Preparation of tissue samples for imaging

Abstract

The present disclosure involves preparing a cryostat tissue section for use with electron microscopy and other high resolution imaging techniques. A tissue sample is soaked in a solution having a predefined concentration of freeze protectant that prevents creation of freeze damage artifacts in the tissue sample that are greater than 10 nanometers in at least one dimension. The tissue sample is frozen in order to perform a cryostat sectioning. The cryostat sectioning is performed on the tissue sample to produce a cryostat tissue section. The cryostat tissue section is mounted onto a slide, and the cryostat tissue section is covered on the slide with the solution having the predefined concentration of freeze protectant.

Description

PREPARATION OF TISSUE SAMPLES FOR IMAGINGInventors: Mike Reichelt and Miriam Sagullo BacaCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U. S. Provisional Patent Application number 63 / 614,004 filed on 22 December 2023.BACKGROUND

[0002] Electron microscopy of biological samples can provide structural information about cell and tissue architecture with unparalleled detail and context in the nanometer range. However, sizes of tissue samples that are viewable by electron microscopy are still very small. For example, the size of such tissue samples are less than .5 square millimeters and are not large enough to be able to target specific structures to be viewed.SUMMARY

[0003] In accordance with the present disclosure, a method is provided that comprises soaking a tissue sample in a solution having a predefined concentration of freeze protectant that prevents a formation of at least one freeze damage tissue artifact in the tissue sample that is greater than 10 nanometers in at least one dimension, freezing the tissue sample in order to perform a cryostat sectioning, and performing the cryostat sectioning on the tissue sample to produce a cryostat tissuesection. The method further comprises mounting the cryostat tissue section onto a slide, and covering the cryostat tissue section on the slide with the solution having the predefined concentration of freeze protectant.

[0004] In addition, a method is provided that comprises soaking a tissue sample in a solution having a concentration of at least 60% sucrose, freezing the tissue sample for a cryostat sectioning, and performing the cryostat sectioning on the tissue sample to produce a cryostat tissue section. The method further comprises mounting the cryostat tissue section onto a slide, and covering the cryostat tissue section on the slide with the solution having the concentration of at least 60% sucrose.

[0005] Still further, an arrangement is provided that comprises a slide, a cryostat tissue section mounted on the slide, the cryostat tissue section having a cross sectional area of at least 1 square millimeter, and the cryostat tissue section being infiltrated with a solution having a concentration of at least 60% sucrose. An amount of the solution covers an exposed side of the cryostat tissue section on the slide.

[0006] Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described features are usable in all portions of the disclosuretaught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the present disclosure are combinable and interchangeable with one another.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Many features and examples of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, with emphasis instead being placed upon clearly illustrating the principles of the disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.

[0008] FIG. 1 illustrates the generation of a tissue sample from a raw tissue sample in accordance with the present disclosure.

[0009] FIGS. 2A and 2B illustrate a soaking of a tissue sample in accordance with the present disclosure.

[0010] FIG. 3 depicts a flash freezing of a tissue sample that is subjected to cryostat sectioning in accordance with the present disclosure.

[0011] FIGS. 4A, 4B, and 4C depict further processing of a cryostat tissue section obtained from the tissue sample of FIG. 1 in accordance with the present disclosure.

[0012] FIGS. 5A and 5B depict examples of tissue samples taken from a cryostat tissue section as depicted in FIGS. 4A, 4B, and 4C in accordance with the present disclosure.

[0013] FIGS. 6A, 6B, and 6C depict examples of tissue samples illustrating imaging of a cryostat tissue section in accordance with the present disclosure.

[0014] FIG. 7 is a flow chart of one example method for generating and storing cryostat tissue sections in accordance with the present disclosure.DETAILED DESCRIPTION

[0015] Disclosed are various approaches for creating cryostat tissue sections of a raw tissue sample from which further sections may be taken that can be used for high resolution imaging such as electron microscopy, immunogold electron microscopy or other high resolution imaging technologies. The cryostat tissue sections can provide for the ability to create wide scale tissue sections that are suitable for high resolution imaging such as electron microscopy or other high resolution imaging technologies. Such wide scale tissue sections provide for much better orientation in terms of finding regions of interest, greater visibility into the histology of a given sample, and superior ultrastructural preservation to facilitate imaging detail in tissue on a nanometer scale. In accordance with the present disclosure, the freeze damage and / or other damage is minimized or prevented to such wide scale tissue samples so that, ultimately, finer tissue sections can be created therefrom that are suitable for use with high resolution imaging such as electron microscopy or other high resolution imaging technologies.

[0016] Although the following discussion provides illustrative examples of the operation of various components of the present disclosure, the use of the followingillustrative examples does not exclude other implementations that are consistent with the principles disclosed by the following illustrative examples.

[0017] With reference to FIG. 1 , shown is a raw tissue sample 103 that is to be processed in accordance with the present disclosure. The raw tissue sample 103 may comprise, for example, tissue taken from a kidney, brain, liver, tonsil, or other organs or structures. The raw tissue sample 103 may be taken from organs of a human, an animal, plant, or other living organism.

[0018] The raw tissue sample 103 is subjected to a chemical fixative to promote morphological preservation. According to one example of the present disclosure, the raw tissue sample 103 is subjected to a “light” fixation to prevent the fixative from interfering with later processes as will be described. This may entail subjecting the raw tissue sample 103 to a fixative such as 4% paraformaldehyde (PFA), which is compatible with downstream immunohistochemistry applications, or 2% paraformaldehyde with 2% glutaraldehyde for downstream morphology imaging applications such as electron microscopy. Given that such a light fixation is applied to the raw tissue sample 103, the time from when the raw tissue sample 103 is harvested to the time it is processed as described herein may be reduced to ensure minimal morphological degradation such that resulting tissue sections are stable for at least 1 month at 4° Celsius.

[0019] The raw tissue sample 103 is subjected to raw tissue sectioning 106 to cut the raw tissue sample 103 into tissue samples 109 for further processing. The tissue samples 109 are cut so that a resulting tissue sample 109 comprises at least one dimension that is less than 5 millimeters. The tissue sample 109 may be a slicewith a maximum thickness T of approximately 5 millimeters or less, where the thickness T of the slice comprises the dimension that is 5 millimeters or less. For example, the tissue samples 109 can be cut or sliced so that the resulting tissue sample 109 comprises at least one dimension that is less than 2 millimeters.

[0020] Alternatively, the raw tissue sample 103 may be separated into sections having at least one dimension that is greater than 1 millimeter given adjustments to the procedure as will be described.

[0021] In addition, given that the tissue sample 109 results from slicing the raw tissue sample 103, the tissue sample 109 is also subjected to the fixative such as 4% paraformaldehyde (PFA), glutaraldehyde, or other fixative as mentioned above.

[0022] Referring next to FIGS. 2A and 2B, shown is a vessel 113 that contains an amount of solution that includes a predefined concentration of freeze protectant. The freeze protectant within the solution may comprise, for example, sucrose, glycerol, or another appropriate freeze protectant. A solution that includes a predefined concentration of freeze protectant is referred to herein as freeze protectant solution 116.

[0023] In a case where the freeze protectant comprises sucrose, the corresponding freeze protectant solution 116 may have a concentration of sucrose within a range having a lower limit of 60% up to the point of saturation, where the concentration of sucrose is approximately 79% or 2.3 Molar. That is to say, the freeze protectant solution 116 may have a concentration of sucrose that ranges from 60% to a point of a maximum concentration possible up to a point of saturation of the freeze protectant solution 116 with sucrose, which is approximately 2.3 Molarsucrose. As an additional alternative, the concentration of sucrose in the freeze protectant solution 116 may be greater than or equal to 60%. In one specific example, the concentration of sucrose in the corresponding freeze protectant solution 116 comprises substantially 79% or 2.3 Molar where the concentration may vary by 1 % below saturation.

[0024] If other freeze protectants are used, then the concentration of such freeze protectants in the respective freeze protectant solutions 116 may be determined by way of an iterative process to achieve a minimum of freeze damage to the tissue sample 109.

[0025] In one example, the concentration of freeze protectant in an example of freeze protectant solution 116 may be a predefined concentration that prevents creation of one or more freeze damage artifacts that are greater than 10 nanometers in at least one dimension in the tissue sample 109. For example, such freeze damage artifacts may comprise a gap of at least 10 nanometers in the tissue sample 109 formed due to the expansion of water as the tissue sample 109 is frozen. Without freeze protection as set forth herein, intracellular water may form hexagonal ice crystals that expand in volume and ultimately distort membranes and organelles forming "holes” that destroy cell ultrastructure.

[0026] Also note that the solvent in the freeze protectant solution 116 may be water, phosphate buffered saline (PBS), or other liquid.

[0027] Once a tissue sample 109 has been obtained by slicing or otherwise dividing the raw tissue sample 103 (FIG. 1 ), the tissue sample 109 can then be soaked or submerged in the freeze protectant solution 116 for a predefined periodof time to facilitate a predefined level of infiltration 119 of the freeze protectant solution 116 into the tissue sample 109. In one example, the predefined period of time is specified so that the infiltration 119 of the freeze protectant solution 116 is a full infiltration 119 as will be described below. Such predefined period of time may comprise, for example, 24 hours or other period of time to cause the desired infiltration 119 as may be indicated by the tissue sample 109 sinking in the freeze protectant solution 116 as will be described further below.

[0028] Once the tissue sample 109 is placed in the freeze protectant solution 116 in the vessel 113, the freeze protectant solution 116 is maintained at a temperature of approximately 4° Celsius to help preserve the tissue sample 109 during the process of infiltration of the freeze protectant solution 116 into the tissue sample 109. This may be accomplished by placing the vessel 113 containing the freeze protectant solution 116 and the tissue sample 109 into a refrigerator that is set for 4° Celsius. Alternatively, the freeze protectant solution 116 and the tissue sample 109 may be maintained at a temperature other than approximately 4° Celsius such as any temperature within the range of 4 ° Celsius to 25° Celsius provided that such temperature provides for adequate preservation of the tissue sample 109 during the process of infiltration 119 of the freeze protectant into the tissue sample 109. In one example, it is noted that small tissue pieces measuring 1 cubic millimeter can be infiltrated within 1 hour at room temperature.

[0029] As contemplated herein, it is understood that a refrigerator set at 4° Celsius may vary within a predefined tolerance of plus or minus up to 3° Celsius, where 4° Celsius is a target temperature. Accordingly, for all temperature targetsstated herein, it is understood that each such temperature target may vary within an acceptable predefined tolerance of, for example, plus or minus 3° Celsius.However, to minimize the possibility that freeze damage artifacts are formed in the tissue sample 109, the temperature at which the vessel 113 containing the freeze protectant solution 116 and the tissue sample 109 can be maintained above freezing or 0° Celsius.

[0030] FIG. 2B shows a portion of the tissue sample 109 that illustrates the infiltration 119 of the freeze protectant solution 116 into the tissue sample 109. As contemplated herein, the infiltration 119 of the freeze protectant solution 116 includes the diffusion of the freeze protectant into the tissue sample 109. Also, as the freeze protectant solution 116 infiltrates into the tissue sample 109, it is understood that the freeze protectant in the freeze protectant solution 116 also infiltrates the tissue sample 109.

[0031] In one example, a period of time in which the tissue sample 109 is soaked in the freeze protectant solution 116 is such that the infiltration 119 of the freeze protectant solution 116 into the tissue sample 109 is full or complete infiltration such that the freeze protectant solution 116 infiltrates all regions or volumes of the tissue sample 109 that can be infiltrated. This reflects the fact that there may be some elements of a given tissue sample 109 that cannot be infiltrated.

[0032] In one example, a degree of infiltration 119 of the freeze protectant into the tissue sample 109 may be deemed adequate when the tissue sample 109 sinks into the freeze protectant solution 116 below an initial floating level 123a (FIG. 2A) to a lower floating level 123b (FIG. 2B). In this respect, the tissue sample 109 maybe suspended in the middle of the freeze protectant solution 116. The sinking of the tissue sample 109 below the initial floating level 123a reflects the fact that the infiltration 119 has reached such a point that the buoyancy of the tissue sample 109 in the freeze protectant solution 116 is reduced given that the tissue infiltrated with sucrose solution is heavier than the sucrose solution, thereby causing the tissue sample 109 to sink in the freeze protectant solution 116. Accordingly, in one example, the tissue sample 109 is soaked at least until the tissue sample 109 sinks below the initial floating level 123a in the freeze protectant solution 116. A specific sinking threshold may be established where the tissue sample 109 is deemed adequately infiltrated when the tissue sample 109 sinks below such threshold. In another example, the soaking of the tissue sample 109 is performed until the tissue sample 109 sinks to the bottom of the vessel 113, thereby indicating that full infiltration has occurred. For many tissue samples 109, this may be accomplished by soaking the tissue sample 109 for 24 hours.

[0033] As an additional alternative, the tissue sample 109 is soaked in the freeze protectant solution 116 for a predefined period of time, thereby resulting in a predefined degree of infiltration 119 that may be less than a full or complete infiltration 119 if such degree of infiltration 119 is adequate to protect the tissue sample 109 from freeze damage as will be described. The period of time specified for the soaking of a tissue sample 109 in freeze protectant solution 116 may be determined by an iterative process and is affected by the size of the tissue sample 109 and the type of tissue that makes up the tissue sample 109.

[0034] Further, the period of time that a tissue sample 109 is soaked in the freeze protectant solution 116 may be specified based on a maximum thickness T of the tissue sample 109 (FIG. 1 ) in one dimension. That is to say, a maximum thickness T of given tissue sample 109 may vary. The period of time that a tissue sample 109 is soaked in the freeze protectant solution 116 may vary depending on the actual thickness T of the tissue sample 109.

[0035] For example, if the thickness T of the tissue sample 109 is 2 millimeters or less, the period of time that the tissue sample 109 is soaked in the freeze protectant solution 116 with a concentration of, for example, 79% sucrose or a concentration just before saturation, may be at least 60 minutes. Ultimately, the period of time that a tissue sample 109 is soaked in the freeze protectant solution 116 is specified to achieve a predefined degree of infiltration 119. Ultimately, the period of time a tissue sample 109 is soaked in a given freeze protectant solution 116 of a given formulation to achieve the desired infiltration 119 of freeze protectant solution 116 into the tissue sample may be determined by way of an iterative process.

[0036] As mentioned above, the tissue sample 109 is cut from the raw tissue sample 103. To achieve a full or complete infiltration 119 of the freeze protectant solution 116 into the tissue sample 109, the smallest width or thickness of the tissue sample 109 in a single dimension cannot be too great such that the freeze protectant solution 116 is prevented from infiltrating through to the center of the tissue sample 109. Thus, when slicing the raw tissue sample 103, care is taken to ensure that the maximum width or thickness in one dimension is not so large as toprevent a desired infiltration 119 of the tissue sample 109. In one example, the slicing of the raw tissue sample 103 results in slices of tissue samples 109 that are up to 5 millimeters.

[0037] In addition, as mentioned above, when subjecting the raw tissue sample, 103 to the chemical fixative to promote morphological preservation, a light fixation is used as mentioned above. The light fixation is performed, for example, to preserve tissue structure and antigens for immunohistochemistry. According to one example, the percent concentration of the fixative is specified so that the fixative that infiltrates the raw tissue sample 103, and the tissue sample 109 as part of the raw tissue sample 103, does not interfere with the infiltration 119 of the freeze protectant solution 116 and the corresponding freeze protectant into the tissue sample 109. Examples of a light fixative are set forth above.

[0038] With reference to FIG. 3, once the tissue sample 109 has been soaked for the required period of time such that the freeze protectant solution 116 and the freeze protectant have infiltrated the tissue sample 109 in accordance with the specifications set forth above, next the tissue sample 109 is placed in a bath of Optimal Cutting Temperature (OCT) compound 133 in a mold 136 that is compatible with a given cryostat. In doing so, the tissue sample 109 is suspended in the OCT compound 133 in the mold 136.

[0039] Thereafter, the mold 136 containing the OCT compound 133 and the tissue sample 109 is subjected to flash freezing 139, for example, by submerging the same in liquid nitrogen. Due to the fact that the freeze protectant has infiltrated the tissue sample 109 as described above, any damage to the morphology of thetissue sample 109 is minimized or prevented. Such damage might typically occur due to the expansion of water during freezing. Freezing damage is particularly visible on a nanometer scale when using an electron microscope or other such high-resolution imaging technologies. To the extent that damage occurs, in one example such damage is below a threshold so that sections may be obtained from the tissue sample 109 that are ultimately suitable for electron microscopy or other high resolution imaging technologies.

[0040] Thereafter, cryostat sectioning 143 is performed on the mold 136 with the frozen OCT compound 133 and the tissue sample 109 in a suitable cryostat to produce one or more cryostat tissue sections while preserving the ultrastructural detail. Such a cryostat may comprise, for example, a Leica Cryostat model CM3050S manufactured by Leica Biosystems headquartered in Nussloch, Germany. The cutting temperature of the cryostat may be, for example, in a range of -35° Celsius to -45° Celsius and the resulting cryostat tissue sections may comprise slices of the tissue sample 109 with a thickness that falls, for example, within a range of 2 to 10 micrometers.

[0041] In order to ensure that the resulting cryostat tissue sections are suitable for electron microscopy and other similar purposes, care is taken to minimize sectioning artifacts when slicing the tissue sample 109 into cryostat tissue sections. According to one example, a knife angle setting of a cryostat is specified as 5 degrees.

[0042] Referring next to FIG. 4A, once a cryostat tissue section 146 has been produced by virtue of the cryostat sectioning 143 as described above, next suchcryostat tissue section 146 is mounted onto a slide 149. The slide 149 may be gold plated, or other type of slide. In one example the slide 149 may be Superfrost Plus Gold Microscope Slides manufactured by Thermo Fisher Scientific of Waltham, Massachusetts.

[0043] Then, as shown in FIG. 4B, the cryostat tissue section 146 is covered on the slide 149 with an amount of the freeze protectant solution 116 as soon as possible after the cryostat tissue section 146 adheres to the slide 149. The freeze protectant solution 116 is placed onto the cryostat tissue section 146 on the slide 149 in a manner such that the freeze protectant solution 116 completely covers the cryostat tissue section 146. Thereafter, as shown in FIG. 4C, the next task is to store the slide 149 with the cryostat tissue section 146 covered by or submerged in the freeze protectant solution 116 in a refrigerator 153.

[0044] With reference to FIGS. 4A, 4B, and 40, the mounting of the cryostat tissue section 146 to the slide 149 and the application of the freeze protectant solution 116 is performed as quickly as possible after the cryostat tissue section 146 is sliced from the tissue sample 109 to minimize any drying that may occur to the cryostat tissue section 146. Adsorption may occur from 10 seconds to 120 seconds depending on the thickness of the cryostat tissue section 146. If multiple cryostat tissue sections 146 are produced, then multiple slides 149 may be needed for the mounting of the individual cryostat tissue sections 146. A cryostat tissue section 146 is mounted on the slide 149 in a manner so as to adhere to the slide 149 as can be appreciated.

[0045] In one example, the mounting of a cryostat tissue section 146 onto the slide 149 and the covering of the cryostat tissue section 146 with the freeze protectant solution 116 is performed within a time period of a few seconds after the cryostat tissue section 146 is available for mounting from the cryostat. In one example, this time period may be 10 seconds or less for cryostat tissue sections 146 of 2 urn thickness or more. In another example, this time period may be up to 120 seconds for cryostat tissue sections 146 having a thickness of 10 urn thickness or more. A good indicator for adsorption of a cryostat tissue section 146 without completely drying out is if the Optimal Cutting Temperature (OCT) compound that surrounds the cryostat tissue section 146 turns opaque.

[0046] In another example, the time period is specified so that drying is minimal such that the morphology of the cryostat tissue section 146 is substantially preserved for purposes of viewing under an electron microscope or other devices that can view an item at a resolution of less than 200 nanometers. That is to say, the time period beginning at a first time when the cryostat tissue section 146 is mounted onto the slide 149 and ending at a second time when the freeze protectant solution 116 covers the cryostat tissue section 146 is short enough to prevent drying of the cryostat tissue section 146, thereby preserving a morphology of the cryostat tissue section.

[0047] According to one example, the cryostat sectioning of the tissue sample 109 produces cryostat tissue sections 146 having a cross-sectional area of the cryostat tissue sections 146 that can be greater than 1 square millimeter. Specifically, the cross-sectional area of the cryostat tissue sections 146 in terms ofsquare millimeters may be greater than, for example, 1 mm2, 2 mm2, 3 mm2, 4 mm2, 5 mm2, or 6 mm2, 10 mm2, 15 mm2, 20 mm2, 25 mm2, or higher. Such cross- sectional areas are termed “wide scale” herein as they are greater than 1 mm2.Such wide scale cross-sectional areas allow target tissue samples to be viewed to find target structures or areas, where tissue samples that are below 1 mm2are generally difficult to view using, for example, electron microscopy on a scale of 200 nanometers or below due to the fact that certain structures within tissue samples that one wishes to view may not be captured in such a tissue sample having such a small cross-sectional area.

[0048] In addition, the refrigerator 153 may be set to maintain each slide 1 9, cryostat tissue section 146, and the amount of freeze protectant solution 116 covering the cryostat tissue section 146 at 4° Celsius. Alternatively, the freeze protectant solution 116 and the tissue sample 109 may be maintained at a temperature other than approximately 4° Celsius such as any temperature within the range of 4-10° Celsius provided that such temperature provides for adequate preservation of the cryostat tissue section 146 for future use. Also, as contemplated herein, it is possible that the temperature inside the refrigerator 153 may vary over time, where the 4° Celsius comprises on example of a target temperature.

[0049] The slide 149 that includes the cryostat tissue section 146 covered by or submerged in the freeze protectant solution 116 may be stored in the refrigerator 153 at around 4° Celsius for up to 2 months, where the refrigerator may ultimately fluctuate between 2° Celsius and 6° Celsius. Alternatively, the storage can be any time period within which the morphology of the cryostat tissue section 146 isadequately preserved. By virtue of the fact that the cryostat tissue section 146 is covered or submerged in the freeze protectant solution 116 on the slide 149, the cryostat tissue section 146 is prevented from drying by the freeze protectant solution 116 during storage in the refrigerated compartment.

[0050] In addition, the process described herein that includes the soaking of the tissue sample 109, freezing of the tissue sample 109, performing the cryostat sectioning on the tissue sample 109 to create one or more cryostat tissue sections 146, mounting a cryostat tissue section 146 onto the slide 149, and covering the cryostat tissue section 146 with the freeze protectant solution 116 are performed without an intervening step that results in a drying of the tissue sample 109 or the cryostat tissue section 146. For example, during the process no deliberate action is taken that would result in drying of the tissue sample 109 or the cryostat tissue section 146. Stated further, neither the tissue sample 109 nor the cryostat tissue section 146 are exposed to acetone or other solution that would promote drying. Also, no deliberate step is taken such as allowing the tissue sample 109 or the cryostat tissue section 146 to stand in ambient air for more time than is necessary to cause the cryostat tissue section 146 to adhere to the slide 149 as described above.

[0051] At resolutions of 200 nanometers or less, very small variations in the structural details of a tissue sample look very large. Also, very small artifacts caused by damage to tissue samples due to freezing or drying can be very significant such that such tissue samples are not suitable for applications that operate at a resolution of below 200 nanometers. The processes described hereinto result in cryostat tissue sections 146 that retain ultrastructural morphology on a nanometer scale such that the tissue in the cryostat tissue sections 146 is suitable for viewing on a system having a resolution below 200 nanometers over a relatively wide-field area.

[0052] As such, the cryostat tissue sections 146 are suitable for further processing for several different purposes including, for example, electron microscopy morphology (morphology EM), immunogold electron microscopy (immuno-EM), correlated immunofluorescence and immunogold electron microscopy (also known as correlative light and electron microscopy (CLEM)), energy dispersive X-ray (EDX) (also known as X-ray Energy Dispersive Spectroscopy (XEDS), or other high resolution applications.

[0053] With reference to FIGS. 5A and 5B, shown are examples of human kidney tissue 156a and 156b. The specific tissue sections depicted were magnified by 200X (optical microscope setting) and the image size is 200 micrometers by 200 micrometers or 40,000 square micrometers.

[0054] The tissue section depicted in FIG. 5A was taken from a tissue sample that was soaked in a 20% concentration of freeze protectant solution that included 20% sucrose. The tissue section depicted in FIG. 5B was taken from a tissue sample that was soaked in a 79% concentration of freeze protectant solution (nearly saturated) as described above. As shown, the tissue section depicted in FIG. 5A includes freeze damage artifacts 159 comprising gaps between tissue elements due to the expansion of water during freezing. However, the tissue section depicted in FIG. 5B does not show similar freeze damage artifacts 159.

[0055] Thus, the cryostat tissue sections 146 provide a significant advantage in that they make possible wide scale tissue samples that are acceptable for electron microscopy or other high resolution imaging technologies.

[0056] Referring next to FIG. 6A, shown is an example of a cryostat tissue section 146 (FIG. 4A) imaged with wide-field light microscopy with fluorescent stain comprising 4’,6-diamidino-2-phenylindole (DAPI). As noted, the cryostat tissue section 146 is over 2.5 mm wide.

[0057] FIG. 6B shows an example of the same cryostat tissue section 146 as depicted by FIG. 6A that has been processed for electron microscopy and imaged with a wide-field back-scattered electrons (BSE) scanning electron microscope that shows much greater detail than is depicted in the image of FIG. 6A with the same scale of 2.5 mm as shown.

[0058] FIG. 6C shows a further example of an image of a cryostat tissue section 146 that has also been processed for and imaged using electron microscopy at high resolution. As shown, the image generated by the electron microscope reveals the morphology of extended reticular membranes of follicular dendritic cells.

[0059] As the images set forth in FIGS. 6A, 6B, and 6C depict, a wide scale view of the cryostat tissue section 146 is possible with little perceivable freeze damage given that the cryostat tissue section 146 is prepared in a manner as described herein.

[0060] Referring next to FIG. 7, shown is a flowchart of one example method160 according to the present disclosure. The method 160 depicted in FIG. 6 provides for one or more advantages or benefits including the creation of “widescale” tissue samples 109 (FIG. 1 ) that can be used for high resolution imaging such as electron microscopy or other high resolution imaging technologies. Specifically, the method 160 and other procedures and structures described herein minimize or prevent freeze damage to such wide scale tissue samples 109 so that they are suitable for use with high resolution imaging such as electron microscopy or other high resolution imaging technologies.

[0061] Beginning with step 163, the method 160 begins by soaking a tissue sample in a solution having a predefined concentration of freeze protectant that prevents a formation of at least one freeze damage tissue artifact in the tissue sample 109 (FIG. 1 ) that is greater than 10 nanometers in at least one dimension.

[0062] Thereafter, step 166 involves freezing the tissue sample in order to perform a cryostat sectioning 143 (FIG. 3). Then, in step 169, the method 160 further comprises performing the cryostat sectioning 143 on the tissue sample 109 to produce a cryostat tissue section 146 (FIG. 4A).

[0063] Next, in step 173, the method 160 comprises mounting the cryostat tissue section 146 onto a slide 149 (FIG. 4A). Then, in step 176, the method 160 includes covering the cryostat tissue section 146 on the slide 149 with the solution having the predefined concentration of freeze protectant. Thereafter, the process ends as shown.

[0064] With reference back to FIG. 4C, an arrangement is provided that includes the slide 149 having a cryostat tissue section mounted on the slide, the cryostat tissue section having a cross sectional area of at least 2 square millimeters, the cryostat tissue section being infiltrated with a solution having a concentration of at least 60%sucrose, and an amount of the solution covering an exposed side of the cryostat tissue section on the slide.

[0065] With reference to FIGS. 1 through 7, in view of the foregoing discussion, below is a description of various example features and examples of the present disclosure. It is understood that the below examples and features are not an exhaustive recitation of those possible in the present disclosure.

[0066] Clause 1 is a method, comprising soaking a tissue sample in a solution having a predefined concentration of freeze protectant that prevents a formation of at least one freeze damage tissue artifact in the tissue sample that is greater than 10 nanometers in at least one dimension. The method further comprises freezing the tissue sample in order to perform a cryostat sectioning, and performing the cryostat sectioning on the tissue sample to produce a cryostat tissue section. Further, the method comprises mounting the cryostat tissue section onto a slide, and covering the cryostat tissue section on the slide with the solution having the predefined concentration of freeze protectant.

[0067] Clause 2 is an method as set forth in clause 1 , wherein the cryostat sectioning on the tissue sample produces the cryostat tissue section having a cross- sectional area of greater than 2 square millimeters.

[0068] Clause 3 comprises a method as set forth in any one of clauses 1 or 2, further comprising storing the slide with the cryostat tissue section covered with the solution having the predefined concentration of freeze protectant in a refrigerator.

[0069] Clause 4 is a method, comprising soaking a tissue sample in a solution having a concentration of at least 60% sucrose, freezing the tissue sample for acryostat sectioning, performing the cryostat sectioning on the tissue sample to produce a cryostat tissue section, mounting the cryostat tissue section onto a slide, and covering the cryostat tissue section on the slide with the solution having the concentration of at least 60% sucrose.

[0070] Clause 5 is a method as set forth in clause 4, wherein the cryostat sectioning on the tissue sample produces the cryostat tissue section having a cross- sectional area of greater than 3 square millimeters.

[0071] Clause 6 is a method as set forth in clause 4, wherein the cryostat sectioning on the tissue sample produces the cryostat tissue section having a cross- sectional area of greater than 2 square millimeters.

[0072] Clause 7 comprises a method as set forth in any one of clauses 4 through 6, further comprising storing the slide with the cryostat tissue section covered with the solution having the concentration of at least 60% sucrose in a refrigerator.

[0073] Clause 8 comprises a method as set forth in any one of clauses 4 through 6, wherein the refrigerator is set at a temperature of 4° Celsius.

[0074] Clause 9 comprises a method as set forth in any one of clauses 7 or 8, wherein the cryostat tissue section is stored for less than 3 months.

[0075] Clause 10 comprises a method as set forth in any one of clauses 4 through 9, further comprising subjecting the tissue sample to a chemical fixation using a 4% paraformaldehyde (PFA) solution.

[0076] Clause 11 comprises a method as set forth in any one of clauses 4 through 10, wherein the soaking of the tissue sample, freezing of the tissue sample,performing the cryostat sectioning on the tissue sample, mounting the cryostat tissue section onto the slide, and covering the cryostat tissue section are performed without an intervening step that results in a drying of the tissue sample or the cryostat tissue section.

[0077] Clause 12 comprises a method as set forth in any one of clauses 4 through 11 , wherein the tissue sample is soaked for a predefined period of time to facilitate a full infiltration of the solution having the concentration of at least 60% sucrose into the tissue sample.

[0078] Clause 13 comprises a method as set forth in any one of clauses 4 through 11 , wherein the tissue sample is soaked at least until the tissue sample sinks below an initial floating level in the solution having the concentration of at least 60% sucrose into the tissue sample.

[0079] Clause 14 comprises a method as set forth in any one of clauses 4 through 13, wherein the tissue sample comprises at least one dimension that is less than 2 millimeters.

[0080] Clause 15 comprises a method as set forth in any one of clauses 4 through 14, further comprising minimizing a time period beginning at a first time when the cryostat tissue section is mounted onto the slide and ending at a second time when the solution having the concentration of at least 60% sucrose covers the cryostat tissue section to prevent drying of the cryostat tissue section.

[0081] Clause 16 comprises a method as set forth in clause 15, wherein the time period is less than 120 seconds.

[0082] Clause 17 comprises a method as set forth in any one of clauses 4 through 16, wherein the solution has a concentration of sucrose within a range of 60% up to a point of saturation of the solution.

[0083] Clause 18 comprises a method as set forth in any one of clauses 4 through 16, wherein a concentration of sucrose in the solution is as high as possible before saturation.

[0084] Clause 19 is an arrangement, comprising a slide, a cryostat tissue section mounted on the slide, the cryostat tissue section having a cross sectional area of at least 1 square millimeters; the cryostat tissue section being infiltrated with a solution having a concentration of at least 60% sucrose, and an amount of the solution covers an exposed side of the cryostat tissue section on the slide.

[0085] Clause 20 is an arrangement as set forth in clause 19, further comprising a frigerated compartment, and the slide having the cryostat tissue section mounted thereon is stored in the refrigerated compartment.

[0086] Clause 21 is an arrangement as set forth in clause 20, wherein the cryostat tissue section is prevented from drying by the solution during storage in the refrigerated compartment.

[0087] Clause 22 comprises a method as set forth in any one of clauses 20 or 21 , wherein the refrigerated compartment is maintained at a temperature substantially falling within a range of 2° Celsius to 6° Celsius.

[0088] Disjunctive language such as the phrase “at least one of X, Y, or Z,” unless specifically stated otherwise, is otherwise understood with the context as used in general to present that an item, term, etc., can be either X, Y, or Z, or anycombination thereof (e g., X; Y; Z; X or Y; X or Z; Y or Z; X, Y, or Z; etc.). Thus, such disjunctive language is not generally intended to, and should not, imply to require at least one of X, at least one of Y, or at least one of Z to each be present.

[0089] It should be emphasized that the above-described features and examples of the present disclosure are merely possible examples of implementations set forth for a clear understanding of the principles of the disclosure. Many variations and modifications can be made to the above-described features and examples without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.

Claims

CLAIMSTherefore, the following is claimed:

1. A method, comprising: soaking a tissue sample in a solution having a predefined concentration of freeze protectant that prevents a formation of at least one freeze damage tissue artifact in the tissue sample that is greater than 10 nanometers in at least one dimension; freezing the tissue sample in order to perform a cryostat sectioning; performing the cryostat sectioning on the tissue sample to produce a cryostat tissue section; and mounting the cryostat tissue section onto a slide; and covering the cryostat tissue section on the slide with the solution having the predefined concentration of freeze protectant.

2. The method of claim 1 , wherein the cryostat sectioning on the tissue sample produces the cryostat tissue section having a cross-sectional area of greater than 2 square millimeters.

3. The method of claim 1 , further comprising storing the slide with the cryostat tissue section covered with the solution having the predefined concentration of freeze protectant in a refrigerator.

4. A method, comprising: soaking a tissue sample in a solution having a concentration of at least 60% sucrose; freezing the tissue sample for a cryostat sectioning; performing the cryostat sectioning on the tissue sample to produce a cryostat tissue section; and mounting the cryostat tissue section onto a slide; and covering the cryostat tissue section on the slide with the solution having the concentration of at least 60% sucrose.

5. The method of claim 4, wherein the cryostat sectioning on the tissue sample produces the cryostat tissue section having a cross-sectional area of greater than 3 square millimeters.

6. The method of claim 4, wherein the cryostat sectioning on the tissue sample produces the cryostat tissue section having a cross-sectional area of greater than 2 square millimeters.

7. The method of claim 4, further comprising storing the slide with the cryostat tissue section covered with the solution having the concentration of at least 60% sucrose in a refrigerator.

8. The method of claim 7, wherein the refrigerator is set at a temperature that falls within a range of 4° Celsius and 10° Celsius.

9. The method of claim 8, wherein the cryostat tissue section is stored for less than 3 months.

10. The method of claim 4, further comprising subjecting the tissue sample to a chemical fixation using a 4% paraformaldehyde (PFA) solution.11 . The method of claim 4, wherein the soaking of the tissue sample, freezing of the tissue sample, performing the cryostat sectioning on the tissue sample, mounting the cryostat tissue section onto the slide, and covering the cryostat tissue section are performed without an intervening step that results in a drying of the tissue sample or the cryostat tissue section.

12. The method of claim 4, wherein the tissue sample is soaked for a predefined period of time to facilitate a full infiltration of the solution having the concentration of at least 60% sucrose into the tissue sample.

13. The method of claim 4, wherein the tissue sample is soaked at least until the tissue sample sinks below an initial floating level in the solution having the concentration of at least 60% sucrose into the tissue sample.

14. The method of claim 4, wherein the tissue sample comprises at least one dimension that is less than 2 millimeters.

15. The method of claim 4, further comprising minimizing a time period beginning at a first time when the cryostat tissue section is mounted onto the slide and ending at a second time when the solution having the concentration of at least 60% sucrose covers the cryostat tissue section to prevent drying of the cryostat tissue section.

16. The method of claim 15, wherein the time period is less than 120 seconds.

17. The method of claim 4, wherein the solution has a concentration of sucrose within a range of 60% up to a point of saturation of the solution.

18. The method of claim 4, wherein a concentration of sucrose in the solution is as high as possible before saturation.

19. An arrangement, comprising: a slide; a cryostat tissue section mounted on the slide, the cryostat tissue section having a cross sectional area of at least 1 square millimeter;the cryostat tissue section being infiltrated with a solution having a concentration of at least 60% sucrose; and an amount of the solution covers an exposed side of the cryostat tissue section on the slide.

20. The arrangement of claim 19, further comprising: a refrigerated compartment; and the slide having the cryostat tissue section mounted thereon is stored in the refrigerated compartment.21 . The arrangement of claim 20, wherein the cryostat tissue section is prevented from drying by the solution during storage in the refrigerated compartment.

22. The arrangement of claim 20, wherein the refrigerated compartment is maintained at a temperature substantially falling within a range of 2° Celsius to 6°Celsius.

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