for reflection of a pressure wave generated by the transducer 906. The chamber 903 also includes O-rings 908A, 908B, fixture plates 907A, 907B and a transducer 906. In particular, as shown in FIG. 6, the first fixture plate 907A is configured as a separate part which is insertable into the stationary chamber block 901A. In particular, the transducer 906 is attached to the second fixture 907B and the transducer 906 and second fixture 907B form a further part that is insertable into the stationary chamber block 901 A.

In particular, the stationary chamber block 901 A extends along a first axis A1 , and the sample holder 902, the mobile chamber block 901 B and the transducer 906 are characterized by a cylindrical shape with a central cylinder axis and a circular cross-section perpendicular to the cylinder axis, wherein the sample holder 902, the mobile chamber block 901 B, and the transducer 906 are configured to be inserted into or arranged with the stationary chamber block, so that the respective cylinder axis is parallel to the first axis A1 . More particularly, the mobile chamber block 901 B, the sample holder 902 and the transducer 906 are configured to be arranged in the stationary chamber block in a coaxial arrangement. FIG. 7 shows a cross-sectional view of the chamber 903 after assembly, including suitable electrode ports for mounting electrodes (e.g. platinum electrodes) such that relative to the propagation of the acoustic waves the electric field is orthogonal or axial, via the orthogonal or axial electrodes, respectively.

According to the embodiment shown in FIG. 7, the components of the system or device 900 are arranged, particularly coaxially, along the first axis A1 in the stationary chamber block 901A in the following arrangement (viewed from left to right in FIG. 7): first fixture plate 907A, mobile chamber block 901 B, sample holder 902, O-ring 908B, transducer 906, second fixture plate 907B. The fixture plates 907A, 907B exert a force on the arrangement in the axial direction in respect of the first axis A1 , in particular to tightly couple the transducer 906 to the O-ring 908B.

FIG. 8 illustrates the system 900 in operation, during the application of a pressure wave US, particularly an acoustic ultrasonic wave US, and directional electric fields E1 and E2, upon the sample 901 held in the sample holder 902 (cast in paraffin as described previously), with a gel embedded dye positioned in the sample holder 902. As indicated, voltage is applied through electrodes engaging with the chamber block via the electrode ports illustrated in FIG. 7.

Therein, as shown in FIG. 8, the pressure wave US is traveling from the transducer 906 in the axial direction in respect of the first axis A1 and is reflected by the reflection plate 905.

A first electrode 904A is inserted into the (radially extended) axial cathode port 909A (see FIG. 7), and a second electrode 904B is inserted into the (radially extended) axial anode port 909B, such that the first and the second electrode 904A, 904B are arranged in a radial direction in respect of the first axis A1 and the tips of the first and second electrodes 904A, 904B are arranged along the first axis A1 , resulting in an electric field E2 parallel to the first axis A1 when a potential difference is applied between the first and the second electrode 904A, 904B by means of a voltage source 1 1 1.

Furthermore, a third electrode 904C and a fourth electrode 904D are inserted into orthogonal electrode ports 909C, such that their tips are arranged perpendicular (radially) to the first axis A1 . When an electric potential difference is applied between the third electrode 904C and the fourth electrode 904D by means of a further voltage source 1 1 1 , an electric field E1 is generated, which is perpendicular to the first axis A1.

This arrangement results in a combination of an axial pressure wave US, a radial electric field E1 and a further axial electric field E2, particularly for sono-electro-osmosis.

Described herein are experimental results from example usage of the systems and methods disclosed, showing that the sonication-induced structural effects and improved local reaction kinetics combined with the electrophoresis of hydrogel-embedded dyes through tissue, result in significantly enhanced throughput for staining large tissue blocks.

FIG. 9 illustrates the use of sonication, using a system similar to the system described herein, on tissue samples. Panel A shows comparison of sample images of human lymph node slices (1 mm thick) stained with nuclear dyes (To-Pro-3 and DAPI), taken at a depth of at 10 pm, the slices being treated with sonication at 1.7 MHz (labelled US on the right) and without being treated with sonication (labelled CTRL, on the left). Panel B shows control (CTRL) and treated (US) images similar to panel A, but with images obtained from the respective tissue samples (treated and untreated with sonication at 1 .7MHz) from a depth of 650 pm. Panel C shows images of an orthogonal slice of the same stacks of tissue, illustrating the depth of staining from the surface (top) in control or untreated tissue sample (CTRL on the left) and tissue sample treated with sonication (US on the right). As shown in Panels A-C, treatment with sonication indicates a significantly enhanced extent of staining in all dimensions.

FIG. 10 illustrates quantification of average pixel intensity per z-plane as a function of depth along the z-axis of the image stack shown in FIG.9. The quantification is shown for tissue labelled with dyes To-Pro-3 (top row) and DAPI (bottom row) when untreated with sonication (CTRL on the left column) and when treated with sonication (US on the right column). The quantification shows a drastic increase in staining intensity both on the surface and deep in tissue upon sonication. The different colors corresponding to effects of treatment duration of 5, 15 and 30 minutes. The defined lines depict the mean values, and the shaded portions of the same but fainter color show the standard deviation, of data obtained from three samples for each treatment.

FIG.1 1 shows a set of images of tissue phantoms (HeLa cells solubilized and fixed in acrylamide- paraformaldehyde gel in 200 pi laboratory tubes) stained from the gel surface (red lines) with fluorescent anti-cytokeratin antibodies for 30 minutes. The depth of stain is assessed by looking at the width of the fluorescent cell-band (yellow arrows). The combination of focused electrophoresis with sonication increases staining depth. Tissue in the presence of the fluorescent antibodies but without exposure to an electric field or ultrasound (top left panel) shows little or no staining. Application of ultrasound only (top right panel) resulted in limited staining, and slightly better staining results were obtained with focused electrophoresis only (bottom left panel). The combination of both ultrasound and focused electrophoresis (bottom right panel) provided a greater extent of staining compared to each approach alone.

FIG.12 illustrates a flowchart of an example method 1200 to process a tissue sample, such as by using a sono-electro-osmosis system as described herein, for example. The method 1200 includes, at 1251 , an optional step (indicated by the dashed lines) of preparation of a tissue sample for processing. In some instances, the tissue sample can be naturally occurring biological tissue that is suitably extracted. For example, the tissue sample can be an excised or sliced tissue sample, a whole-mount tissue sample, or a tissue sample generated from a biopsy procedure, or a tissue sample generated via a punching process. In some instances, the tissue sample can be a freshly extracted tissue sample. In some other instances, the tissue sample can be previously extracted and stored, for example as a pre-processed tissue sample, or a frozen tissue sample. In some instances, the tissue sample can be an engineered sample or otherwise synthetically generated using suitable processes. In some instances, the preparation of the tissue sample can include procedures like formaldehyde fixation, hydrogel embedding, mounting, and the like.

At 1252 the prepared tissue sample is disposed in a sample holder that can be configured to be removably disposed in an apparatus configured for sono-electro-osmosis, such as the systems100, 900, described herein. The sample holder can be substantially similar in structure and/or function to the sample holders 102, 202, 302, 402, 502, 602, 702, 802, and/or 902 described herein. At 1253 the sample holder is removably disposed in the second chamber portion of the system.

At 1254 and 1255, a first and second buffer are flowed into the first and third portions of the chamber of the apparatus respectively. In some instances, the first and second buffers can be the same or substantially similar, while in other instances they can be of different composition or constituents. As described previously, the first and/or the second buffers can include ionic detergents and non-ionic detergents. In some instances, the first and/or the second buffer can include one or more enzymatic agents for digesting one or more components of the tissue sample. The first and second buffers can be flowed with a suitable predetermined or monitored flow rate, and the buffers can be suitably recirculated using one or more pumps. For example, in some instances, the first buffer can be flowed at a rate of from about 10 ml/min to about 100 ml/min. Similarly, the second buffer can be flowed at a rate of from about 10 ml/min to about 100 ml/min.

In some instances of processing a tissue sample using the method 1200, optionally, a staining agent can be disposed in the chamber, as described at 1256 and indicated by the dashed lines. The staining agent can be any suitable dye or staining molecule including one or more proteins, and one or more nucleic acids. Based on one or more properties of the staining agent, in some instances, the staining agent can be disposed in the chamber by dissolving the staining agent in the first buffer in the first chamber portion or the second buffer flowed in the third chamber portion or both. Based on properties of the staining agent, in some other instances, the staining agent can be disposed by entrapping the staining agent in a non-crosslinking gel and disposing the non-crosslinking gel in a first or third portion of the chamber. In some instances, the noncrosslinking gel can be cast on a surface of the tissue sample. In some other instances, the noncrosslinking gel can be cast in a capsule and the capsule can be disposed in a portion of the chamber of the apparatus. In some instances, the method 1200 can include one or more additional steps, not shown in the flowchart in FIG.12, of introducing additional buffers after the steps of flowing the first and/or the second buffer at 1254 and/or 1255, and before the disposing of the staining agent at 1256. For example, in some instances, the method can include introducing a washing buffer to wash various portions of the apparatus (e.g. the first, second or third chamber portions) and/or tissue sample using a third buffer (e.g., washing buffer) before the disposing of the staining agent at 1256. The introduction of the washing buffer can be configured to remove most or all of the detergents that may be included in the first and/or second buffer from the apparatus and/or tissue sample before the disposing of the staining agent.

At 1257, the method 1200 includes applying an electrical signal to a set of electrodes in the apparatus to generate a directional electric field that can be applied onto the tissue sample. The electric field can be applied via a set of electrodes including a cathode and an anode, each positioned in a predetermined chamber portion of the apparatus. For example, the electric field can be applied via an electrical signal applied to a cathode positioned in the first chamber portion and an anode in the third chamber portion. At 1258, the method includes inducing electrophoresis in the tissue sample via the applied electric field at 1257, such that the electric field lines pass only or predominantly through the tissue sample in the second chamber portion of the apparatus.

The staining agent can be disposed, at 1256, in consideration with the application of electric field at 1257. For example, in some instances, the staining agent can be disposed in one or more predetermined portions of the chamber (e.g. first chamber portion or the third chamber portion, or both) based on the properties of the staining agent such as net charge and the configuration of the electrodes in the apparatus, as described below.

For example, in some instances, the staining agent can carry a net negative charge. In some such instances, the negatively charged staining agent can be disposed by entrapping in a noncross-linking gel directly on the surface of the tissue sample or in a capsule. In some other such instances, the negatively charged staining agent can be dissolved in a first buffer flowed into the first chamber portion of the apparatus. The negatively charged staining agent either dissolved in the first buffer or entrapped in a non-cross-linking gel can be disposed in the first chamber portion of the apparatus wherein the first electrode in the first chamber portion is configured to be a cathode and the second electrode in the third chamber portion is configured to be an anode. The negatively charged staining agent can be disposed such that upon application of a directional electric field, the negatively charged staining agent moves or migrates from near the cathode in the first chamber portion towards the anode in the third chamber portion via the tissue sample in the second chamber portion thus staining the tissue sample.

The electric signal can, in some instances, be applied via a power source operating at a voltage between about 5 V and about 500 V, in constant voltage mode. In some instances, the electrical signal can be applied such that the power source is configured to generate an output current of at most between about 30 mA and 1000 mA. In some instances, the electrical signal can be applied via a power source operating for a predetermined duration such as a time period of between about 10 minutes and about 1000 minutes, for more than 1000 minutes, and/or the like.

In some instances, the method 1200 can include, at 1259, the application of a directional pressure wave to the sample, via a transducer included in the apparatus. The application of pressure wave at 1259 can be made to induce sono-osmosis in the tissue sample at 1260, with the pressure wave passing through the tissue sample in the second chamber portion of the apparatus. The pressure wave can be a continuous wave, pulse waves, a standing wave, an acoustic wave or an ultrasonic wave. For example, a directional ultrasonic wave can be applied at 1259 via a suitably positioned ultrasonic transducer included in the apparatus. The direction of the pressure wave can be configured based on/accounting for the direction of the electrical signal applied at 1257. For example, in some instances, the pressure wave can be applied in a direction parallel to the electric field vector of the electric field. In some other instances, the pressure wave can be applied in a direction orthogonal to the direction of the electric field. In some instances, the method 1200 can include one or more steps (not shown in FIG.12) after the induction of electrophoresis at 1258, and before the application of directional pressure wave to the sample at 1259. For example, in some instances, the method can include the flowing of one or more buffers (e.g. a fourth and/or a fifth buffer flowed through the first and/or the third chamber portions of the apparatus) before the application of the pressure wave at 1259. In certain embodiments, the method includes the flowing of a washing buffer.

The application of the electrical signal at 1257 and the application of the pressure wave at 1258 can be optional. That is, in some instances, only the electrical signal can be applied at 1257 without the application of the pressure wave at 1258. In some other instances, only the pressure wave can be applied at 1258, omitting the application of electrical signal at 1257. In some other instances, both the electrical signal and the pressure wave can be applied at 1257 and at 1258 while processing the tissue sample.

In the instances of application of both the electrical signal and the pressure wave, any suitable approach can be followed to configure or time the application of the electrical signal at 1257 and the application of the pressure wave at 1258, including interleaved application, near concurrent application, overlapping application, and simultaneous application. The process of applying an electric field at 1257 and inducing electrophoresis at 1258, and/or applying pressure wave at 1259 and inducing sono-osmosis at 1260, can be repeated over multiple runs until a predetermined time or number of runs or a predetermined amount of staining is achieved. In some instances, alternative cycles can include application of electric field at 1257 to induce electrophoresis at 1258 and the remaining cycles can include application of the pressure weave at 1259 to induce sono-osmosis at 1260.

Following an indication of completion, based on, for example, when a suitable time duration is passed or a suitable number of cycles are run, or when a predetermined amount of staining is achieved, the method 1200 can be terminated at 1262 and the sample holder can be removed from the apparatus and the tissue sample can be retrieved from the tissue holder.

FIG. 13 illustrates a flowchart of another example method 1300 of processing a tissue sample, such as by using a sono-electrosmosis system as described herein. Steps and aspects of the method 1300 can be substantially similar to similarly named/referenced steps and aspects of the method 1200 of FIG. 12 and described above. Method 1300 includes an optional step of preparing a tissue sample at 1351 indicated by the dashed lines. The method 1300 includes disposing a tissue sample in a sample holder at 1352. As described previously with respect to the method 1200 above, the sample holder can be configured to be removably disposed in an apparatus configured for sono-electro-osmosis, such as the systems 100, 900, described herein. At 1353, the sample holder is removably disposed in the second chamber portion of the system. The sample holder can be substantially similar in structure and/or function to the sample holders 102, 202, 302, 402, 502, 602, 702, 802, and/or 902 described herein.

At 1354, a first buffer is flowed into the first chamber portion of the apparatus such that the first buffer is in contact with the tissue sample. At 1355, a second buffer is flowed into the third chamber portion of the apparatus such that the second buffer is in contact with the tissue sample.

As described previously, with reference to the method 1200 illustrated in FIG. 12, in some instances, the method 1300 can include one or more additional steps, not shown in the flowchart in FIG.13, of introducing additional buffers after the steps of flowing the first and/or the second buffer at 1354 and/or 1355, and before the application of electrical signal to induce electrophoresis at 1356. For example, in some instances, the method can include introducing a washing buffer to wash various portions of the apparatus (e.g., the first, second or third chamber portions) and/or tissue sample using a third buffer before inducing electrophoresis at 1356.

At 1356, an electrical signal is applied to the sample to induce electrophoresis in the sample. The electrical signal is applied via a first electrode disposed in the first chamber portion of the apparatus and a second electrode disposed in the third chamber portion of the apparatus. The electrical signal is applied such that an electric field is applied between the first and second electrodes and on the sample wherein the electric field lines pass through only the tissue sample. At 1357, following the electrophoresis of the sample, the sample holder is removed from the apparatus and the tissue sample is retrieved.

Some embodiments described herein relate to a computer storage product with a non-transitory computer-readable medium (also may be referred to as a non-transitory processor-readable medium) having instructions or computer code thereon for performing various computer- implemented operations. The computer-readable medium (or processor-readable medium) is non-transitory in the sense that it does not include transitory propagating signals per se (e.g., a propagating electromagnetic wave carrying information on a transmission medium such as space or a cable). The media and computer code (also may be referred to as code or algorithm) may be those designed and constructed for the specific purpose or purposes. Examples of non-transitory computer-readable media include, but are not limited to, magnetic storage media such as hard disks, floppy disks, and magnetic tape; optical storage media such as Compact Disc/Digital Video Discs (CD/DVDs); Compact Disc-Read Only Memories (CD-ROMs), and holographic devices; magneto-optical storage media such as optical disks; solid state storage devices such as a solid state drive (SSD) and a solid state hybrid drive (SSHD); carrier wave signal processing modules; and hardware devices that are specially configured to store and execute program code, such as Application-Specific Integrated Circuits (ASICs), Programmable Logic Devices (PLDs), Read-Only Memory (ROM), and Random-Access Memory (RAM) devices. Other embodiments described herein relate to a computer program product, which may include, for example, the instructions and/or computer code disclosed herein.

The systems, apparatuses, and/or methods described herein may be performed by software (executed on hardware), hardware, or a combination thereof. Hardware modules may include, for example, a general-purpose processor (or microprocessor or microcontroller), a field programmable gate array (FPGA), and/or an application specific integrated circuit (ASIC). Software modules (executed on hardware) may be expressed in a variety of software languages (e.g., computer code), including C, C++, Java®, Python, Ruby, Visual Basic®, and/or other object-oriented, procedural, or other programming language and development tools. Examples of computer code include, but are not limited to, micro-code or micro-instructions, machine instructions, such as produced by a compiler, code used to produce a web service, and files containing higher-level instructions that are executed by a computer using an interpreter. Additional examples of computer code include, but are not limited to, control signals, encrypted code, and compressed code.

In some embodiments, the systems, apparatuses, and methods may be in communication with other computing devices (not shown) via, for example, one or more networks, each of which may be any type of network (e.g., wired network, wireless network). A wireless network may refer to any type of digital network that is not connected by cables of any kind. Examples of wireless communication in a wireless network include, but are not limited to cellular, radio, satellite, and microwave communication. However, a wireless network may connect to a wired network in order to interface with the Internet, other carrier voice and data networks, business networks, and personal networks. A wired network is typically carried over copper twisted pair, coaxial cable and/or fiber optic cables. There are many different types of wired networks including wide area networks (WAN), metropolitan area networks (MAN), local area networks (LAN), Internet area networks (IAN), campus area networks (CAN), global area networks (GAN), like the Internet, and virtual private networks (VPN). Hereinafter, network refers to any combination of wireless, wired, public and private data networks that are typically interconnected through the Internet, to provide a unified networking and information access system.

Cellular communication may encompass technologies such as GSM, PCS, CDMA or GPRS, W- CDMA, EDGE or CDMA2000, LTE, WiMAX, and 5G networking standards. Some wireless network deployments combine networks from multiple cellular networks or use a mix of cellular, Wi-Fi, and satellite communication. In some embodiments, the systems, apparatuses, and methods described herein may include a radiofrequency receiver, transmitter, and/or optical (e.g., infrared) receiver and transmitter to communicate with one or more devices and/or networks. In certain embodiments, the disclosure includes a method for processing a biological sample comprising: (i) fixing the sample by contacting the biological sample with a fixation agent; (ii) clearing the fixed sample; and, optionally, (iii) labeling the cleared fixed sample with one or more first detectable marker. In another embodiment, the disclosure includes a method for processing a biological sample comprising: (i) fixing the sample by contacting the biological sample with a fixation agent in the presence of hydrogel subunits; (ii) polymerizing the hydrogel subunits to form a hydrogel-embedded sample; (iii) clearing the hydrogel-embedded sample; and, optionally, (iv) labeling the cleared hydrogel-embedded sample with one or more first detectable marker. In certain embodiments, the clearing and optionally, the labeling is performed by focused electrophoresis and, optionally, sonication. In particular embodiments, the clearing and/or labeling is performed in an apparatus disclosed herein, e.g., an apparatus configured to allow focused electrophoresis. In particular embodiments, during the focused electrophoresis, the electric field lines of the electric field pass only through or predominantly through the sample. In certain embodiments, sonication is performed by applying a pressure wave to the sample via a transducer. In certain embodiments, the clearing and the labeling are performed during the same, overlapping, or different time periods. For example, the clearing may be performed first by electrophoresis, optionally in combination with sonication, and then the labeling may be performed after the clearing, by electrophoresis, optionally in combination with sonication, e.g., after providing a staining agent to the apparatus. In particular instances, the staining agent, e.g., molecular dye, is embedded and concentrated in a non-crosslinking hydrogel medium located at one side of the sample. In certain embodiments, when the fixing is performed in the presence of hydrogel subunits, the hydrogel subunits are cross-linked to biomolecules (e.g., polypeptides and/or nucleic acids) within the biological tissue specimen to produce biomolecule-bound hydrogel subunits. In certain embodiments, during the clearing, a plurality of one or more cellular components, e.g., lipids, are removed from the tissue sample. In particular embodiments, the biological sample is stained, e.g., with an antibody. Illustrative methods of fixing, cross-linking, and staining tissue samples are disclosed in PCT Application Publication Nos. WO2017/096248 and WO2014/025392, which are incorporated herein by reference in their entireties.

Any convenient fixation agent, or "fixative," may be used to fix the biological sample, optionally in the presence of hydrogel subunits, such as, for example, formaldehyde, paraformaldehyde, glutaraldehyde or any other aldehyde, acetone, ethanol, methanol, aminoalcohols, etc. In some embodiments, the fixative can be diluted in a buffer, e.g., saline, phosphate buffer (PB), phosphate buffered saline (PBS), citric acid buffer, potassium phosphate buffer, etc., e.g., at a concentration of about 1 -10%, e.g. 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 10%, for example, 4% paraformaldehyde/0.1 M phosphate buffer; 2% paraformaldehyde/0.2% picric acid/0.1 M phosphate buffer; 4% paraformaldehyde/0.2% periodate/1 .2% lysine in 0.1 M phosphate buffer; 4% paraformaldehyde/0.05% glutaraldehyde in phosphate buffer; etc. The type of fixative used and the duration of exposure to the fixative can depend on the sensitivity of the molecules of interest in the tissue sample to denaturation by the fixative and can be readily determined using conventional histochemical or immunohistochemical techniques, for example as described in Buchwalow and Bocker. Immunohistochemistry: Basics and Methods. Springer-Verlag Berlin Heidelberg 2010.

In some embodiments, fixing includes exposing the sample, e.g., cells of the sample, to a fixation agent such that the cellular components become crosslinked to one another. In particular embodiments, a tissue sample is fixed in the presence of hydrogel subunits, e.g., hydrogel monomers. The hydrogel/hydrogel network can include any suitable network of polymer chains that are water-insoluble, sometimes found as a colloidal gel in which water is the dispersion medium. In other words, hydrogels can belong to a class of polymeric materials that can absorb large amounts of water without dissolving. In certain embodiments, hydrogels can contain over 99% water and may comprise natural or synthetic polymers, or a combination thereof. Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content. A detailed description of suitable hydrogels may be found in published U.S. patent application 20100055733, herein specifically incorporated by reference and as detailed below. Examples of suitable hydrogels include acrylamide. Examples of hydrogel subunits/monomers include, but are not limited to, poly(ethylene glycol) and derivatives thereof (e.g., PEG-diacrylate (PEG-DA), PEG-RGD), polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohols, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate), and poly(hydroxyethyl methacrylate), dimethylaminoethyl methacrylate, 2-acylamido-2-methyl- propanosulfonic acid, collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose and the like. In some embodiments, the hydrogel subunits may be modified to add specific properties to the hydrogel; for example, peptide sequences can be incorporated to induce degradation (see, e.g., West and Hubbell, 1999, Macromolecules, 32:241 ) or to modify cell adhesion (see, e.g. Hem and Hubbell, 1998, J. Biomed. Mater. Res., 39:266). Agents such as hydrophilic nanoparticles (e.g., poly-lactic acid (PLA), poly-glycolic acid (PLG), poly(lactic-co-glycolic acid) (PLGA), polystyrene, poly(dimethylsiloxane) (PDMS), etc.) may be used to improve the permeability of the hydrogel while maintaining patternability (see, e.g., US Patent Application No. 13/065,030; Lee W. et al. 2010 Proc. Natl. Acad. Sci. 107, 20709-20714). Materials such as block copolymers of PEG, degradable PEO, poly(lactic acid) (PLA), and other similar materials can be used to add specific properties to the hydrogels (see, e.g., Huh and Bae, 1999, Polymer, 40:6147). Crosslinkers (e.g., bis-acrylamide, diazirine, etc.) and initiatiors (e.g., azobisisobutyronitrile (AIBN), riboflavin, L-arginine, etc.) may be included to promote covalent bonding between interacting macromolecules in later polymerization steps.

Hydrogel subunits or hydrogel precursors can encompass hydrophilic monomers, prepolymers, or polymers that can be crosslinked, or "polymerized", to form a three-dimensional (3D) hydrogel network. In certain embodiments, the hydrogel monomer solution includes a cross-linker, such as, e.g., a bis-acrylamide or diethylene glycol dimethacrylate. The hydrogel subunit/monomer solution may further comprise an initiator, such as, e.g., VA-044 Initiator. The hydrogel subunit/monomer solution may further comprise paraformaldehyde. Concentrations of hydrogel subunits and modifiers that provide desired hydrogel characteristics may be readily determined by methods in the art. Without being bound by scientific theory, it is believed that this fixation of the biological sample in the presence of hydrogel subunits crosslinks the components of the sample to the hydrogel subunits, thereby securing molecular components in place, preserving the tissue architecture and cell morphology.

In some embodiments, following fixation of the tissue sample in the presence of hydrogel monomers or subunits, the hydrogel subunits are polymerized, i.e., covalently or physically crosslinked, to form a hydrogel network. Polymerization may be by any method including, but not limited to, thermal crosslinking, chemical crosslinking, physical crosslinking, ionic crosslinking, photo-crosslinking, irradiative crosslinking (e.g., x-ray, electron beam), and the like, and may be selected based on the type of hydrogel used.

After fixation or polymerization, the tissue sample may be cleared, i.e., one or more tissue components removed. Clearing can include ensuring that the sample is made substantially permeable to light, i.e., transparent. In other words, about 70% or more of the visual (i.e., white) light, ultraviolet light or infrared light that is used to illuminate the sample will pass through the sample and illuminate only selected cellular components therein, e.g., 75% or more of the light, 80% or more of the light, 85% or more of the light, in some instances, 90% or more of the light, 95% or more of the light, 98% or more of the light, e.g. 100%, including all values and sub ranges in between, of the light will pass through the sample. This change in the optical properties of the sample provides for the visualization of cellular and subcellular structures internal to the tissue.

An apparatus or method disclosed herein may be used to clear a tissue sample. In some instances, clearing is using focused electrophoresis, optionally in combination with sonication. In some embodiments, clearing is performed using a solvent that does not quench fluorescent proteins. Examples of organic solvents that are known to quench fluorescent proteins include tetrahydrofuran, hexane, benzylalcohol/benzylbenzoate (BABB), and dibenzyl ether. Accordingly, in order to preserve the fluorescence of various proteins, in some embodiments clearing is conducted using solvents other than those listed above, e.g., non-organic solvents.

In some instances, clearing is conducted using a buffer comprising an ionic surfactant, e.g., SDS, in order to expedite the clearing process by actively transporting charged ionic micelles out of the sample that is being cleared. Clearing may be performed in any convenient buffer that is compatible with the selected clearance method, e.g., saline, phosphate buffer, phosphate buffered saline (PBS), sodium borate buffer, boric acid buffer, citric acid buffer, etc., as known in the art. In some embodiments, a clearing solution comprises a buffer, such as a borate buffer, and SDS. One particular clearing solution comprising 200 mM borate buffer, pH 8.5 and 8% SDS. In some embodiments, optimal clearing time may be readily determined by visual inspection of the sample for clarity.

After clearing, a tissue sample will generally be substantially free of lipids. In some embodiments, the original amount of lipid present in the sample before clearing can be reduced by approximately 70% or more, such as by 75% or more, such as by 80% or more, such as by 85% or more, such as by 90% or more, such as by 95% or more, such as by 99% or more, such as by 100%, including all values and sub ranges in between.

Tissue samples may be labeled, e.g., by contacting them with a detectable probe or label. These include, but are not limited to: nucleic acid stains like DAPI and Hoechst, which bind the minor groove of DNA, thus labeling the nuclei of cells; drugs or toxins that bind specific cellular structures and have been derivatized with a fluorescent reporter may be employed; nucleic acids probes that bind specific nucleic acid target molecules, such as DNA or RNA; one or more polypeptides, e.g., antibodies, labeled peptides, and the like, that are specific for and will bind to particular cells or cellular biomolecules, optionally for either direct or indirect labeling by color or immunofluorescence (i.e. probes). In particular embodiments, a detectable probe is a fluorescent molecule or protein. Immunofluorescence can include any suitable technique that uses the highly specific binding of an antibody to its antigen or binding partner in order to label specific proteins or other molecules within the cell. A sample is treated with a primary antibody specific for the biomolecule of interest. A fluorophore can be directly conjugated to the primary antibody or peptide or conjugated to a secondary antibody. In another example, a tissue sample may be contacted with an antisense RNA that is complementary to and specifically hybridizes to a transcript of a gene of interest, e.g., to study gene expression in cells of the sample.

In particular embodiments, the biological sample was obtained from a mammal. In certain embodiments, the biological sample is a tumor tissue sample, a previously frozen biological sample, or a cell line. In particular embodiments, the biological sample is a cell line pellet, e.g., a frozen cell line pellet. In certain embodiments, the biological sample has a length of greater than 10 microns and/or a thickness of greater than 10 microns. In certain embodiments, the biological sample has a length of greater than 20 microns and/or a thickness of greater than 20 microns.

FIG. 14-15 show parts of a device 100 or system 100 for electrophoretic clearing, washing and/or staining of a tissue sample 101 , 201 according to the invention. FIG. 14 is a perspective view and FIG. 15 is a cross-section along the first axis A1 indicated in FIG. 14 and 15.

The device 100 comprises a first block 130A, a second block 130B and a sample holder 102 which can be assembled and combined with electrodes 104A, 104B (not shown) to form the functional device 100.

The first block 130A forms or defines the first electrolyte chamber 103A configured to contain the first electrolyte 1 10A, the second block 130B forms or defines the second electrolyte chamber 103C configured to contain the second electrolyte 1 10B, and the sample holder 102 comprises a sample volume 121 in which the tissue sample 101 , (not shown in FIG. 14 and 15) can be arranged and held by the sample holder 102. When the device 100 is assembled, e.g. using bolts 135 inserted into axial guidances 134A, 134B, 134C, the first electrolyte chamber 103A, the sample volume 121 and the second electrolyte chamber 103C form a continuous chamber extending along the first axis A1.

In particular, when electrodes 104A, 104B (e.g. formed as a straight needle, rod or capillary) are inserted into through-holes 138A, 138B of electrode holders 137A, 137 comprised in the lids 136A, 136B and the lids 136A, 136B are placed on top of the respective first and second block 130A, 130B, an electric field generated by an electric potential difference between the electrodes 104A, 104B will be oriented parallel to the first axis A1 and will drive electrophoresis (e.g. of a detergent or staining preparation) along the first axis A1 .

The first block 130A and the second block 130B each comprise a respective first half shell 131 A, 131 B and a respective second half shell 132A, 132B, wherein the first half shell 131 A, 131 B and the second half shell 132A, 132B can be joined at a joining surface parallel to the first axis A1 and assembled by placing bolts 135 into the lateral guidances 133A, 133B, respectively. Thereby, the resulting first electrolyte chamber 103A and second electrolyte chamber 103C are jointly formed by the respective first and second half shell 131 A, 132A, 131 B, 132B.

When viewed in a cross-section perpendicular to the first axis A1 , the first block 130A and the second block 130B have a rectangular cross-section, which facilitates placement on an even surface during operation. Likewise, the sample holder 102 depicted in FIG. 14 and 15 has a rectangular, particularly square-shaped, cross-section perpendicular to the first axis A1 . In particular, the sample volume 121 of the sample holder 102 is circular in cross-sectional view perpendicular to the first axis A1. The sample holder 102 comprises axial guidances 134C for insertion of a bolt 135 to connect the sample holder 102 with the first block 130A and the second block 130B.

In the cross-sectional view illustrated in FIG. 15, the first block 130A and the second block 130B are L-shaped and the first and second electrolyte chamber 103A, 103C each comprise a respective first section 140A, 140B extending along the first axis A1 from a respective axial opening 142A, 142B to a respective lateral wall 144A, 144B (the lateral wall particularly stretching perpendicular to the first axis A1 ) and a respective second section 141A, 141 B extending along a second axis A2 perpendicular to the first axis A1 between the respective first section 140A, 140B towards a respective top opening 143A, 143B. This arrangement results in an L-shape of the first and second electrolyte chamber 103A, 103C. The border between the respective first and second sections 140A, 141A, 140B, 141 B is shown as a dot-dashed line in FIG. 15.

According to the invention, the device 100 comprising the parts shown in FIG. 14 and 15 can particularly be used for electrophoretic staining of a tissue sample 101 . Therein, in particular, the first block 130A and/or the second block 130 is assembled, placed such that the first axis A1 is horizontally arranged and the second axis A2 is vertically arranged, and the axial opening 142A, 142B is closed by a liquid-tight seal. Subsequently, a monomer solution able to form a cross- linking hydrogel (such as acrylamide/bisacrylamide mixture containing suitable polymerization initiators) may be poured into the first section 140A, 140B of the respective electrolyte chamber 103A, 103C from the respective top opening 143A, 143B (wherein particularly the entire first section 140A, 140B is filled with the monomer solution), and the monomer solution may be allowed to polymerize to form a cross-linked hydrogel. Thereafter, the respective electrolyte solution 1 10A, 1 10B can be poured into the respective second section 141A, 141 B, and electrophoresis can be performed after assembling the first and second block 130A, 130B with the sample holder 102 holding the tissue sample 101 , 201 , removing the liquid-tight seal, placing the electrodes 104A, 104B into the electrode holders 137A, 137B and applying an electric potential difference between the electrodes 104A, 104B.

In particular, in case a hydrogel preparation 223 comprising a staining preparation (see, e.g. FIG 3D and FIG. 4) is to be used to stain the tissue sample 101 , a liquid-tight seal comprising a protrusion extending into the first section 140A, 140B of the respective electrolyte chamber 103A, 103C can be used to close the axial opening 142A, 142B of one or both of the blocks 130A, 130B. When the cross-linking hydrogel is polymerized in the respective first section 140A, 140B, a recess is formed in the cross-linked hydrogel at the surface formed within the axial opening 142A, 142B. Advantageously, it is possible to place a small piece of a hydrogel preparation 223, particularly a non-cross-linking hydrogel, such as low-melt agarose, comprising a staining preparation, into the recess. During electrophoresis, this hydrogel preparation 223 is arranged next to the tissue sample 101 in the sample holder 102, such that the staining preparation is drawn into and through the tissue sample 101 to stain the sample effectively. Therein, the electric potential applied to the electrodes 104A, 104B is chosen such that the staining preparation is drawn towards the tissue sample 101. For example if the staining preparation is negatively charged at the pH of the buffer used for electrophoresis, the hydrogel preparation 223 comprising the staining preparation is placed in the first block 130A harboring the cathode, such that the staining preparation migrates towards the anode in the second block 130B on the opposite side of the sample holder 102.

In particular, the hydrogel preparation 223 containing the staining preparation may be generated by filling, more particularly pouring or pipetting, the hydrogel preparation (in its liquid state) directly into the recess in the cross-linked hydrogel in the electrolyte chamber 103A, 103C. To this end, the first or second block 130A, 130B may be placed such that the axial opening 142A, 142B is pointing upwards (such that the first axis A1 is vertically oriented and the second axis A2 is horizontally oriented, thus turned 90° compared to the preferred positioning during electrophoresis). Thereby, a tight fit of the hydrogel preparation 223 comprising the staining preparation in the recess of the cross-linked hydrogel in the first and/or second block 130A, 130B is achieved, which facilitates effective transport of the staining preparation during electrophoresis. Alternatively, the hydrogel preparation may be filled into a mold shaped as a negative of the protrusion of the liquid-tight plug, allowed to solidify and then inserted into the recess.

After electrophoresis, the first block 130A and the second block 130B can be easily disassembled into the half shells 131 A, 132A, 131 B, 132B, particularly for removing the cross- linked hydrogel and cleaning the electrolyte chambers 103A, 103C.

As best seen in FIG. 15, the first block 130A and the second block 130B each comprise a lid 136A, 136B to be placed on the top opening 143A, 143B to close the top opening 143A, 143B. The lids 136A, 136B each comprise an electrode holder 137A, 137B comprising a through-hole 138A, 138B for inserting an electrode 104A, 104B (see e.g. Fig. 1 ) and an internal thread 139A, 139B for inserting a screw to fix the electrode 104A, 104B inserted into the through-hole 138A, 138B.

In addition, an inlet and/or outlet for the electrolyte solution 1 10A, 1 10B may be provided in the first block 130A and/or second block 130B, e.g. in the respective first half shell 131 A, 131 B, second half shell 132A, 132B and/or in the respective lid 136A, 136B. Such inlets and outlets may be used in particular for recirculation of the electrolyte solution 1 10A, 1 10B such as e.g. shown in Fig. 1.

FIG. 16 to 19 show an embodiment of a sample holder 102 for use in clearing, washing and/or staining of a tissue sample 101 comprising four parts which can be assembled: a bottom part 150, an outer cylinder part 160, an inner cylinder part 170 and a fixing ring 180.

The sample holder 102 extends along a first axis A1 and is characterized by a cylindrical shape, wherein the first axis A1 is the central cylinder axis, and wherein the sample holder 102 comprises a circular cross-section perpendicular to the first axis A1.

The bottom part 150 comprises an internal thread 156 configured to engage an external thread 162 of the outer cylinder part 160 as illustrated in the detailed view of FIG. 19A showing detail B indicated in FIG. 18 right panel to connect the bottom part 150 and the outer cylinder part 160 by screwing.

As shown in FIG. 19A, a first membrane 191 , particularly a semi-permeable membrane, is held at the interface of the bottom part 150 and the outer cylinder part 160, such that the first membrane 191 extends perpendicular to the first axis A1 in an internal cavity formed by the bottom part 150 and the outer cylinder part 160.

The first membrane 191 is tightly held by circumferential teeth 157 of the bottom part 150 engaging circumferential notches 163 of the outer cylinder part 160 when the two parts are connected by screwing. As a alternative, the bottom part 150 and/or the outer cylinder part 160 may comprise an O-ring, particularly from rubber or other suitable material, at the interface of the bottom part 150 and the outer cylinder part 160 to tightly fix the first membrane 191 when the two parts are screwed together. Furthermore, as shown in FIG. 19B showing detail C indicated in FIG: 18 right panel, the fixing ring 180 comprises an external thread 181 configured to engage an internal thread 173 of the inner cylinder part 170, such that the fixing ring 180 and the inner cylinder part 170 may be connected by screwing.

The fixing ring 180 and the inner cylinder part 170 hold a second membrane 192, particularly a semipermeable membrane, at their interface, such that the second membrane 192 extends perpendicular to the first axis A1 in an internal cavity formed by the fixing ring 180 and the inner cylinder part 170 (FIG. 19B).

The second membrane 192 is tightly held by circumferential teeth 182 of the fixing ring 180 engaging circumferential notches 174 of the inner cylinder part 170 when the two parts are connected by screwing. Alternatively, the inner cylinder part 170 and/or the fixing ring 180 may comprise an O-ring, particularly from rubber or other suitable material, at the interface of the inner cylinder part 170 and the fixing ring 180 to tightly fix the second membrane 192 when the two parts are screwed together.

Finally, the inner cylinder part 170 comprises an external thread 171 configured to engage an internal thread 161 of the outer cylinder part 160 to connect the inner cylinder part 170 and the outer cylinder part 180 by screwing.

Thereby the first membrane 191 and the second membrane 192 are positioned parallel to each other and at an appropriate distance along the first axis A1 , such that a tissue sample 101 , 201 may be arranged between and held in place by the first membrane 191 and the second membrane 192.

The bottom part 150 comprises a ring-shaped base 152 extending in a circumferential direction in respect of the first axis A1 and a lateral ring 151 extending in a circumferential direction in respect of the first axis A1 and protruding from the base 152 in a radial direction in respect of the first axis A1 (see FIG. 16). The base 151 surrounds a space 158 and comprises two semicircular recesses 153 positioned opposite of each other and connecting the space 158 to the exterior of the bottom part 150. Furthermore, the bottom part 150 comprises an end face 159, particularly a conical end face 159, stretching essentially perpendicular to the first axis A1 and surrounded by the base 152. The end face 159 comprises the second opening 1 17B of the sample holder 102.

On the end face 159, an electrode holder 154 for holding the second electrode 104B is disposed. Likewise, the inner cylinder part 170 comprises an electrode holder 172 for holding the first electrode 104 A.

The inner cylinder part 170 is hollow, the inside of the inner cylinder part forming a first electrolyte chamber 103A for containing the first electrolyte solution 1 10A. Advantageously, this eliminates the need for a separate first electrode chamber 103A, thus reducing the size of the device. Furthermore, the bottom part 150 is configured to be placed in a liquid reservoir (not shown), the reservoir forming the second electrolyte chamber 103C for containing the second electrolyte solution 1 1 OB. The recesses 153 of the ring-shaped base 152 allow entry of the second electrolyte solution 1 10B into the space 158.

Furthermore, in particular, the recesses 153 serve as openings to educt gases generated during electrophoresis (due to electrolysis).

To avoid the build-up of such gases (e.g. in the form of bubbles which may block current flow) below the first membrane 191 , end face 159 particularly has a conical shape, wherein the tip of the conus points towards the base 152 (particularly to the bottom of the vertically arranged sample holder 102 during the preferred operational mode).

By means of the base 152 of the bottom part 150, the sample holder 102 is configured to be arranged such that the first axis A1 is oriented in a vertical direction. In this configuration, the first opening 1 17A of the sample holder 102 (see FIG. 17A) is facing upwards and the second opening 1 17B of the sample holder (see FIG. 17B) is facing downwards, such that electrophoresis can be performed in the vertical direction.

In the method according to the invention, the tissue sample 101 is often displaced or shifted by electroosmosis, which complicates focused electrophoresis. Due to the possibility to perform electrophoresis in a vertical direction, this can be prevented by the sample holder 102 described above and shown in FIG. 16-19, if the sample holder 102 is set up such that gravity counteracts the electroosmosis effect. For example, assuming conditions where the tissue sample 101 is negatively charged, this is the case if the anode is placed above the tissue sample 101 (in the electrode holder 172 of the inner cylinder part 170.

In addition, the first membrane 191 and the second membrane 192 tightly hold the tissue sample 101 in place. In particular, the first membrane 191 and/or the second membrane 192 is non- permeable for at least one dye molecule comprised in a staining preparation used in the staining step of the method according to the invention. Thus, these dye molecules are retained between the two membranes and the dilution of the staining preparation by the electrolyte solution 1 10A, 1 10B (buffer) is prevented. This significantly improves the efficiency of the staining step. In addition, the staining preparation can also be used efficiently in liquid phase, eliminating the need for embedding the staining preparation in a hydrogel.

FIG. 20 shows an array of hollow microneedles 193 configured to stain a tissue sample 101 according to the invention. The tissue sample 101 is sandwiched between a membrane 190, particularly a semipermeable membrane, and a conductive polymer 196, such as polyacrylamide. The conductive polymer 196 is in fluid communication with a first electrolyte solution 1 10A disposed in a first electrolyte chamber 103A. Likewise, the membrane 190 is in fluid communication with a second electrolyte solution 1 10B in a second electrolyte chamber 103C. The microneedles 193 are arranged parallel to each other along a first axis A1 in the conductive polymer 196, with their tips 199 inserted into the tissue sample 101 . In particular, the first axis A1 may be vertically oriented. The microneedles 193 contain a staining preparation 195 and a respective microneedle electrode 194 is inserted into the hollow space inside each microneedle 193 in contact with the staining preparation 195.

The microneedle electrodes 194 are electrically connected in parallel to a first voltage source 197A by electric connection 198. Furthermore, the first voltage source 197A is electrically connected to the second electrolyte solution 1 10B contained in the second electrolyte chamber 103C, wherein particularly the electrolyte solution 1 10B is at ground potential and the microneedle electrodes 194 exhibit a positive or negative electric potential. Alternatively, the second electrolyte solution 1 10B may exhibit a positive or negative electric potential in respect of ground potential, wherein there is an electric potential difference between the microneedle electrodes 194 and the tissue sample 101 and/or the second electrolyte solution 1 10B.

In particular, if an appropriate positive or negative potential at a given composition of the first and second electrolyte solution 1 10A, 1 10B is applied to the microneedle electrodes 194, the staining preparation 195 is forced into the tissue sample 101 by iontophoresis. Therein, dosing of the staining preparation may be controlled by setting an appropriate electric potential.

Subsequently, in a second step, a second voltage source 197B electrically connected to said first electrolyte solution 1 10A and said second electrolyte solution 1 10B may be used to apply a potential difference between the first electrolyte solution 1 10A and the second electrolyte solution 1 10B. Accordingly, an electric field will be generated between the membrane 190 and the conductive polymer 196 in contact with the tissue sample 101 , and the staining preparation 195 may be distributed within the tissue sample 101 , particularly evenly, by iontophoresis and/or electroosmosis.

As used herein, the terms “about” and/or “approximately” when used in conjunction with numerical values and/or ranges generally refer to those numerical values and/or ranges near to a recited numerical value and/or range. In some instances, the terms“about” and“approximately” may mean within ± 10% of the recited value. For example, in some instances,“about 100 [units]” may mean within ± 10% of 100 (e.g., from 90 to 1 10). The terms“about” and“approximately” may be used interchangeably.

In addition, any combination of two or more such features, structure, systems, articles, materials, kits, steps and/or methods, disclosed herein, if such features, structure, systems, articles, materials, kits, steps and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. Moreover, some embodiments of the various inventions disclosed herein may be distinguishable from the prior art for specifically lacking one or more features/elements/functionality found in a reference or combination of references (i.e. , claims directed to such embodiments may include negative limitations).

Any and all references to publications or other documents, including but not limited to, patents, patent applications, articles, webpages, books, etc., presented anywhere in the present application, are herein incorporated by reference in their entirety. Moreover, all definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The invention is further illustrated by, but not limited to, the following items:

1 . An apparatus, comprising:

a chamber including a first chamber portion, a second chamber portion, and a third chamber portion, the second chamber portion configured to hold a tissue sample, the first chamber portion configured to hold a first buffer in contact with the tissue sample, the third chamber portion configured to hold a second buffer in contact with the tissue sample; and

a set of electrodes coupled to the chamber, the set of electrodes configured to apply an electrical signal to the sample to induce electrophoresis in the sample, a first electrode of the set of electrodes disposed in the first chamber portion and a second electrode of the set of electrodes disposed in the third chamber portion, wherein during application of an electric field between the first electrode and the second electrode, the electric field lines of the electric field pass only through or predominantly through the sample in the second chamber portion.

2. The apparatus of item 1 , wherein the second chamber portion is further configured to removably hold a sample holder including the tissue sample.

3. The apparatus of item 2, wherein the sample holder includes a first opening to interface with the first buffer in the first chamber portion and a second opening to interface with the second buffer in the second chamber portion.

4. The apparatus of any of items 1-3, wherein the first chamber portion includes a first inlet port and a first outlet port to permit recirculation of the first buffer, and wherein the third chamber portion includes a second inlet port and a second outlet port to permit recirculation of the first buffer.

5. The apparatus of any of items 1 -4, further comprising a power source coupled to the set of electrodes.

6. The apparatus of any of items 1-5, wherein the set of electrodes includes a first group of electrodes including the first electrode disposed in the first chamber portion and a second group of electrodes including the second electrode disposed in the third chamber portion.

7. The apparatus of item 6, further comprising a controller coupled to the set of electrodes and configured to sequentially activate two or more electrodes of the first group of electrodes as anodes and to sequentially activate two or more electrodes of the second group of electrodes as cathodes to change a direction of an electric field vector of the electric field.

8. The apparatus of item 6, further comprising a controller coupled to the set of electrodes and configured to selectively activate, at a first time, one or more electrodes of the first group of electrodes as anodes and to selectively activate one or more electrodes of the second group of electrodes as cathodes.

9. The apparatus of item 8, the controller configured to selectively activate, at a second time, one or more electrodes of the first group of electrodes as cathodes and to selectively activate one or more electrodes of the second group of electrodes as anodes to reverse the polarity of the electric field.

10. The apparatus of any of items 1-9, further comprising a transducer coupled to the chamber, the transducer configured to apply a pressure wave to the sample.

1 1 . The apparatus of item 10, wherein the pressure wave is a continuous wave.

12. The apparatus of item 10, wherein the pressure wave is a pulse wave.

13. The apparatus of item 10, wherein the pressure wave is a standing wave.

14. The apparatus according to any one of the previous items 10 to 13, wherein the pressure wave is an acoustic wave.

15. The apparatus according to any one of the previous items 10 to 14, wherein the transducer is an ultrasonic transducer.

16. The apparatus of any of items 10-15, wherein the pressure wave is applied in a direction parallel to the electric field vector of the electric field.

17. The apparatus of any of items 10-16, wherein the pressure wave is applied in a direction orthogonal to the electric field vector of the electric field

18. The apparatus according to any one of the previous items 10 to 17, further comprising a controller coupled to the transducer and the set of electrodes, the controller configured to apply the electric field and the pressure wave to the tissue sample simultaneously.

19. The apparatus according to any one of the previous items 10 to 17, further comprising a controller coupled to the transducer and the set of electrodes, the controller configured to apply the electric field and the pressure wave to the tissue sample in an overlapping manner. 20. The apparatus according to any one of the previous items 10 to 17, further comprising a controller coupled to the transducer and the set of electrodes, the controller configured to apply the electric field and the pressure wave to the tissue sample in an interleaved manner.

21 . The apparatus according to any one of the previous items, wherein each electrode of the set of electrodes includes a platinum electrode having the same surface area as a face of the tissue sample facing that electrode.

22. A method, comprising:

removably disposing a sample holder comprising a tissue sample in a second chamber portion of a chamber of an apparatus, the chamber including a first chamber portion, the second chamber portion, and a third chamber portion;

flowing a first buffer in the first chamber portion, the first buffer in contact with the tissue sample;

flowing a second buffer in the third chamber portion, the second buffer in contact with the tissue sample;

applying an electrical signal (an electrical field) to the sample to induce electrophoresis in the sample via a first electrode and a second electrode of a set of electrodes of the apparatus, the first electrode of the set of electrodes disposed in the first chamber portion and the second electrode of the set of electrodes disposed in the third chamber portion, wherein during application of an electric field between the first electrode and the second electrode, the electric field lines of the electric field pass only through, or predominantly through, the sample in the second chamber portion.

23. The method of item 22, wherein the tissue sample is a naturally occurring biological tissue or an engineered tissue.

24. The method of item 22 or item 23, wherein the tissue sample is selected from the group consisting of a sliced tissue sample, a whole-mount tissue sample, a tissue sample generated from a biopsy procedure, and a tissue sample generated via a punching process.

25. The method of any of items 22-24, wherein the tissue sample is selected from the group consisting of a fresh tissue sample, a pre-processed tissue sample, and a frozen tissue sample.

26. The method of item 22, wherein the tissue sample is prepared using at least one of aldehyde (particularly formaldehyde or glutaraldehyde) fixation, and hydrogel embedding. 27. The method of any of items 22-26, wherein the first buffer and the second buffer are the same.

28. The method of any of items 22-27, wherein the first buffer and the second buffer include ionic detergents and non-ionic detergents.

29. The method of any of items 22-28, the flowing the first buffer including flowing the first buffer at a rate of from about 10 ml/min to about 100 ml/min, particularly approx.. 30ml/min,, and the flowing the second buffer including flowing the second buffer at a rate of from about 10 ml/min to about 100 ml/min.

30. The method of any of items 22-29, further comprising maintaining the temperature of the first buffer and the second buffer between about 4 degrees Celsius and about 80 degrees Celsius, particularly between 37°C and 44°C, more particularly between 39°C and 42°C.

31 . The method of any of items 22-30, the applying including applying the electrical signal via a power source operating at a current value between about 30 mA and about 1000 mA, in constant current mode.

32. The method of item 31 , wherein the power source generates an output voltage of at most between about 10 V and about 200 V, particularly wherein the voltage applied to the sample is between 10 and 60V, more particularly between 20 and 50V.

33. The method of any of items 22-32, the applying including applying the electrical signal (field) via a power source operating at a voltage between about 5 V and about 500 V, in constant voltage mode.

34. The method of item 31 , wherein the power source generates an output current of at most between about 30 mA and 1000 mA, particularly of 50 to 200mA, more particularly 100 to 150mA.

35. The method of any of items 22-34, the applying including applying the electrical signal via a power source operating for about 10 minutes or more, particularly between 15 min and 500 min, more particularly between 15 min and 200 min.

36. The method of any of items 22-35, further comprising disposing a staining agent in the chamber, such that the applying results in the staining of the tissue sample with the staining agent.

37. The method of item 36, the disposing including dissolving the staining agent in the first buffer, in the second buffer, or both. 38. The method of item 36 or 37, wherein the staining agent carries a net negative charge, the disposing including dissolving the staining agent in the first chamber portion, the applying including configuring the first electrode in the first chamber portion as a cathode and configuring the second electrode in the third chamber portion as an anode.

39. The method of item 36 or 37, wherein the staining agent carries a net positive charge, the disposing including dissolving the staining agent in the first chamber portion, the applying including configuring the first electrode in the first chamber portion as an anode and configuring the second electrode in the third chamber portion as a cathode.

40. The method of any of items 36-39, wherein the staining agent is selected from the group consisting of one or more proteins, and one or more nucleic acids.

41 . The method of item 36 to 40, the disposing including entrapping the staining agent in a non-crosslinking gel and disposing the non-crosslinking gel in the chamber.

42. The method of item 41 , the disposing including casting the non-crosslinking gel on a surface of the tissue sample.

43. The method of item 41 , the disposing including casting the non-crosslinking gel in a capsule and disposing the capsule in the first chamber portion or in the third chamber portion.

44. The method of any of items 36-43, wherein at least one of the first buffer and the second buffer includes an enzymatic agent for digesting one or more components of the tissue sample.

45. The method of item 44, wherein the one or more components of the tissue sample were previously introduced into the tissue sample.

46. The method of any of items 36-45, the applying including applying the electrical signal via a power source operating at a voltage between about 5 V and about 500 V, particularly of 10 to 100 V, more particularly of 20 to 60V, in constant voltage mode.

47. The method of any of items 36-46, the applying including applying the electrical signal via a power source operating for about 10 minutes or more.

48. The method of any of items 22-47, further comprising applying a pressure wave to the sample via a transducer of the apparatus.

49. The method of item 48, wherein the pressure wave is a continuous wave.

50. The method of item 48, wherein the pressure wave is a pulse wave.

51. The method of item 48, wherein the pressure wave is a standing wave. 52. The method of item 48, wherein the pressure wave is an acoustic wave.

53. The method of any of items 48-52, wherein the transducer is an ultrasonic transducer.

54. The method of any of items 48-53, the applying the pressure wave including applying the pressure wave in a direction parallel to the electric field vector of the electric field.

55. The method of any of items 48-53, the applying the pressure wave including applying the pressure wave in a direction orthogonal to the electric field vector of the electric field.

56. The method of any of items 48-55, the applying the pressure wave including applying the pressure wave to the tissue sample simultaneously with the applying the electric field.

57. The method of any of items 48-55, the applying the pressure wave including applying the pressure wave to the tissue sample in an overlapping manner with the applying the electric field.

58. The method of any of item 48-55, the applying the pressure wave including applying the pressure wave to the tissue sample in an interleaved manner with the applying the electric field.

59. A method, comprising:

at a first time:

flowing a first buffer in the first chamber portion and flowing a second buffer in the third chamber portion, the first buffer and the second buffer including ionic detergents and non-ionic detergents;

applying a first electrical signal to the sample to induce electrophoresis in the sample via a first electrode and a second electrode of a set of electrodes of the apparatus, the first electrode of the set of electrodes disposed in the first chamber portion and the second electrode of the set of electrodes disposed in the third chamber portion, wherein during application of an electric field between the first electrode and the second electrode, the electric field lines of the electric field pass only through the sample in the second chamber portion;

applying a first pressure wave to the sample via a transducer of the apparatus such that the applying the first electrical signal and the applying the first pressure wave collectively results in at least partial lipid extraction from the tissue sample;

at a second time different than the first time:

flowing a third buffer in the first chamber portion and flowing a fourth buffer in the third chamber portion;

disposing a staining agent in the chamber;

applying a second electrical signal to the sample to induce electrophoresis in the sample; applying a second pressure wave to the sample via the transducer of the apparatus, such that the applying the second electrical signal and the applying the second pressure wave collectively results in the staining of the tissue sample with the staining agent.

List of reference numerals

Claims

1 . A method for preparing a tissue sample (101 , 201 ) for microscopy, comprising the steps of

a. providing a tissue sample (101 , 201 );

b. submitting said tissue sample (101 , 201 ) to a chemical fixation treatment, yielding a fixed tissue sample (101 , 201 );

c. mounting said fixed tissue sample (101 , 201 ) inside of an electrically insulating sample holder (102, 202, 902), said sample holder (102, 202, 902) defining a sample volume (121 , 221 ) configured to receive, particularly to hold, the tissue sample (101 , 201 ) within the sample holder (102, 202, 902), the sample holder (102, 202, 902) further having a first opening (1 17A, 217A) and a second opening (1 17B, 217B), with the sample volume (121 , 221 ) disposed between the first opening (1 17A, 217A) and the second opening (1 17B, 217B);

d. mounting said sample holder (102, 202, 902) between a first electrolyte chamber (103A) comprising a first electrode (104A, 904A) and a second electrolyte chamber (103C) comprising a second electrode (104B, 904B), and wherein i. said first electrolyte chamber (103A) is configured to comprise an electrolyte solution (1 10) in contact with said tissue sample (101 , 201 ); ii. said second electrolyte chamber (103C) is configured to comprise an electrolyte solution (1 10) in contact with said tissue sample (101 , 201 ); e. in a clearing step, applying an electric potential difference (voltage) to said first electrode (104A, 904A) and said second electrode (104B, 904B) wherein

said first electrolyte chamber (103A) is filled with a first electrolyte solution (1 10A) comprising an ionic detergent having an amphiphilic ion and a counterion, and

said second electrolyte chamber (103C) is filled with a second electrolyte solution (1 10B), and

wherein said second electrode (104B, 904B) is an anode if said amphiphilic ion has a negative charge, and wherein said second electrode (104B,

904B) is a cathode if said amphiphilic ion has a positive charge.

2. The method according to claim 1 , wherein

a. the electric potential difference is in the range of 10V to 100V, particularly in the range of 20V to 60V; and/or b. the electric potential difference is adjusted, particularly throughout the clearing step, to yield an electric current of 5 mA to 200 mA, particularly of 100-150 mA; and/or

c. a diameter of the sample volume (121 , 221 ) is between 3mm and 30mm; and/or d. a distance between the first electrode (104A, 904A) and the second electrode (104B, 904B) is 10 mm to 150 mm, particularly between 20 mm and 100 mm; and/or

e. the fixation step is conducted by keeping the tissue sample (101 , 201 ) in a solution comprising 2 % to 6% (particularly 4%) (w/v) acrylamide comprising, in relation to the acrylamide content, 0,5% to 2% (particularly 1 ,2%) (w/w) bisacrylamide, and/or 0,5% to 2% (w/v) formaldehyde or glutaraldehyde; and/or f. the concentration of the ionic detergent is 2% to 8% (w/v);

g. the concentration of ions inside of the sample volume (121 , 221 ) of the first electrolyte solution (1 10A) and/or the second electrolyte solution (1 10B) without counting the concentration of the ionic detergent, is 150 to 250 mOsm; and/or h. the clearing step is conducted for <24h to achieve full migration of the ionic detergent from the first electrolyte chamber (103A) to the second electrolyte chamber (103C).

3. The method according to claims 1 or 2, wherein the clearing step is followed by a

washing step, in which said first electrolyte chamber (103A) is filled with a third electrolyte solution devoid of detergent, and said second electrolyte chamber (103C) is filled with a fourth electrolyte solution devoid of detergent, and an electric potential difference (voltage) is applied to said first electrode (104A, 904A) and said second electrode (104B, 904B).

4. The method according to any one of the preceding claims, wherein subsequent to the clearing step, in a staining step a staining preparation comprising a dye specific for a biomolecule comprised in the tissue sample (101 , 201 ) is introduced into the first electrolyte chamber (103A) and/or into the tissue sample (101 , 201 ), and an electrical potential difference is applied effecting the dye being drawn towards the second electrode (104B, 904B).

5. The method according to claim 4, wherein the staining preparation is applied

a. comprised in, or in the form of, an electrolyte solution and/or b. comprised in, or in the form of, a hydrogel preparation (223), particularly a noncrosslinking hydrogel preparation, positioned between the first electrode (104A, 904A) and the tissue sample (101 , 201 ).

6. The method according to claim 4, wherein the staining preparation is applied by inserting a hollow microneedle having a diameter < 1/100 of the diameter of the sample volume (particularly a diameter of 20 pm to 150 pm) into the tissue sample (101 , 201 ) and injecting the staining preparation into the tissue sample (101 , 201 ) by action of a micropump or by applying an electric potential difference between the inside of the hollow microneedle and the tissue sample (101 , 201 ).

7. The method according to claim 6, wherein an array comprising 9 to 81 of said

microneedles is employed to apply the staining preparation.

8. The method according to any one of the preceding claims, wherein pressure waves are applied to the sample during the clearing step, the washing step and/or the staining step, particularly wherein the pressure waves are acoustic waves in the range of 200kHz to 5MHz, more particularly in the range of 1 to 2 MHz.

9. The method according to any one of the preceding claims, wherein the clearing step, the washing step and/or the staining step are conducted maintaining a temperature of 37°C to 42°C, particularly a temperature of 38,5°C to 40,5°C.

10. The method according to any one of the preceding claims, wherein the electrolyte

solution (1 10) comprised in the first electrolyte chamber (103A) and/or the second electrolyte chamber (103C) is circulated or exchanged during application of said electric potential difference.

1 1. The method according to any one of the preceding claims, wherein the sample holder (102, 202, 902) is

a tube and/or

separable into two parts (202A, 202B), a first part (202A) comprising the first opening (217A) and a second part (202B) comprising the second opening (217B), with the sample volume (221 ) being confined or defined by the first part (202A) and the second part (202B).

12. The method according to any one of the claims 4 to 1 1 , wherein prior to the staining step a cross-linking hydrogel, particularly poly-acrylamide, is provided in a first section (140A, 140B) of said first electrolyte chamber (103A) and/or said second electrolyte chamber (103C) wherein a second section (141 A, 141 B) of said first and/or second electrolyte chamber (103A, 103C) is filled with said first or second electrolyte solution (1 10A, 1 10B), particularly wherein a hydrogel preparation, more particularly from a non-crosslinking hydrogel (such as low-melt agarose), comprising a staining preparation is embedded in the cross-linking hydrogel, more particularly adjacent to said sample volume (121 ) and/or adjacent to said tissue sample (101 , 201 ).

13. The method according to any one of the preceding claims, wherein prior to said clearing step, washing step and/or staining step, said tissue sample is arranged between a first membrane (191 ) and a second membrane (192), wherein particularly said first membrane (191 ), said tissue sample (101 , 201 ) and said second membrane (192) are arranged, particularly stacked, in a vertical direction, wherein said electric potential difference (in said clearing step, washing step and/or staining step) is applied in said vertical direction, more particularly wherein a positive electric potential is applied above the tissue sample (101 , 201 ) (in other words the electrode above the tissue sample is the anode).

14. A device (100, 900) for electrophoretic clearing, washing and/or staining of a tissue

sample (101 , 201 ), said device (100, 900) comprising

- a sample holder (102, 202, 902) made of an electrically insulating material, said sample holder (102, 202, 902) defining a sample volume (121 , 221 ) configured to receive, particularly to hold, a tissue sample (101 , 201 ), particularly a tissue sample (101 , 201 ) embedded in a hydrogel, within the sample holder (102, 202, 902), the sample holder (102, 202, 902) further having a first opening (1 17A, 217A) and a second opening (1 17B, 217B), with the sample volume (121 , 221 ) disposed between the first opening (1 17A, 217A) and the second opening (1 17B, 217B),

wherein said first opening (1 17A, 217A) is configured to be brought into fluid communication with a first electrolyte chamber (103A) and said second opening (1 17B, 217B) is configured to be brought into fluid communication with a second electrolyte chamber (103C) and

- a first electrode (104A, 904A) and a second electrode (104B, 904B), wherein the first electrode (104A, 904A) is configured to be brought in electrically conductive contact with a first electrolyte solution (1 10A) contained in said first electrolyte chamber (103A), and wherein said second electrode (104B, 904B) is configured to be brought in electrically conductive contact with a second electrolyte solution (1 10B) contained in the second electrolyte chamber (103C), said first electrode (104A, 904A) and said second electrode (104B, 904B) being configured to be connected to a voltage source.

15. The device (100, 900) according to claim 14, wherein said device (100, 900) further comprises a generator (106, 906) for generating pressure waves, particularly acoustic waves, more particularly ultrasound waves, wherein said generator (106) is configured to transmit said pressure waves to said sample holder (102, 202, 902), particularly to said sample volume (121 , 221 ), such that said pressure waves are transmittable to a tissue sample (101 , 201 ) arranged in said sample volume (121 , 221 ) of the sample holder (102, 202, 902).

16. The device (100, 900) according to claim 15, wherein said generator (106, 906) is

configured to generate acoustic waves in the range of 200kHz to 5MHz, particularly in the range of 1 to 2 MHz.

17. The device (100, 900) according to any one of the claims 14 to 16, wherein said sample holder (102, 202, 902) comprises a third opening (217C) and a fourth opening (217D) configured to be brought in fluid communication with said sample volume (221 ), particularly wherein said third opening (217C) and said fourth opening (217D) are arranged along a first axis (A1 ) and said first opening (217A) and said second opening (217B) are arranged along a second axis (A2) which is non-parallel to the first axis (A1 ), more particularly wherein said first axis (A1 ) is perpendicular to said second axis (A2).

18. The device (100, 900) according to claim 17, wherein said sample holder (102, 202, 902) defines a first compartment (225A), a second compartment (225B), a third compartment (225C) and/or a fourth compartment (225D), wherein said first opening (217A) leads to said first compartment (225A), said second opening (217B) leads to said second compartment (225B), said third opening (217C) leads to said third compartment (225C), and said fourth opening (217D) leads to said fourth compartment (225D), particularly wherein said first compartment (225A), said second compartment (225B), said third compartment (225C) and/or said fourth compartment (225D) is delimited by a respective conical inner wall tapering towards said sample volume (221 ).

19. The device (100, 900) according to claim 18, wherein said first compartment (225A), said second compartment (225B), said third compartment (225C) and/or said fourth compartment (225D) is configured to receive, particularly hold, a hydrogel preparation (223), particularly from a non-crosslinking hydrogel, comprising a staining preparation.

20. The device (100, 900) according to any one of the claims 14 to 19, wherein

- said first electrolyte chamber (103A) comprises an axial opening (142A) configured to be brought in fluid communication with said first opening (1 17A) of said sample holder (102, 202, 902) and said second electrolyte chamber (103C) comprises an axial opening (142B) configured to be brought in fluid communication with said second opening (1 17B) of said sample holder (102, 202, 902), and wherein

- said first electrolyte chamber (103A) and/or said second electrolyte chamber (103C) comprises a first section (140A, 140B) extending along a first axis (A1 ) between said respective axial opening (142A, 142B) and a respective lateral wall (144A, 144B) of the first and/or second electrolyte chamber (103A, 103C) and a second section (141A,

141 B) adjacent to said respective first section (140A, 140B), wherein said second section (141A, 141 B) extends along a second axis (A2) or third axis (A3) perpendicular to said first axis (A1 ) from said respective first section (140A, 140B) to a respective top opening (143A, 143B) of said first and/or second electrolyte chamber (103A, 103C), particularly wherein said first electrolyte chamber (103A) and/or said second electrolyte chamber is L-shaped when viewed in a cross-sectional plane defined by the first axis (A1 ) and the second axis (A2), and/or the first axis and the third axis (A3).

21. The device (100, 900) according to claim 20, wherein said first electrolyte chamber (103A) is comprised in a first block (130A) comprising a first half shell (131 A) and a second half shell (132A), wherein said first half shell (131 A) and said second half shell (132A) jointly form said first electrolyte chamber (103A) when assembled, and/or wherein said second electrolyte chamber (103C) is comprised in a second block (130B) comprising a first half shell (131 B) and a second half shell (132B), wherein said first half shell (131 B) and said second half shell (132B) jointly form said second electrolyte chamber (103C) when assembled.

22. The device (100, 900) according to any one of the claims 14 to 21 , wherein said sample holder (102, 202, 902) comprises a first membrane (191 ), particularly a semi-permeable membrane, and a second membrane (192), particularly a semi-permeable membrane, wherein said tissue sample (101 , 201 ) is arrangeable between said first membrane (191 ) and said second membrane (191 ).

23. The device according to claim 22, wherein said sample holder (102, 202, 902) comprises a bottom part (150) configured to be placed in a liquid reservoir, particularly the second electrolyte chamber (103C), such that said first membrane (191 ), said tissue sample (101 ) and said second membrane (191 ) are arranged in a vertical direction, particularly wherein said sample holder (102, 202, 902) extends along a first axis (A1 ), wherein the bottom part (150) comprises a ring shaped base (152) extending in the circumferential direction in respect of the first axis (A1 ), wherein said base (152) defines a space (158), more particularly wherein said base (152) comprises at least one recess (153) configured to allow entry of said second electrolyte (1 10B) from said liquid reservoir into said space (158) via said recess (153).

24. The device (100, 900) according to any one of claims 14 to 23, wherein the sample

holder (102, 202, 902) is a hollow tube.

25. The device (100, 900) according to any one of claims 14 to 23, wherein the sample

holder (102, 202, 902) is separable into two parts (202A, 202B), a first part (202A) comprising the first opening (217A) and a second part (202B) comprising the second opening (217B), particularly wherein the sample volume (221 ) is confined or defined by the first part (202A) and the second part (202B).

26. The device (100, 900) according to any one of claims 14 to 25, further comprising at least one hollow microneedle (193), particularly a microneedle (193) having a diameter < 1/100 of a diameter of the sample volume (121 , 221 ) (more particularly a diameter of 20 pm to 150 pm), said microneedle (193) being configured to be inserted into said sample volume (121 , 221 ) such that a tissue sample (101 , 201 ) arranged in the sample volume (121 ,

221 ) is penetrable by said hollow microneedle (193), particularly such that a staining preparation (195) is injectable into said tissue sample (101 , 201 ) by said microneedle (193).

27. The device (100, 900) according to claim 26, said hollow microneedle (193) comprising a microneedle electrode (194) capable of applying an electrical potential difference between a tip (199) of said microneedle (193) and said sample volume (121 , 221 ) of the sample holder (102, 202, 902) or relative to said first electrode (104A, 904A) and/or said second electrode (104B, 904B).

28. The device (100, 900) according to any one of the claims 14 to 27, wherein said device (100, 900) further comprises a third electrode (904C) and a fourth electrode (904D), wherein the third electrode (904C) is configured to be brought in electrically conductive contact with a first electrolyte (1 10A), contained in said first electrolyte chamber (103A), and wherein said fourth electrode (904D) is configured to be brought in electrically conductive contact with a second electrolyte (1 10B), particularly contained in the second electrolyte chamber (103C), said third electrode (904C) and said fourth electrode (904D) being configured to be connected to a voltage source.