CA1239290A - Apparatus and method for cryopreparing biological tissue for ultrastructural analysis - Google Patents

Abstract

APPARATUS AND METHOD FOR CRYOPREPARING
BIOLOGICAL TISSUE FOR ULTRASTRUCTURAL ANALYSIS

ABSTRACT

This invention relates to apparatus for the cryo-preparation of biological tissue samples for ultrastruc-tural analysis. The use of the apparatus comprises vitrifying a biological tissue sample under cryogenic temperature conditions and ultra low vacuum conditions.
The depressurized, vitrified tissue sample is brought to equilibrium in a sample holder at a temperature of less than -140°C. The tissue sample is then dehydrated while maintained in a state of thermal equilibrium. After reaching equilibrium the tissue sample is optionally infiltrated with a degassed resin followed by a polymeri-zation of the resin to form an embedded tissue sample.

Description

~39~9~ UTSH:041 APPARATUS AND METHOD FOR CRYOPREPARING BIOLOGICAL TISSUE
_ FOR ULTRASTRUCTURAL ANALYSIS

The subject matter in this application is related in part to the subject matter in my copending application Canadian Serial No. 460,541 filed August 8, 1~84 and assigned to the same as~iqnee.

This invention relates to apparatus and the method for preparing biological tissue samples for ultrastruc-tural analysis or other medical use, i.e. transplantation, by avoiding significant modification of the ultrastructure of tissue during preparation of the samples themselves.
It is well known in the medical arts that to examine tissue samples, and determine the cellular structure and function thereof, the tissue must be t'fixed" prior to the application of nearly all analytical methodologies.

Although the phrase "tissue samples" is used through-out this disclosure, the term should be understood to include small ~issue samples appropriate for microscopic examination and larger tissue masses such as corneas which are appropriate for transplantation. The definition of tissue samples should be understood to include without limitation sperm, eggs, embryos and blood components. The contemplated utility of the apparatus of this invention is not limited to speciic types or sizes of tissue, rather it should be understood to refer to any tissue made up from cells. The apparatus of this invention can be designed or adapted to any size, shape or type of cellular tissue. Therefore, the terms "tissue" and "tissue samples" are used interchangeably and are not limiting on the uses to which the method and apparatus of this invention can b~ placed.

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For purposes of this application, the term "vitreous" or "vitrification" or "vitreous phase" should be understood to refer to the physical condition o~ tissue upon ultrarapid cooling at a rate and under conditions in which resolvable ice crystals are not present and/or are not being nucleated at a rate which will result in the formation of resolvable ice crystals.
Although the examination of tissue by ~se of various microscopes or related magnifying apparatus has been practiced for many years, there has been an inherent problem in preparing tissue for use with conte~porary high resolution analytical microscopes, such as the STEM electron microscopes, which permit the examination of sample constituents via X-ray analysis at powers of from 500X to 500,000X with point to point xesolution of

2 to 3 Angstrom units.
Specifically, it is difficult to interpret the results of tissue analysis while concomitantly assessing the extent of various artifacts produced during the tissue preparation processes. It is thus essential that artifacts be avoided wherever possible. The term "artifact" refers to a product o~
artificial character due to extraneous agency. Another problem results from physical shrinkage of the tissue sample itself when subjected to the extreme, but necessary for successful preparation, procedures extant in current dogma. In most currently used tissue preparation steps, tissue shrinkage is in the order of 40% to ~0%. This shrinkage inevitably results in alteration of ultrastructure and massive rearrangement of infrastructural resolution. The net result of this is ultrastructural translation damage and inaccurate detail in descriptions via existing analytical procedures.
During the so~called "Golden Age of Morphology"
the predominant underlying goal in gualitative and quantitative microscopy has been an aesthetically pleasing image.
This goal is readily attainable with the fixation methods and apparatus which are currently available. However, it has become essential that the aesthetically pleasing 1~
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image, which is produced by the preparation process, also yield a tissue sample which accurately reflects the true condition of tissue in the living organism, i.e. approaching the "living state". This is the problem which the apparatus of this invention addresses and solves. Magnification apparatus which are currently available for analytical use are technically more advanced than are current tissue preparation techniques which have been previously employed. The method of this invention results in the preparation of tissue samples which are readily usable on known magnification and analytical apparatus.
Although the primary thrust of this application is in the preparation of tissue samples for analysis by current magnification apparatus, the inven~ion is not intended to be so limited. More specifically, the "preparation" of tissue should be understood to refer to preparation of tissue for analysis as well as the cryofixation of tissue in anticipation of transplantation, modi~ication, in vitro or in vivo cellular growth, fertilization, animated suspension or the more typical resin impregnation, setting, infiltration and analysis. The apparatus of this invention can be used to prepare tissue for any medical or analytical procedure without the ultrastructural damage previously thought to be inevitable in cryopreparation.
The invention in one aspect pertains to a method of cryopreparing biological tissue which comprises rapidly cooling the tissue at a rate and to a temperature sufficient to vitrify the water int~-etissue, and removing the water from the tissue by vaporization directly from its vitrified state.
Another aspect of the invention comprehends a method for cryopreparing biological tissue comprising vitrifyiny biological tissue, equilibrating the vitrified biological tissue under temperature and vacuum conditions that preclude the formation of resolvable cell water`ice crystals, and dehydrating the vitrified biological tissue while the tissue is maintained in a state of equilibrium.
A still further aspect of the invention pertains to a method o~ cryopreparing biological tissue which comprises rapidly cooling the tissue at a rate and to a temperature sufficient to

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vitrify the water in ~he tissue, subjecting the tissue containing the vitrified water to a combination of vacuum and temperature conditions at which the water is capable of vaporizing directly from its vitrified state, and slowly adding energy to the tissue at a rate and in a quantity sufficient to dehydrate the tissue by vaporizing the vitrified water directly from the vitrified state.
The invention also comprehends apparatus for cryopreparing vitrified biological tissue comprising a sample holder for retaining vitrifi~d biological tissue, means for maintaining the sample holder at a cxyogenic temperature, ultra-high vacuum means for depressurizing the atmosphere in the sample holder, and means for dehydrating the vitrified biological tissue while maintaining the tissue at a cryogenic temperature of less than -80C.
The invention still further comprehends a method of cryopreparing corneal tissue for use in surgical procedures comprising providing a corneal tissue sample, suitable for its intended end use, vitrifying the corneal tissue sample in less than one second, bringing the vitrified corneal tissue sample to equilibrium at a temperature of less than -1~0C, and dehydrating the vitrified corneal tissue sample by sublimation and under vacuum while the corneal tissue sample is maintained in a state of equilibrium, the vacuum reaching a level of from 1 x 10 Torr 1 10~1 T
Various other aspects of the invention will become apparent from the further description and preferred embodiments set forth herein.
The apparatus of this invention is to be dlstinguished from contemporary freeze--drying apparatus. Freeze-drying is a technique which is well known in the art together with the equipment necessary to implement such freezs drying. See, for example, U.S. patent No. 4,232,453.
~lthough in certain freeze-drying techniques liquid nitrogen is used as a cooling medium, the tissue or sample itself does not attain such temperature.
Freeze-drying norm~lly contemplates sample temperatures of -50C
to -~0C. In contrast, the cryopxeparation of this ~i 5 ~P~

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invention contemplates sample temperatures of -120C or below. Therefore, for purposes of this application the terms "cryopreparation" and "cryofixation" are used in distinction to conventional "freeze drying" technology (-50C to -80C).

The extreme low temperatures and vacuums used in the practice of the apparatus of this invention have generated unique problems not associated with freeze-drying appa-ratus. For example, sealing devices such as squeezable0-rings made from elastomeric material do not function efectively at these anticipated cryopreparation tempera-tures and vacuums. Therefore, it is necessary that cryopreparation apparatus be designed to seal various structures at the extremes of temperature and pressure encountered, i.e., the sample chamber to the rest of the apparatus, outside the liquid nitrogen environment. This is but one example of problems which have been encountered in the design of and which are unique to cryopreparation apparatus.

The vacuum levels disclosed and used in the apparatus of this invention cannot be achieved safely with prior art freeze drying equipment. Typical of previous methods for drawing vacuums in freeze drying methods and apparatus is the above-mentioned U. S. Patent 4,232,453 which discloses the use of molecular sieves in glass containers. Molec-ular sieves in easily compromised containers cannot be used safely to create and maintain the required vacuum levels to achieve the partial pressures required for sub-limation of water at the anticipated temperatures ~-120~C
or below) created by the apparatus of the disclosed invention.

The most common prior art method for preparation of tissue samples for analysis is by means of chemical fixa-~:35~2~3 tion and organic solvent dehydration. Inherent in prior art processes is the concomitant artifact creation, sample shrinkage and resultant damage to and modification of tissue characteristics. These tissue characteristic modifications, whether in the form of artifacts or the like, require interpretation by the individual or appa ratus analyzing or evaluating the sample. This intro-duces, in many instances, an unsatisfactor~ risk of error.

Chemical fixation is a well known technique and has served the analytical biologist well for many years and undoubtedly will continue to do so in certain limited applications. However, as the use of tissue sample analysis becomes more complex and the use of such analysis becomes more widespread, alternatives to chemical fixation are demanded. This is especially true as advances are being made in the magnification and analytical apparatus which are available. It is necessary that tissue prepara-tion methods and the apparatus necessary to prepare tissuesamples be equally advanced as the analytical tools, i.e., electron microscopes, which are being used to analyze the samples. Obviously, i~ the technology for tissue sample preparation is behind the technology of microscopy then the advanced microscopes cannot be used to full advantage by the morphologist or other tissue examiner.

Similarly, it is essential that cryopreparation methods and apparatus develop concurrently with other medical t~chnology, i.e., surgical transplant techniques, bio-engineering and biogenetics. In short, cryopreparation is an essential intermediate step in evolving processes using or analy~ing cells or tissue. If cryopreparation apparatus does not evolve then the thrust of medical technology into ~Inexplained and unexplorad medical arts will be blunted. The apparatus of this ~3~

invention represents the cryopreparation breakthrough that will permit research into the use and preparation of biological tissue to keep pace with other advances in medical ~echnology.

The most common alternative for preparing corneal tissue and to chemical fixation and organic solvent dehydration is free~e drying cryofixed samples. Freeze-drying following cryofixation is a well documented and well known technique for tissue preservation. It has several advantages. Freeze-drying results in a near-instantaneous arrest of cellular metabolism. There is also a stabilization and retention of soluble cell constituents through elimination of solvent contact with the sample. These are significant advantages to cryofixation freeze-drying that have resulted in a great deal of research in attempting to apply cryofixation and freeze-drying techniques to known t:issue preparation processes.

Unfortunately, freeze-drying technology inherently possesses a number of disadvantages relevant to tissue preparation methodologies. The primary disadvantage in currently available freeze-drying techniques and apparatus is the inherent formation of ice crystals. As can be readily appreciated, the formation of ice crystals destroys the ultrastructural integrity of the tissue sample being reviewed. The image is distorted and the cytoplasm becomes reticulated. The formation of ice crystals in the sample can also result in a change in p~ within microcompartments of the tissue (eutectic formation) which possibly can result in abnormal tertiary conformation of macromolecules. There is also the possibility that proteins will denature and precipitate. These are but a few of the disadvantages which are inherent in the freeze-drying process.

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This general topic is discussed in some detail together with other prior art methods in an article entitled Freezin~ and Drying of Biological Tissues for Electron Microscopy, Louis Terracio and Karl G. Schwabe, published in The Journal of ~istochemistry and Cytochem-istry, Volume 29, No. 9 at pp. 1021-1028 (1981). Problems associated with artifact formation are described in Understanding the Artefact Problem in Free~e-Fracture Replication: A Review, The Royal Microscopial Society, (1982) at pp. 103-123.

A general principle found applicable to freezing techniques, which has demonstrated utility in the prepa-ration of tissue samples, is that as the cooling rate increases, tissue fluids can be vitrified without the separation of water to extracellular spaces. It has been postulated that regardless of the rate of cooling, ice crystals may still be formed, but as the cooling rates increase the size of the intracellular ice crystals ~0 decreases. The small size or absence of ice crystals at high freeze rates is of course a substantial advantage in morphology retention as this results in minimal artifact creation and minimal ultrastructural damage during tissue dehydration. The apparatus of this invention requires the rapid supercocling of tissue samples to the vitreous phase in less than one second followed by dehydration of the tissue sample while in the state of reduced partial pressure of water vapor, all without substantial ultrastructural damage to the tissue cells.
Historically, the criteria by which the techniques for rapid supercoolin~ have been Judged was not the cool-ing rate of the system but simply the temperature of the environment in which the tissue was frozen. Thus, the term rapid supercooling has been applied to any system in which the supercooling agent has a temperature of -150C

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or ~elow. The effectiveness of a cooling system is depen-dent upon the rate at which heat is removed from the sample. ~eat transfer is dependent not only on the tem-perature of the freezing system but also on its physical and therrnal characteristics, as well as the size and thermal characteristics o the tissue.

The most commonly used technique for rapid super-cooling is to immerse or "quench" the sample in a ~luid cooling bath. ~he most commonly used fluids for ~uenching are liquid nitrogen, isopentane, propane and fluorocarbons such as Freon 12 and Freon 22. Although liquid nitrogen is generally regarded as an ideal ~uenching fluid due to its low temperature (-196C), there are inherent disad-vantages in the use of liquid nitrogen due to the occur-rence of tissue surface film boiling caused at least in part by the low heat of vaporization of liquid nitrogen.
Film boiling is a characteristic of liquid nitrogen that inhibits the heat transfer rates by actually insulating the sample.

An alternate prior method for rapid supercooling is free~ing on the polished surface of a chilled metal block.
This typically involves opposing the tissue sample to a polished flat metal surface by pressing it firmly against the surface of the metal. Silver and copper are typically used as the polished metal blocks. This method is designed to take advantage of the high thermal conductiv-ities and heat capacities of these metals when cooled to liquid nitrogen or liguid helium temperatures. The crit-ical step in chilling on the surface of a metal is making firm contact with the dry, chilled metal surface with no rotational, translational or rebounding motion. Certain commercially available apparatus having known utility in the medical arts address and provide "bounce-free" freez-ing. Credit for the development of this apparatus is 9 ~3~2~

generally accorded to Dr. Alan Boyne of the University of Maryland School of Medicine.

There has recently been verification that there is a direct correlation between cooling rate and ultrastruc-tural preservation in quenching fluids. As the freezing rate increases over the range of 100C to 4100UC per second (liquid nitrogen - propane~, there is a corre-sponding decrease in the size of ice crystals formed and thus an improvement in morphological preservation. Use of such quenching fluids or other supercooling apparatus to vitrify a tissue sample in less than 1 second is preferred.

The critical steps in the subsequent tissue prepara-tion process are invariably stimulated sublimation -dehydration of the supercooled tissue fluids, which have recently been described as a stimulated "molecular dist.illation" process. Once the appropriate supercooling method has been chosen and implemented, it is sometimes necessary to further process the tissue for microscopic evaluation, since electron microscopes or other magnification apparatus that al~ow the viewing of frozen hydrated specimens are not readily available. Thus, dehydration is an essential step in the preparation of biological tissue samples for storage and a step which oftentimes results in the destruction via reticulation of the infrastructure and ultrastructure of the tissue.
Tissue cell destruction from dehydration not only impairs analysis by magnification apparatus but also adversely affects the functional characteristics and viability of tissue masses being used, i.e. transplanted.

In certain prior drying techniques, the tissue sample had not been entirely solidified due to eutectic formation as the cellular fluid solutes were concentrated in bound ~3~

water compartments. This transfer of solute occurs while the materials are in the fluid state when slow cooling is employed. When rapid cooling techniques are used, unique procedures which are distinct from those characteristic of freeze-drying, must be employed in the dehydration step.
Problems result from the fact that dehydration must take place (the water must be removed) in the solid rather than the liquid state, i.e., via sublimation. An alternate procedure which has been used successfully is stimulated molecular distillation. Stimulated molecular distillation refers to a process in which the amount of energy in the antibonding orbitals of surface molecules is elevated, enabling them to escape to the gas phase and not be recaptured by the solid phase.
In the prior art, the freeze substitution approach has involved the removal of tissue water by substituting a solvent or solvent-fixative mixture for the solid phase water at -50 to -80C. This introduces less severe solvent phase separation and chemical alteration artifacts to a tissue sample than past routine chemical fixation methodologies. From a practical standpoint freese-drying is complicated by the requirement that the tissue sample be warmed to increase the vapor pressure of the supercooled water and allow sublimation to proceed in a reasonable period of time. The increased temperature, in addition to incre~sing vapor pres~ure can produce a series of physical events leading to the expansion of ice crystals and concomitant damage to the ultrastructural morphology of the tissue sample. Many of the physical events which occur during the warming process have to do with transitions in the physical state of the water which is present. Changes which are typically encountered are glass transition, devitrification and recrystallization with an ensuing series of crystal lattice configuration transitions.

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Thus it can be appreciated that freeze-drying tech-nology and cryopreparation techniques present an excep-tional opportunity for the preparation of tissue samples for morphological examination. Howevex, inherent in the use of freeze-drying techni~ues are problems associated with dehydration and fixation of samples. These are the problems which are addressed by the process and apparatus of this invention.

The cryopreparation process o this invention has demonstrated an extraordinary application in the trans-planting of corneal tissue. Prior to this invention attempts to transplant corneas which involved a necessary freezing or freeze-drying of the corneas after removal lS from the donor invariably resulted in a clouded cornea upon transplanting. This physical condition of the transplanted cornea was caused by crystal formation in the cornea itself and concomitant damage to the stroma. Use of the apparatus of this invention has enabled ophthal-mologists to cryoprepare corneas and to then transplantthose corneas to recipients with virtually negligible clouding or crystal formation. The ability to so trans-plant corneas represents an exceptional advantage to the process of this in~ention as well as a medical break-through in corneal transplant surgery.

One advantage of the apparatus of this invention isthe ability to cryoprepare tissue without overt disruption or destruction of the morphological characteristics of the ultrastructure of tissue cells. The apparatus of this invention permits the cr~opreparation of tissue by dehydrating tissue maintained in the solid, vitreous phase without creating unnecessary artifacts which restrict interpretation by conventional analytical apparatus.

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Figure 1 is a schematic flow diagram of a method associated with the use of the apparatus of this inven-tion.

Figure 2 is a schematic drawing of the apparatus of this invention.

Figure 3 is an exploded schematic drawing of the portion of the apparatus of this invention connecting the vacuum means to the sample chamber.

Figure 4 is an exploded schematic drawing of the s~mple ch~r and sample holder of this invention; as sh~wn with Fi ~ e 1.

Figure S is a schematic view of the sample holder of this inventi Otl .

Figure 6 is a schematic view of the tissue reservoir co~er used in the sample holder of this invention.
~0 This invention relates to apparatus and the method for the cryopreparation of biological tissue samples. The apparatus includes components for implementing the stimulated dehydration of biological tissue under severely depressurized conditions. The depressurized, vitrified tissue sample is brought to equilibrium at a temperature of less than -14~C. The tissue sample is then dehydrated while maintained in a state of equilibrium. After removal of tissue ~ater, the tissue sample is optionally infil-trated with a degassed resin followed by a polymerizationof the resin to form an embedded tissue sample. In other applications of the apparatus and method of this invention the dehydrated tissue sample can be used, i.e. trans-planted, without any infiltration or degassing steps.

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The apparatus of this invention includes a sample holder for retaining vitrified biological tissue. The sample holder and the vitrified tissue are maintained at cryogenic temperatures while the tissue sample is being dehydrated. Vltra-high vacuum means are used to depressurize the atmosphere of the sample holder to permit the desired sublimation; equilibration and dehydration procedures.
The apparatus of the invention is used in combination with conventional apparatus to vitrify (ultrarapid cooling at a rate and under conditions such that resolvable ice crystals are not formed) biological tissue~ The preferred vitrifying apparatus is a metal rod adapted to transform the tissue to the vitreous phase at a temperature of -123C or below. The vitrified tissue is inserted in a sample holder which is fittably received by a sample chamber which in turn can be inserted or withdrawn from a cryogenic bath.
The ultra high vacuum assembly used to depressurize the sample chamber provides a pressure of from 1 x 10 Torr to 1 x 10 10 Torr. The ultra-high vacuum assemblyis removably attached to the sample chamber.
In practice the apparatus of this invention is used to cryoprepare biological tissue for analysis or other medical end use, i.e. transplantation. The apparatus is adaptable to an infinite variety of tissue shapes, sizes and configurations. The apparatus of this invention results in the cryopreparation of biological tissue resulting in a final product whose ultrastructure is substantially unmodified and which is ready for analysis and end uses which have been heretofore impossible in the medical arts.
In the apparatus of this invention it is a fundamental prerequisite that the desired tissue is obtained.

~issue samples are collected by a variety of means, i.e., surgical extraction, withdrawn blood samples, binders and any of a variety of other techniques which are well known and conventional. The particular method of obtaining the biological sample is not limiting on the apparatus of this invention. However, the preparation of the tissue sample in the apparatus of this invention is enhanced if the tissue sample is processed as soon after excising as is possible.
The preparation of the tissue sample takes place immediately as it is received. The tissue sample cannot be retained in a fixative, i.e., formaldehyde, or another biologically active stabilizing solution, in an attempt to maintain the sample during shipping, storage or other necessary operations. It is also critical that the sample not be routinely frozen or otherwise physically modified prior to preparation according to the method of this invention. The sample may later be physically sectioned or otherwise physically prepared for long-term storage in apparatus or use with various currently available com-mercial analytical apparatus.

In one application of the apparatus of this invention a tissue sample is prepared for analysis. The preferred optimum biological sample for preparation in the apparatus of this invention is a fresh one cubic millimeter biopsy sample. This sample must be vitrified as soon as pos-sible. By vitrifying or vitrification it is intended to make reference to a process which results in cryofixation of the sample which is different from "frozen." In the process of vitriying, the cooling apparatus which is used renders the sample in the vitreous phase such that soluble and insoluble moities contained in the tissue sample are not disturbed, translated, or altered nor are they concen-trated ~as eutectics). By definitlon, a vitrified liquid - 15 - ~ ~3~

will shatter when undergoing a shear stress, e.g., window glass. The vitreous phase involves the conversion o~
liquid water into an amorphous or "glass" phase. This is accomplished by rapidly supercooling the tissue sample by opposing it "bounce-free" onto the highly polished (mirror-like) condensate-free surface of a metal rod maintained at about -196C. These operations have been discussed previously in the prior art section of this disclosure. It is preferred that such rapid-supercooling be completed in less than one second.

Of particular utility in the process and apparatus of this invention is a "bounce-free" freezing apparatus which has been identified in association with Dr. Alan Boyne of the University of Maryland ~chool of ~edicine. In this freezing apparatus, a copper block is used to vitrify the tissue sample. This vitrlfication in conjunction with a supercooled fluid such as li~uid nitrogen, helium, propane or the various ~reons will cause the tissue sample fluids to supercool to the amorphous state before and/or without the formation of noticeable or resolvable cell water ice crystals. It is desirable in the preferred embodiment that the now vitrified tissue sample be maintained at a temperature of less than about -120C and preferably less - 25 than -140C during storage and transfer operations prior to removal of the tissue water.

Temperature control is essential to prevent ice crystallization. It is thought that ice crystallization begins to occur at about -123C. This is, however, dependent on the chemical constituents of the cellular water. Applicant has therefore selected -140C as the pre~erred temperature. It should be understood that the desired result is to maintain the temperature ~elow that at which ice begins to crystallize and that -~23C and - 16 ~ 2~

-140C have been selected based on current experimentation. Therefore, for purposes of this application, the preferred tissue sample temperature to be maintained is below -123C while the more preferred temperature is below -140C and the most preferred temperature is -196C or below.

Depending on the anticipated time lag between super-cooling of the sample and dehydration of the sample, it may be stored submerged in a liquid nitrogen dewar. Once the sample has bsen dried and embedded properly it may be stored virtually indefinitely without cytoplasmic reticu-lation or other forms of cellular catabolism which will cause modifications and crystal lattice transitions resulting in undesirable artifacts which render the tissue uninterpretable as analytical data.

After vitrifying, and while maintaining the tissue sample at a temperature of less than -140DC it is trans-ferred via a specimen transport and fed to a specimenholder in vacuo. The specimen holder (also commonly referred to as a sample holder) is maintained in a temper-ature controlled container. The container and specimen holder are both preferably maintained at temperatures below -140C. In the most preferred embodiment of this invention, liquid nitrogen temperatures of -196C are maintained. The reason that -140C is preferred is that pure water, existing in the vitreous phase when at liquid nitrogen temperatures, will begin to initiate cubic ice crystallization at -123C. As discussed in the prior art section of this disclosure, ice crystallization causes ultrastructural damage, i.e., reticulation to the mor-phology of tissue samples.

Next, the atmosphere surrounding the tissue sample, specimen holder and container is depressurized. This is - 17 - ~ ~3~

typically done by drawing a vacuum on the sample holder with conventional mechanical vacuum apparatus. The vacuum is drawn to a level of 3 x 10 9 Torr in less than 300 minutes. In other embodiments of this invention, the vacuum which is drawn is from 1 x 10 8 Torr to 1 x 10 10 Torr accomplished in less than 300 minutes. These pres-sures remain at approximately 3 x 10 9 Torr throughout the remainder of the prescribed routine until all the tissue water has been removed. Throughout equilibration of the system (10-lO0 hours), the specimen temperature is main-tained by liquid nitrogen or other suitable cooling means while the vacuum is being drawn and maintained.

At this time the tissue sample is at ultra low pressure and exceptionally low equilibrium cryo-tempera-ture. After equilibration i.s obtained (with equilibrium temperature below -140C), the vitreous water which is found in the tissue sample will begin to sublime as energy equal to the heat of sublimation is intermittantly and incrementally supplied to the sublimation front found in the tissue. This is a slow process but one which is critical to the preparation of the sample. It is an important requirement that the sample be permitted enouyh time to allow it to reach equilibrium after each addition of energy. By equilibrate it is meant that the tempera-ture of the tissue sample no longer changes over a 1 to 5 hour time period and preferably a 2 to 4 hour time interval. In a typical tissue preparation process the sample is rapidly vitrified to -196C and maintained below -140C during storage/transfer to the sample holder in the sublimation (dryinq) apparatus. After appropriate equili-bration time the equilibrium temperature will be somewhere between 140C and -196C. During this entire e~uilibra-tion process a critical ultra-low pressure is maintained at 3 x 10 9 Torr or below.

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After the equilibration process, it would take an exceptional length of time ~or any appreciable amount of water to evaporate from the sample if no energy (heat) of sublimation were added to the system. Estimates are in terms of years for the water to evaporate at temperatures and pressures which are associated with the method of this invention. Therefore, in the most preferred embodiment of this invention, a secondary energy source (heating) is added to excite the sublimating water molecules without causing damage to the ultrastructur~ of the dry tissue sample. Radiant photon energy, having a particular wavelength, is thought to be an especially useful approach to accomplish this goal. Sublimation energy via microwave, laser systems and magnetic energy are also appropriate. The most preferred secondary source is tne nuclear magnetic resonance or electron spin resonance approach in combination with the above. At equilibrium, the temperature of the tissue will not change unless the a~bient parameters of the immediate environment (radiant energy predominates, i.e., room temperature is 27C) change. This is the general identification of the end point of system equilibrium.

Subsequent to the tissue sample reaching e~uilibrium, it is necessary to remove the supercooled solid water and/or presently unresolvable ice crystals (20 nanometers diameter or less) which have formed in the tissue during the vitrification operation. This portion of the dehydra-tion process is absolutely critical and is the step where most potential disruption and reticulation of the ultra-structure in the tissue will express itself. This is accomplished by gradually replacing the energy of sublima-tion in the sample by minimal increments of stimulated energy per hour. The optimal condition is to have no tissue temperature increases.

By so raising the thermal energy equivalent to the latent heat of sublimation all of the solid water, whether micro-ice crystals or amorphous supercooled water, is effectively removed from the tissue sample by the sur-rounding cryosystem. This drying may be accomplished attemperatures between -150C and -80C. This regimen of greater temperature latitude will provide variable results and is possible due to elevation of devitrification temperatures by the solutes that are dissolved in cell water at varying concentrations. With appropriate instru-mentation, i.e. residual gas analyzers, it is possible to determine when all cell water has been removed. At that point, the energy increase can be accelerated to produce a final specimen temperature 3C above room temperature (28C - 30C). Thus, with this instrumentation a signifi-cant advantage in the process of this invention is obtained.

The now dehydrated tissue sample has been permitted to reach room temperature plus 3~C. Even though reaching room temp~rature the vacuum is maintained at the original exceptionally ultra-low levels as has the temperature surrounding the sample. Room temparature for purposes of this application should be understood to be approximately 24C - 27C. There may logically be variations in this temperature level.

A person of ordinary skill in the art can readily appreciate that control of tempera~uresthroughout the processes of vitrification, equilibration, sublimation and dehydration are essential. The precise temperatures at 30 which the tissue is maintained and the rate that the tissue ternperature is changed are crucial although varied for different cellular structures. A typical routine for a cell mass such as a cornea would reg~ire the initial vitrification of the cornea tissue at -190C or below.
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The sample is immediately heated to -150C in approximately 4 hours. During the equilibrium, sublimation, dehydration stage the tissue sample is heated from -150C to -70C in 60 hours (rate = 1.333C/hr.). The drying process begins at approximately -119C and is completed before devitrification at -80C. The sample is then heated from -70C to -~25C in 4 hours. Generally the sample is heated to slightly above room temperature to prevent water condensation from invading the sample.
At thisjuncture, the investigator has the option of exposing the tissue to osmium vapors for approximately one hour to provide contrast enhancement via electron density. This may be omitted if proven to be deleterious to the moiety of interest or if the ultimate goal is clinical use such as in corneal surgical transplantation. In this case, the tissue sample would be slowly lS brought to atrnospheric pressure and stored as necessary for later rehydration and use.
If the investigator's option is to expose the tissue to osmium vapors, the osmium vapour is removed by recrystallization by cryoprecipitation. In other established fixation processes, paraformaldehyde and/or gluteraldeh~de in buffer solution is used. These materials are typically referred to as chemical-fixative materials. The most preferred material which is typically added is osmium tetraoxide. This material will enhance the resolution and contrast of the various constituents of the tissue for the ~arious analytical apparatus which might be used to interpret the tissue sample.
For samples prepared for analysis a degassed resin is then added to the tissue while still maintaining the depressurized condition. This is typically referred to as resin infiltration and results in an embedded tissue sample. Resins which have shown utility in past methods are equally applicable to the method of this invention. See for example United States patent Nos. 3,679,4S0; 4,100,158; 4,120,991 and 4,278,701.

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Subsequent to these steps the tissue sample and resin are brought to atmospheric pressure by slowly admitting air through the resin port. The embedded tissue sample which has resulted from the resin application process is removed and the resin is polymerized at its prescribed temperature. The particular method of polymerization is largely dependent on the resin that is used. Typically, the tissue sample is pol~erized by heat application in an oven for 12 hours. A normal temperature would be 60C, but may be as low as -80C if necessary. It is essential that the polymerization step be accomplished without damage to the tissue ultra-structure.

Following polymerization the tissue sample can then be stored at room temperature, thin sectioned, stained or further prepared for other analysis. However, having been dehydrated in the fashion disclosed by this invention the sample is maintained in a cryofixed state which is readily interpretable by conventional ultramicrotomes and electron ~0 microscopes and provides the basis for exceptionally meaningful analysis of tissue samples with a significant alteration o~ and reduction of artifacts conco~itantly reducing or eliminating past constraints thought to be ubiquitous in fixation and/or tissue preparation for visual analysis.

The actual relating of structure to function in these biolo~ical tissues is done by routine ultrathin sectioning with an enormous expansion of applicable staining methods heretofore deemed unapproachable via conventional electron microscopy, (i.e., immunological analysis of any soluble moieties, sugars, lipids and soluble proteins~, enzyme cytochemistry, X-ray dispensive STEM analysis, tissue transplant preparations, microprobe analysis, autoradio-~raphy (especially of soluble compounds) and pharma-ceutical preparations.

- 22 - ~3~

Qther apparatus are available for the execu~ion of this hierarchy, but none have produced the result expected as they do not incorporate in totality the required, defined parameters discussed earlier. The apparatus which is used in the practice of the method of this invention is illustrated schematically in Figures 2 through 6.

The rapid freezing attained by the apparatus of the Alan Boyne type is preferred to the practice of the process of this invention. Liquid nitrogen and other types of quenching baths in conjunction with chilled metal applications are used in the process of this invention to the extent they provide the vitrified phase of cell water in less than one second. A liquid nitrogen quenching bath is used to lower and maintain the temperature of the tissue sample which is included in the tissue holder. It should be noted that while the tissue sample is maintained in the liquid nitrogen condition, it is necessary that tubulation access the various staining and fixation materials which are optionally preferred in the process of this invention, as well as the various resins which are ultimately used to embed the tissue samples of this invention prior to polymerization. Again, each of these functions is illustrated schematically in the attached figures. However, it should be understood that these are not intended to be limiting features of this invention but merely illustrative of available technology.

In designing the apparatus or in selectin~ the apparatus for use in the method of this invention, it is necessary to understand the effects of the exceptionally low temperatures and pressures on various materials. For that reason, portions of the apparatus of this invention used to treat the material while in the vitriied state are typically made from stainless steel. Other materials may well ~e equally viable. Likewise, portions of the ~2~2 ~
- 23 - ~

apparatus of this invention are made from or coated with Te~lon+, a Dupont manufactured material which consists in a major portion of tetrafluorans.

Figure 2 illustrates schematically the apparatus of this invention. As shown in Figure 2, the apparatus is broadly categorized into a control panel 10 and the remainder of the apparatus used to vitrify, sublime and equilibrate the tissue. Microprocessor 11 of control panel 10 controls a turbomolecular pump 30. The control by microprocessor 11 is primarily o~ the revolutions per minute at which the components of the turbomolecular pump 30 are rotated and the temperature of the two main bear-ings in the turbo-molecular pump.
Digital vacuum gauge 12 of control panel 10 is connected to the apparatus in several places. In addi-tion, the digital vacuum gauge 1~ is attached to mechan~
ical pumps to provide digital readings of both the low vacuum caused by the mechanical pumps and the ultra high vacuum caused by the turbomolecular pump.

The next component of control panel 10 is a residual gas analyzer 13. Residual gas analyzer 13 functions by reading the partial pressure of each gas in the sample chamber 90. Included in the analyzer 13 is a quadrapole mass spectrometer. This instrument can read the atomic weight of each gas present in the sample chamber 90. In addition, residual gas analyzer 13 is used to determine the water vapor levels in the chamber which can be used to determine the end point for dehydration.

Microprocessor 14 is the component of control panel 10 used to read and control the temperature of the tissue samples in sample holder 100 (see Fiyure 4). Micro-processor 14 reads the temperature of the metal supporting ~3~

the tissue sample in sample holder 100 and does not contact the sample itself. The prograrnmable features o microprocessor 14 enable the implementation of a tempera-ture control function as well as a temperature monitoring function.

Component 15 of control panel 10 is a chart recorder for microprocessor 14. Chart recorder 15 provides a graphic illustration of temperatures measured by micro-processor 14.

Mechanical pumps 20 (backing pump) and ~1 (roughpump) a~e located in the control panel lO as well as in conjunction with the main apparatus. Mechanical pump 20 is activated to draw the backing vacuum on the turbo-molecular pump system. I'he initial vacuum is typically lxlO 3 Torr. The mechanical pump 20 is also connected to molecular sieve trap 22 to trap any hydrocarbons that may be going back to the turbomolecular pump 30 from the mechanical pump 20. It is essential that no hydrocarbons reach the turbomolecular pump 30. The mechanical pump 20 and the molecular sieve trap 22 are arranged in series so that no hydrocarbons can bypass molecular sieve trap 22.

Molecular sieve trap 22 is connected to turbomolecu-lar pump 30 by T connection 23. Low vacuum gauge head 24 extends from T connection 23 and is connected to the digital vacuum gauge 12.

In the preferred embodiment of this apparatus a solenoid valve 25 is connected to T connection 23 at the point illustrated by Figure 2. The solenoid valve is used for a backing line (not illustrated) for dry nitrogen gas being connected to the turbomolecular pump 30. In the event that the vacuum or ultra-high vacuum system malfunc-- 25 - ~ ~3~

tion~ and stops, the chamber is filled with inert nitrogen gas instead of moisture and hydrocarbon-containing air.

Turbomolecular pump 30 is used to create the ultra high vacuum of 1 X 10 8 Torr to 1 X 10 10 Torr reg~ired to properly practice the proce~s of this invention. The ultra high vacuum pump 30 can be any of a variety of commercially availabl~ vacuum pumping apparatus. The preferred embodiment is a turbomolecular pump and in particular a turbomolecular pump manufactured by Leybold-Heraeus (Model TMP-360). It is essential that the ultra high vacuum pump, whether it is a turbomolecular pump or not, yield a hydrocarbon free vacuum. As mentioned previously, the mechanical pump 20 is used to pump out gases which are transmitted through the ultra high vacuum pump 30 from sample chamber 90.

In the preferred embodiment of this invention a cooling fan 31 is us~d to cool the bearings of the turbo-molecular pump or other ultra high vacuum pump 30. A
heating bakeout jacket 32 heats the walls of the ultra high vacuum pump 30 while in operation to ensure that gases are desorbed from the inner surfaces of the ultra high vacuum pump. These gases and even liguids are converted from condensation on the inner surfaces of the turbomolecular pump to result in gases thus enhancing the vacuum cr~ated by the turbomolecular pump 30. Thermo-couple 33 provides the connection to the energy scurce (not shown) for heating bakeout jacket 32.

'!Conflat"flange 40 is used to seal the turbomolecular pump to a first spool 50. "Conflat"is a trademark o Varian Industrias, I~c. and describes a brand of flange.
The type of flange associated with "Conflat" is well known to those skilled in the art and can generally be described as a first surface having a knife edge designed to pene-,.._.

- 26 ~

trate a second abutting surface which is a soft metal.
Although many state of the art sealing devices will function effectively to seal the members at the ultra high vacuums and temperatures desired, it has been found most preferable to use a 100 cf Conflat flange which îs a stainless ~teel flan~e with a copper 0-ring seal. Of great importance in Conflat flange seal 40 is the fact that it functions effectively at temperatures up to 150C
during bakeout of the apparatus. This permits the effec-tive formation of a seal wit~ relatively standard sealingmeans. It would be virtual'y impossible to form the seal necessary if flange 40 were sealed with conventional, squeezable 0 rings which are typically made from elastomeric material.

Spool 50 provides the conduit from the turbomolecular pump 30 to a gate valve 60. Spool piece 50 includes four '.! Conflat flanges. The first is the common Conflat flange 40 with turbomolecular pump 30. The second is Conflat flange Sl. The third and fourth Conflat flanges are identified by numerals 52 and 53. Conflat flange 52 connects spool 50 to the residual gas analyzer 13 sensing head while Conflat flang2 53 provides the seal between the spool 50 and a Bayard-Alpert gauge.

An electropneumatic, ultra high vacuum pendulum gate valve 60 comprises the main valve isolating the turbomo-lecular pump 30 from the sample chamber 90. A piston contained within piston housing 61 provides the mechanism for opening and closing gate valve 60. Solenoid valve 62 and nitrogen gas are used to actuate the opening and closing of gate valve 60.

A second spool piece 70 is illustrated in Figur~ 2 ~ but in more specific detail by Figure 3. Reference should i b be made to Figure 3. Second spool piece 70 provides - 27 ~ % ~ ~

feedthrough from the pendulum gate valve 60 to the sample chamber 90. Spool piece 70 has extenslons 71, 72, 74 and 78 connected to the main portion of the spool piece housing. Flange 71 provides tubulation for electrical feedthrough to the control panel 10 from the sample chamber 90. Flange 72 is tubulation for the low pressure vacuum head. At the exterior end of tube 72 is located low vacuum gauge head 73. Low vacuum gauge head 73 is connected to digital vacuum gauge 12. An ultra high vacuum valve 75 to mechanical pump 21 is located at the end portion of extension 74 from spool piece 70. The valve 75 acts to control the preliminary or '1rough" vacuum drawn on sample chamber 90. Conflat flanges 76 and 77 are used to seal spool piece 70 to gate valve 60 and to ceramic insulator spool 80. The fourth extension from spool piece 70 is overpressure relief valve 78 shown by phantom line in Figure 3.

Ceramic insulator spool 80 is inserted between spool piece 70 and sample chamber 90. Insulator spool 80 functions to prevent gross heat transfer from the elsments of the apparatus above spool piece 70 to the cryogenic dewar 99 below (see Figure 4). Without insulator spool 80 frost and ice fre~uently develop on the exterior of the ultra-high vacuum pump assembly 30 and other connected elements. Ceramic insulator spool 80 also permits more efficient utilization of the supercooling material, i.e.
li~uid nitrogen.

The sample chamber 90 is used to retain the sample holder 100. These components are illustrated in Eigures 4 and 5. The sample chamber apparatus 90 includes a resin containing chamber 95 and a glass window 96 to provide visual access to the resin containing chamber 95. A gold sealed ultra high vacuum valve 97 and tubulation 98 to provide access for the resin into the sample chamber 90.

- 28 - ~ ~3~

Glass tube 91 is attached -to sample chamber 90 via glass to metal adapter 92 and tubulation 88 which in turn is connected to a metal "T" flange 93. ~alibrated leak valve 94 is used to flush or permeate the sample chamber 90 with dry nitrogen gas or other inert material. The tube 91 is used to include osmium tetraoxide crystals for introduction of osmium vapors into the sample chamber 90 during staining operations. Support members 94 are used to maintain the relative spacing of tubulation ~8 and 9~
from the housing of sample chamber 90. Cryogenic dewar 93 is used to maintain the cryogenic cooling means, i.e. liqu:id nitrogen. It will of course be appreciated that the staining and tixation elements of the apparatus of the invention are not used during cryopreparation of corneal tissue for surgical procedures.
In the most preferred embodiment of the apparatus of this invention a device is provided for sensing and automatically controlling the level of supercoolant, i.e. liquid nltrogen, in cryogenic dewar 99. Inherent in the use of liquid nitrogen or other similar coolants is the boiling off of the coolant over a period of time. Thus the coolant level must be periodically replenished to maintain the desired level of cooling. This can be accomplished manually or a mechanism can be installed for automatically sensing and replenishing the coolant level.
The sample holder 100, as shown in Figures 5 and 6, is used to retain the actual tissue s~mples. Typically the cryogenic bath environment 99 is liquid nitrogen contained by a dewar. The essential characteristic is that the tissuetemperature must not exceed -1~0C. The thermoconductivity of the cryogenic energy from the cryogenic bath environment 99 to the sample holder lQ0 is inherent in the structure. Reference here is made specifically to Figure 4.
In the most preferred embodiment of this invention radiant heating means 125 are provided to permit a source ~239~

of radiant energy to the tissue samples. 'rypically the radiant heating means are con~rolled by rheostats or thermostats.
Tempexature indicating means such as identified as component 14 of control panel 10 are typically used so that the temperature of the environment and tissue samples can be specifically controlled. In the preferred embodiment of this invention the radiant heating means and temperature indicating means are all operated by a microprocessor of a computer within precise defined ranges.
Other forms of energy are equally useful with the apparatus of this invention. More particularly, electromagnetic energy sources such as microwaves, radio waves, acoustic sound waves, visual light waves and ultraviolet or near ultraviolet waves may be used. Magnetic flux is also useful, especially in combination with any of the above enumerated energy forms. Combinations of the above may be used depending on the application and sample to which the apparatus is placed. Direct infrared radiation should be avoided although infrared radiation produced by laser and transmitted by fiber optics is appropriate. Sample characteristics are of paramount importance in determining the energy source which is ultimately selected.
In actual operation a tissue sample is vitrified to liquid nitrogen temperatures, i.e. less than -140C. The tissue is then transferred from a storage dewar to the sample holder 100 under liquid nitro~en temperatures with prechilled forceps.
The sample holder 100 is placed into the precooled sample chamber 90. Thermocouple wire 102 extending from sample holder 100 is then connected to mating wire 104 extending down from spool piece 70 (see Figure 3). Likewise, heater wire 103 is then connected to matin~ flow through wire 105 extending from spool piece 70. The specimen chamber 90 is then connected to the spool 70 via . , 30 ~ 2~

Conflat flange 77. This connection must be accomplished in the liquid nitrogen bath. Mechanical pump 21 is then activated and the spool 70 and sample chamber 90 are evacuated (rough pumped) to approximately 1 ~ 10 3 Torr.
S The valves connecting the mechanical pump to sample chamber 90 is then closed and the main valve 60 between the turbomolecular pump 30 and sample chamber 90 is opened. At this time the drying process begins.

The system is allowed to thermally equilibrate while being constantly monitored by the instrumentation in control panel 10. The turbomolecular pump draws a vacuum of approximately l x lO Torr to 1 x 10 Torr. The samples themselves are monitored by the residual gas analyzer 13 which includes a quadrapole mass spectrometer.

When the tissue is in equilibrium as indicated by no change in temperature for one to five and preferably two to four hours, the temperature controller is raised from -150C to about -70C. Preferably the tempeature is raised at a rate of 1C per hour to 3C per hour or more.
In the most preferred embodiment the temperature is raised at a rate of from 1C per hour to 10C per hour. When the residual gas analyzer 13 shows no increase in water vapor after an increase in tempexatur~ the tissue is determined to be dry (typically at -85C to -70C). The temperature is then increased to 25C. When the tissue has reached 25C the liquid nitrogen level in the dewar is allowed to drop and the outside walls of the sample chamber 90 are warmed to room temperature.

Gptionally osmium vapor may then be introduced through glass to metal adapter 92. Subsequently the osmium vapo~s are removed by recrystallization in a li~uid nitrogen trap. Also, the resin material is added from ~ 31 ~

resin chamber 95 through tubulation 98. The tissue may then be removed to polymerize the resin.

Figure 1 is a schematic illustration of the process for use of the equipment of this invention. The portion of Figure 1 which is included within a dotted line is not asserted to be new or novel, only the apparatus which is used to implement these steps is new and novel. As can be readily understood from the foregoing description and the flow chart o~ Figure l, the essence of this invention amounts to vitrification, molecular distillation, sublima-tion, dehydration and tissue equilibration. This is a process and result which has not been heretofore thought possible. By use of the apparatus of this invention, it is possible to achieve medical goals heretofore thought to be impossible.

Although the preferred embodiment of the apparatus of this invention has been described hereinabove in some ~O detail, it should be appreciated that a variety o embodi-ments will be readily available to a person designing cryopreparation apparatus for a specific end use. The description of the apparatus of this invention is not intended to be limiting on this invention, but is merely illustrative of the preferred embodiment of this inven-tion. Other apparatus an~ components which incorporate modifications or changes to that which has been described herein are equally included within this application.

It is essential to the proper unctioning of the apparatus of this inVention that the sample holder 100 be sized and designed to be ~ittably received by sample chamber 90 and to maintain one or more tissue samples in the proper condition of vitrification during e~uili-bration, sublimation and dehydration. The sample holder lOO, which has shown speciic utility in the apparatus of .
~.~39~

this invention, is illustrated more specifically in Figures 4, 5 and 6.

Referring now specifically to Figure 5, the sample holder 100 consists of a solid block of metal 110, prefer-ably copper, silver, or gold, and combinations or alloysof copper, silver and gold. In the most preferred embodi-ment an alloy of silver and copper plated with gold is used. A plurality of wells lll have been created in one surface of metal block 110. A central aperture 126 is also found in metal block 110. Radiant heat means 125 is inserted in aperture 126. The wells 111 create tissue reservoirs. The cryoprepared tissue samples are individ-ually inserted into tissue reservoir~ lll with prechilled forceps as previously disclosed.

The tissue samples are then covered with reservoir cover 113. Reservoir cover 113 includes a wire mesh section 114 and a side wall 115. The coniguration o reservoir cover 113 is shown with more specificity in Figure 6. Wire mesh section 114 is attached to side wall 115 by special vacuum adhesives. Solder is not appro-priate because of the out-gas properties of most solders.
The finer the mesh of 114 the more effective the desired gas transfer. Reservoir cover 113 also functions to protect the tissue samples from the effects of sudden changes in pressure such as when the gate valve 60 is opened or closed.

Teflon spacers 120 are intermittently spaced around the exterior surface of solid metal block 110 to provide the proper spacing rom the wal ~or other chilled surface of sample chamber 90. A Teflon leeve ll9 is threaded into central aperkure 126 to protact connecting wires 102 ~1 and 103. The thermocouple connection is found at 1~2 on _` the uppermost surface of sample holder lO0. Although the ~ ~3~

sample holder lOO is shown in a size and configuration which is appropriate for cryopreparation of tissue samples for analysis, it should be understood that a person of ordinary skill in the art can egually prepare a sample holder to accommodate larger tissue masses or other for~s of tissue.

In actual practice, individual tissue samples are placed in reservoirs 111 and reservoir cover 113 is inserted over the top of the tissue sample. As illus-trated by Figure 5, the covers 113 extend slightly abovethe surface of sample holder lOO to pro~ide means for grasping the covers 113 during insertion or removal. In the most preferred embodiment of this invention slits 116 are provided in side wall 115 to give some flexibility to the covers 113, again to assist insertion and removal.
The apertured surface 114 of covers 113 permits dehydra-tion and sublimation without orming unnecessary moisture on the walls of reservoirs 111. It should also be under-stood that the preferred material for use in forming solid metal block llO is copper although other materials, i.e.
silver, gold, and alloys or combinations of copper, silver and gold, have been shown to be equally viable. The characterizing function of solid metal block llO is the ability to transmit ultra low temperatures to the tissue samples and to maintain performance characteristics under the ultra high vacuum and ultra low temperature conditions of the cryopreparation apparatus and process of this invention.

Radiant heating means 125 are illustrated in Figure 5 and provide a source of radiant heat to the ti55ue samples. Radiant heating means 125 are controlled b~
control panel lO. Control panel lO permits infinite variability to the radiant heating means. In particular, temperature reader/recorder 14 and chart recorder 15 . .

_ 34 _ ~3~

maintain information and control over the temperature of tissue reservoirs :111 and the tissue samples contained therein. As has been specifically pointed out herein-above, control of sample temperature and the environmental temperature surrounding the tissue samples is absolutely essential to the effective functionality of the apparatus of this invention.

The most preferred embodiment of the sample holder 100 is illustrated in Figure 5. Included in the most prPferred embodiment is radiant heating means 125 which are shown in aperture 126. The most preferred form of radiant heating means 125 is a 220 volt/100 watt cartridge heater. The heating system is made more efficient by coating the interior, polished (spectral~ surface of side wall 115 of reservoir cover 113 with a material which permits the efficient transfer of radiation energy to the specimen. Thus, in the preferred copper embodiment, the walls 115 are treated with potassium sulfide to turn the interior surface walls black and thus provide the mechanism for controlled radiant heating of the tissue sample. In some embodiments the interior surfaces of wells 111 are likewise spectral.

Thus, the radiant heating means, i.e. cartridge heater 125, is controlled by temperature reader/recorder 14. The heating mechanism is selectively activated manually or preferably by a programmable computer or micropro~essor to maintain the desired temperature or temperature rate of change. Upon heating the metal block 110 conducts heat energy to the tissue reservoirs 111 and 30 the heat energy is absorbed by the spectral coating on reservoir cover 113 and/or side wall 115. The spectral coating then acts as the source of radiant heat to the - tissue samples.
`~b,, "`

,.~

_ 35 _ ~3~

Although the preferred embodiment of the specimen holder of this invention has been described hereinabove in some detail, it should be appreciated that a variety of embodiments will be readily available to a person design-ing an apparatus for a specific end use. The descriptionof the preferred sample holder of this invention is not intended to be limiting on this invention, but is merely illustrative of the preferred embodiment of this inven-tion. Other specimen holders which incorporate modifica-tions or changes to that which has been described herein-above are equally included within this application.

~23~

SUPPLEMENTARY DISCLOSURE
.
In the disclosure as originally framed and with reference toFigure 5, it was noted that the sample holder 100 consisted of a solid block of metal 110, preferably copper, silver or gold and combinations or alloys of copper, silver and gold. A more preferred embodiment was indicated to be an alloy of silver and copper plated with gold.
A holder made from or made in part with other thermally conductive materials such as sapphire and diamond is also operable and useful in carrying out the invention.
Accordingly the invention comprehends in a broader aspect apparatus for supporting or retaining biological tissue during cryopreparation comprising a thermally conductive sample holder including one or more reservoirs. In the preferred aspects the sample holder is made from one or more materials selected from the group consisting of copper, silver, gold, sapphire, diamond and combinations thereof.
Further, although direct infrared radiation is (not`a preferred form of providing a source of radiant energy, radiant energy generated by an infrared laser and transmitted by fiber optics to the tissue samples is a suitable source of radiant energy.
The above is a further description of preferred embodiments of an aspect of the invention but is not intended to be limiting on this invention.

Claims (87)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. Apparatus for cryopreparing biological tissue comprising:
(a) apparatus for rapid supercooling of biological tissue, said apparatus including a metal rod adapted to transform said biological tissue to the vitreous phase, contact with said metal rod maintaining said tissue at a temperature of less than -123°C;
(b) a sample chamber assembly adapted to receive a sample holder containing said vitrified biological tissue, said sample chamber assembly being movable to facilitate insertion into and withdrawal from a cryogenic bath;
(c) an ultra high vacuum pump assembly for depressurizing said sample chamber assembly to from 1 x 10-8 Torr to 1 x 10-10 Torr, said ultra high vacuum pump assembly being removably attached to said sample chamber assembly; and (d) a cryogenic bath for maintaining the temperature of said sample chamber assembly at -123°C or below, said cryogenic bath being adapted to receive at least a portion of said sample chamber assembly.

2. The apparatus of claim 1 for cryopreparing biological tissue further comprising a mechanical pump, said mechanical pump being connected to said ultra high vacuum pump assembly.

3. The apparatus of claim 2 for cryopreparing biological tissue further comprising a hydrocarbon trap connected in series with said mechanical pump and said ultra high vacuum pump assembly.

4. The apparatus of claim 3 for cryopreparing biological tissue wherein said hydrocarbon trap is a molecular sieve trap.

5. The apparatus of claim 1 for cryopreparing biological tissue wherein said ultra high vacuum pump assembly includes a turbomolecular pump.

6. The apparatus of claim 5 for cryopreparing biologi-cal tissue wherein said turbomolecular pump is encased within a bakeout jacket.

7. The apparatus of claim 1 for cryopreparing biologi-cal tissue further comprising a gate valve mounted between said ultra high vacuum assembly and said sample chamber assembly.

8. The apparatus of claim 1 for cryopreparing biologi-cal tissue wherein said sample chamber assembly further includes a resin embedding chamber.

9. The apparatus of claim l for cryopreparing biologi-cal tissue wherein said sample chamber assembly includes means for staining said biological tissue.

10. The apparatus of claim 9 for cryopreparing biologi-cal tissue wherein said means for staining said biological tissue includes osmium tetroxide.

11. The apparatus of claim 1 for cryopreparing biologi-cal tissue further comprising a sample holder, said sample holder being fitably received in said sample chamber assembly.

12. The apparatus of claim 1 for cryopreparing biologi-cal tissue further comprising a control panel.

13. The apparatus of claim 12 for cryopreparing biologi-cal tissue wherein said control panel includes vacuum gauge monitoring means, a residual gas analyzer, and temperature monitoring means.

14. Apparatus for cryopreparing biological tissue comprising:

(a) apparatus for rapid supercooling of biological tissue, said apparatus including a copper rod adapted to transform said biological tissue to the vitreous phase, said copper rod maintaining said tissue at a temperature of less than -140°C;

(b) a sample holder designed to hold said vitrified tissue;

(c) a sample chamber assembly adapted to receive said sample holder containing said vitrified biological tissue, said sample chamber assembly being movable to facilitate insertion into and withdrawal from a cryogenic bath;

(d) a low vacuum mechanical pump connected to said sample chamber assembly for depressurizing said sample chamber assembly to 1 x 10-3 Torr;

(e) an ultra high vacuum pump assembly for depres-surizing said sample chamber assembly to from 1 X 10-8 Torr to 1 x 10-10 Torr, said ultra high vacuum pump assembly being removably attached to said sample chamber assembly;

(f) a cryogenic bath for maintaining the temperature of said sample chamber assembly at -140°C or below, said cryogenic bath being adapted to receive at least a portion of said sample cham-ber assembly; and (g) a control panel electrically connected to said sample chamber assembly to monitor the vacuum, temperature and residual gas in said sample chamber assembly.

15. The apparatus of claim 14 for cryopreparing biologi-cal tissue further comprising a molecular sieve trap.

16. The apparatus of claim 15 for cryopreparing biologi-cal tissue wherein said ultra high vacuum pump assembly includes a turbomolecular pump.

17. The apparatus of claim 16 for cryopreparing biologi-cal tissue wherein said turbomolecular pump is encased within a bakeout jacket.

18. The apparatus of claim 14 for cryopreparing biologi-cal tissue further comprising a gate valve interposed between said ultra high vacuum pump assembly and said sample chamber assembly.

19. The apparatus of claim 14 for cryopreparing biologi-cal tissue wherein said sample chamber assembly further includes a resin embedding chamber.

20. The apparatus of claim 14 for cryopreparing biologi-cal tissue wherein said sample chamber assembly includes means for staining said biological tissue.

21. The apparatus of claim 20 for cryopreparing biologi-cal tissue wherein said means for staining said biological tissue is osmium tetroxide.

22. Apparatus for cryopreparing biological tissue comprising:

(a) apparatus for rapid supercooling of biological tissue, said apparatus including a solid copper rod adapted to transform said biological tissue to the vitreous phase, said solid copper rod maintaining said tissue at a temperature of less than -140°C;

(b) a sample holder designed to hold said vitrified tissue, said sample holder being connected to radiant heating means;

(c) a sample chamber assembly adapted to receive said sample holder containing said vitrified biological tissue, said sample chamber assembly being movable to facilitate insertion into and withdrawal from a cryogenic bath, said sample chamber assembly further including a resin embedding chamber and sample staining means;

(d) a low vacuum mechanical pump connected to said sample chamber assembly for depressurizing said sample chamber assembly to 1 x 10-3 Torr;

(e) a turbomolecular pump for depressurizing said sample chamber assembly to from 1 x 10-8 Torr to 1 x 10-10 Torr, said turbomolecular pump being removably attached to said sample chamber assembly;

(f) a gate valve interposed between said turbomo-lecular pump and said sample chamber assembly, said gate valve being sealed;

(g) a bakeout jacket encasing said turbomolecular pump;

(h) a cryogenic bath for maintaining the temperature of said sample chamber assembly at -140°C or below, said cryogenic bath being adapted to receive at least a portion of said sample cham-ber assembly; and (i) a control panel connected to said sample chamber assembly to monitor the vacuum, temperature and residual gas in said sample chamber assembly.

23. The apparatus for cryopreparing biological tissue of claim l, wherein said sample holder comprises:

(a) one or more tissue reservoirs adapted to retain biological tissue samples, said tissue reser-voirs permitting the flow of moisture and energy to and from said tissue samples;

(b) a support assembly for retaining said one or more tissue reservoirs and said biological tissue samples, said support assembly being adapted to maintain said biological tissue samples at cryogenic temperature and at ultra-low vacuums; and (c) radiant heating means associated with said one or more tissue reservoirs, said radiant heating means being adapted to transfer heat energy to said biological tissue samples.

24. The apparatus for cryopreparing biological tissue of claim 23 wherein said sample holder further comprises one or more tissue reservoir covers, said covers being sized to be fittably received by said one or more tissue reser-voirs, at least a portion of each of said one or more covers including apertures which permit the flow of moisture and energy to and from each of said tissue samples.

25. The apparatus for cryopreparing biological tissue of claim 23 wherein said support assembly of said sample holder comprises a metal block with said one or more tissue reservoirs in one surface thereof, said metal blocking being made from material selected from the group consisting of: copper, silver, gold and combinations thereof.

26. The apparatus for cryopreparing biological tissue of claim 11 wherein said sample holder comprises:
(a) one or more tissue reservoirs adapted to retain biological tissue samples, said tissue reservoirs permitting the flow of moisture and energy to and from said tissue samples;
(b) a support assembly for retaining said one or more tissue reservoirs and said biological tissue samples, said support assembly being adapted to maintain said biological tissue samples at cryogenic temperature and at ultra-low vacuums; and (c) radiant heating means associated with said one or more tissue reservoirs, said radiant heating means being adapted to transfer heat energy to said biological tissue samples.

27. The apparatus for cryopreparing biological tissue of claim 26 wherein said sample holder further comprises one or more tissue reservoir covers, said covers being sized to be fittably received by said one or more tissue reser-voirs, at least a portion of each of said one or more covers including apertures which permit the flow of moisture and energy to and from each of said tissue samples.

28. The apparatus for cryopreparing biological tissue of claim 26 wherein said support assembly of said sample holder comprises a metal block with said one or more tissue reservoirs in one surface thereof, said metal blocking being made from material selected from the group consisting of: copper, silver, gold and combinations thereof.

29. The apparatus for cryopreparing biological tissue of claim 14 wherein said sample holder comprises:
(a) one or more tissue reservoirs adapted to retain biological tissue samples, said tissue reservoirs permitting the flow of moisture and energy to and from said tissue samples;
(b) a support assembly for retaining said one or more tissue reservoirs and said biological tissue samples, said support assembly being adapted to maintain said biological tissue samples at cryogenic temperature and at ultra-low vacuums; and (c) radiant heating means associated with said one or more tissue reservoirs, said radiant heating means being adapted to transfer heat energy to said biological tissue samples.

30. The apparatus for cryopreparing biological tissue of claim 29 wherein said sample holder further comprises one or more tissue reservoir covers, said covers being sized to be fittably received by said one or more tissue reser-voirs, at least a portion of each of said one or more covers including apertures which permit the flow of moisture and energy to and from each of said tissue samples.

31. The apparatus for cryopreparing biological tissue of claim 29 wherein said support assembly of said sample holder comprises a metal block with said one or more tissue reservoirs in one surface thereof, said metal blocking being made from material selected from the group consisting of: copper, silver, gold and combinations thereof.

32. The apparatus for cryopreparing biological tissue of claim 22 wherein said sample holder comprises:
(a) one or more tissue reservoirs adapted to retain biological tissue samples, said tissue reservoirs permitting the flow of moisture and energy to and from said tissue samples;
(b) a support assembly for retaining said one or more tissue reservoirs and said biological tissue samples, said support assembly being adapted to maintain said biological tissue samples at cryogenic temperature and at ultra-low vacuums; and (c) radiant heating means associated with said one or more tissue reservoirs, said radiant heating means being adapted to transfer heat energy to said biological tissue samples.

33, The apparatus for cryopreparing biological tissue of claim 32 wherein said sample holder further comprises one or more tissue reservoir covers said covers being sized to be fittably received by said one or more tissue reser-voirs, at least a portion of each of said one or more covers including apertures which permit the flow of moisture and energy to and from each of said tissue samples.

34. The apparatus for cryopreparing biological tissue of claim 32 wherein said support assembly of said sample holder comprises a metal block with said one or more tissue reservoirs in one surface thereof, said metal blocking being made from material selected from the group consisting of: copper, silver, gold and combinations thereof.

35. A method for cryopreparing biological tissue comprising:
(a) rapidly cooling said tissue to a temperature less than about -123°C and at a rate such that vitrification of the water in said tissue takes place without the formation of resolvable ice crystals;
(b) equilibrating said tissue containing said vitrified water at a temperature less than about -140°C and a vacuum pressure less than about 1 x 10-8 Torr at which said vitrified water is capable of changing physical state directly to the vapor phase;
(c) adding energy to said equilibrated biological tissue at a rate sufficient to cause said vitrified water to change physical state directly to the vapor phase without substantially disturbing said equilibration;
(d) removing water from said biological tissue by molecular distillation by the addition of energy until substantially no vaporized water can be detected in the atmosphere surrounding said biological tissue;
and (e) increasing the temperature of said tissue after water has been molecularly distilled from said tissue.

36. The method of claim 35 in which said energy is added incrementally.

37. The method of claim 35 in which the temperature of said biological tissue, after substantially no water vapor can be detected, is incrementally raised to room temperature.

38. The method of claim 35 in which said water in said biological tissue is vitrified by cooling said tissue with liquid nitrogen.

39. The method of claim 35 in which the vitrification of said biological tissue takes place in less than about 1 second.

40. The method of claim 35 wherein said vacuum is created in less than about 300 minutes.

41. The method of claim 35 in which the equilibrating of said biological tissue is indicated by a constant biological tissue temperature for a time period of at least one hour.

42. The method of claim 41 in which said constant biological tissue temperature is between about -140°C and about -196°C
and said vacuum is from about 1 x 10-8 Torr to 1 x 10-10 Torr.

43. The method of claim 36 in which said energy is heat energy.

44. The method of claim 36 in which said energy is radiant energy.

45. The method of claim 44 which includes generating said radiant energy by nuclear magnetic resonance.

46 A method for cryopreparing biological tissue comprising:
(a) cooling said biological tissue under conditions sufficient to vitrify the water contained in said biological tissue, said cooling taking place in less than about 1 second;
(b) equilibrating said biological tissue containing said vitrified water at a temperature and pressure at which said vitrified water is capable of changing physical state directly to the vapor phase, the temperature of said equilibration being less than -140°C and the pressure being less than 1 x 10 -8 Torr; said equilib-rating being indicated by a constant biological tissue temperature for a time period of at least one hour;
(c) adding energy to said equilibrated biological tissue at a rate sufficient to cause sublimation of said water without substantially disturbing said equilibra-tion;
(d) removing water from said biological tissue by molecu-lar distillation by the addition of energy until substantially no vaporized water can be detected in the atmosphere surrounding said biological tissue, and (e) incrementally increasing the temperature of said biological tissue, after water has been removed, until said biological tissue is at substantially room temperature.

47. A method for cryopreparing biological tissue comprising:
(a) rapidly cooling said tissue at a rate such that vitrification of the water in said tissue takes place without the formation of resolvable ice crystals;
(b) equilibrating said tissue containing said vitrified water at temperature and pressure conditions at which said vitrified water is capable of changing physical state directly to the vapor phase;
(c) adding energy to said equilibrated biological tissue at a rate sufficient to cause said vitrified water to change physical state directly to the vapor phase without substantially disturbing said equilibration;
(d) removing water from said biological tissue by molecular distillation by the addition of energy until substantially no vaporized water can be detected in the atmosphere surrounding said biological tissue; and (e) increasing the temperature of said tissue after water has been molecularly distilled from said tissue.

48. The method of claim 47 in which said biological tissue is rapidly cooled to a temperature of less than about -123°C.

49. The method of claim 48 in which said biological tissue containing vitrified water is equilibrated at a temperature of less than about -140°C.

50. The method of claim 47 in which said energy is added incrementally.

51. The method of claim 47 in which the temperature of said biological tissue, after substantially no water vapor can be detected, is incrementally raised to room temperature.

52. The method of claim 47 in which the equilibrium temperature of said biological tissue is lower than the temperature at which the rate of ice crystal formation in said water permits the formation of resolvable ice crystals.

53. The method of claim 48 in which said water in said biological tissue is vitrified by cooling said tissue with liquid nitrogen.

54. The method of claim 47 in which the vitrification of said biological tissue takes place in less than about 1 second.

55. The method of claim 47 in which the step of equilibrating includes drawing a vacuum on the atmosphere surrounding said biological tissue to reach said pressure.

56. The method of claim 55 wherein said vacuum is from about 1 x 10-8 Torr to 1 x 10-10 Torr.

57. The method of claim 56 wherein said vacuum is created in less than about 300 minutes.

58. The method of claim 47 in which the equilibrating of said biological tissue is indicated by a constant biological tissue temperature for a time period of at least one hour.

59. The method of claim 58 in which said constant biological tissue temperature is between about -140°C and about -196°C.

60. The method of claim 50 in which said energy is heat energy.

61. The method of claim 50 in which said energy is radiant energy.

62. The method of claim 61 which includes generating said radiant energy by nuclear magnetic resonance.

63. A method for cryopreparing corneal tissue for use in surgical procedures comprising:
(a) providing a corneal tissue sample, suitable for its intended end use;
(b) vitrifying said corneal tissue sample in less than one second;
(c) bringing said vitrified corneal tissue sample to equilibrium at a temperature of less than -140°C; and (d) dehydrating said vitrified corneal tissue sample by sublimation and under vacuum while said corneal tissue sample is maintained in a state of equilibrium, said vacuum reaching a level of from 1 x 10-8 Torr to 1 x 10-10 Torr.

64. Apparatus for cryopreparing vitrified biological tissue comprising:
(a) a sample holder for retaining vitrified biological tissue;
(b) means for maintaining the sample holder at a cryogenic temperature;
(c) ultra-high vacuum means for depressurizing the atmosphere in the sample holder; and (d) means for dehydrating said vitrified biological tissue while maintaining said tissue at a cryogenic temperature of less than -80°C.

65. The apparatus of claim 64 wherein said tissue in said holder is maintained at a temperature of -140°C or below.

66. The apparatus of claim 64 wherein said temperature is maintained by means of liquid nitrogen.

67, The apparatus of claim 64 wherein said vacuum means maintains a vacuum from 1 x 10-8 Torr to 1 x 10-10 Torr.

68. The apparatus of claim 67 wherein said vacuum means creates said vacuum in less than 300 minutes,

69. The apparatus of claim 64 including means for indicating the equilibrium of said vitrified tissue sample by a constant temperature over a preselected time period of from 1 to 5 hours.

70. The apparatus of claim 69 wherein said constant temperature is from -140°C to -196°C.

71. The apparatus of claim 64 wherein means for said dehydrating is means permitting sublimation or stimulated molecular distillation.

72. The apparatus of claim 71 including means for enhancing dehydration by the addition of energy from a secondary source.

73. The apparatus of claim 72 wherein said secondary source of energy is heat energy.

74. The apparatus of claim 72 wherein said secondary source of energy is radiant energy.

75. The apparatus of claim 74 wherein said radiant energy is by means of nuclear magnetic resonance.

76. The apparatus of claim 64 further comprising means for adding a contrast enhancing material.

77. The apparatus of claim 76 wherein said contrast enhancing material is osmium tetroxide.

78. The apparatus of claim 77 further comprising means for infiltrating said biological tissue with a resin, said resin being polymerized by heat.

79. The apparatus of claim 77 further comprising means for infiltrating said biological tissue with a resin, said resin being photopolymerized by ultraviolet light.

80. A method for cryopreparing biological tissue comprising:
(a) vitrifying biological tissue;
(b) equilibrating said vitrified biological tissue under temperature and vacuum conditions that preclude the formation of resolvable cell water ice crystals; and, (c) dehydrating said vitrified biological tissue while said tissue is maintained in a state of equilibrium.

81. The method of claim 80 wherein said vacuum is at least 3 x 10-9 Torr.

82. The method of claim 80 wherein the equilibrium of said vitrified biological tissue is indicated by a constant temperature.

83. The method of claim 80 wherein said dehydration is by means of sublimation and molecular distillation.

84. A method of cryopreparing biological tissue which comprises:
(a) rapidly cooling said tissue at a rate and to a temperature sufficient to vitrify the water in said tissue; and (b) removing said water from said tissue by vaporization directly from its vitrified state.

85. A method of cryopreparing biological tissue which comprises:
(a) rapidly cooling said tissue at a rate and to a temperature sufficient to vitrify the water in said tissue;
(b) subjecting said tissue containing said vitrified water to a combination of vacuum and temperature conditions at which said water is capable of vaporizing directly from its vitrified state; and (c) slowly adding energy to said tissue at a rate and in a quantity sufficient to dehydrate said tissue by vaporizing said vitrified water directly from said vitrified state.

CLAIMS SUPPORTED BY THE SUPPLEMENTARY DISCLOSURE

86. The apparatus for cryopreparing biological tissue of claim 23 or 26 wherein said support assembly of said sample holder comprises a block of thermally conductive material with said one or more tissue reservoirs in one surface thereof, said material being selected from the group consisting of: copper, silver, gold, sapphire, diamond and combinations thereof.

87. The apparatus for cryopreparing biological tissue of claim 29 or 32 wherein said support assembly of said sample holder comprises a block of thermally conductive material with said one or more tissue reservoirs in one surface thereof, said material being selected from the group consisting of: copper, silver, gold, sapphire, diamond and combinations thereof.