WO2007123684A2

WO2007123684A2 - Methods and devices for thawing and/or imaging frozen biological samples

Info

Publication number: WO2007123684A2
Application number: PCT/US2007/007915
Authority: WO
Inventors: Mark A. Voelker
Original assignee: Biotime, Inc.
Priority date: 2006-04-04
Filing date: 2007-03-30
Publication date: 2007-11-01
Also published as: WO2007123684A3

Abstract

Methods of freezing, thawing and imaging a biological sample, one or more times, in a manner sufficient to freeze, thaw, and/or image the biological sample, while maintaining the structural integrity and/or viability of the sample are provided. Aspects of embodiments of the methods include freezing, thawing and/or imaging a biological sample under pressure in a manner sufficient to maintain the structural integrity and/or viability of the sample. Also provided are devices and systems for use in practicing the methods.

Description

METHODS AND DEVICES FOR THAWING AND/OR IMAGING FROZEN BIOLOGICAL SAMPLES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part application of U.S. patent application Serial No. 11/601 ,434 entitled "Methods and Devices for Thawing a Frozen a Biological Sample," and filed November 17, 2006; and U.S. Application. Serial No. 11/601 ,442 titled "Methods and Devices for Imaging and Manipulating a Biological Sample," filed November 17, 2006; which applications pursuant to 35 U.S.C. § 119 (e), claim priority to the filing date of United States Provisional Patent Application Serial No. 60/789,541 , filed April 4, 2006; which applications are incorporated herein by reference in their entirety.

BACKGROUND

The development of microscopy has allowed scientists to image cells and tissues with increasing levels of detail and with increasing spatiaf and spectral resolution. Improvements in the detail that is visible in microscope images of cells and tissues have helped scientists understand how living organisms function and sometimes malfunction. This has increased the understanding of the structure and composition of various biological cells and tissues and has advanced the development of new protocols for the investigation, screening and diagnosis of disease.

Two techniques available for acquiring high-resolution images of biological samples include: optical microscopy and electron microscopy. Optical microscopy uses photon bombardment to magnify a sample. Optical microscopy allows scientists to image living cells and tissues with a spatial resolution traditionally defined by the Rayleigh criterion. In practical terms, the Rayleigh criterion means spatial resolution of at least about 200 nm for the best oil immersion objectives, but more typically up to about 500 nm for microscopes that do not reach Rayleigh criterion performance. Optical microscopes are easy to use, relatively inexpensive and can image living samples without killing them. However, the Rayleigh criterion spatial resolution attainable with optical microscopes is too large to directly image most of the molecular-scale components of living cells.

The electron microscope uses electron bombardment to magnify a sample. Using electron microscopy, biological cells can be imaged at very high spatial resolution (10 nm or better) and magnified over 2 million times. This allows for the direct imaging of cells and their components in minute detail. Intracellular structures, such as membranes, chromosomes, vesicles, microtubules, and even large protein molecules, may be imaged with the electron microscope. However, sample preparation methods, and the energetic nature of electron bombardment itself, usually causes loss of viability when electron microscopy is used to image biological samples. The tradeoff for being able to achieve such high resolution imaging of biological cells using the electron microscope is that the cells so imaged are kilted in the process of acquiring the images.

There are several ways in which a cell sample can be prepared for imaging by an electron microscope. For instance, the cells can be fixed so as to preserve the sample. Specifically, the sample may be dehydrated and the water in the cell sample replaced with an organic solvent, such as ethanol or aldehyde. The organic solvent may then be replaced with a plastic medium such as a resin (e.g., araldite or epoxy) to fix the fine structure of the sample. The sample may also be stained, for instance, by a heavy metal or dye, so as to generate better contrast between cellular components. Additionally, the sample embedded with a resin, may be sectioned to give very thin slices which can then be separately imaged.

Recently, cryofixation has been used to rapidly freeze a cell sample so as to preserve the cells in their natural state for imaging. Once frozen, embedded in resin, stained and sectioned, the cell sample can then be viewed by electron microscopy. However, cryofixation can lead to the formation of ice crystals that may result in damage or destruction of the structural integrity of the cells to be imaged. Additionally, using cryofixation techniques at ambient pressure only allows for the preservation of a cell sample that is about 15 μm thick or less. There is continued interest in developing methods and devices for imaging the components of cells, tissues and organs with increasing levels of detail and with increasing spatial and spectral resolution.

SUMMARY

Methods of manipulating and imaging a manipulated biological sample are also provided. One embodiment of the methods includes repeatedly freezing, thawing and imaging a biological sample, in a manner sufficient to maintain the structural integrity and/or viability of the sample, wherein the sample may be manipulated once frozen or thawed. The biological sample may be imaged before, during or after the sample is manipulated, frozen and/or thawed.

In certain embodiments, the methods include thawing a biological sample, wherein a previously frozen biological sample is thawed under pressure in a manner sufficient to maintain the structural integrity of the sample. In other embodiments, the methods include thawing a viable biological sample in a manner sufficient to maintain the viability of the biological sample. For instance, in certain embodiments, the methods include the thawing of a biological sample under high pressure. In certain other embodiments, the methods include ambient pressure thawing a viable biological sample, frozen under high pressure, in a manner sufficient to maintain the viability and/or structural integrity of the biological sample. In certain embodiments, the methods include thawing and/or manipulating a biological sample and imaging the thawed and/or manipulated sample. Additionally, in certain embodiments, the methods include freezing and/or manipulating a frozen biological sample and imaging the frozen sample, for instance, wherein the sample has previously been thawed and/or manipulated and/or imaged. In certain embodiments, the methods include refreezing a previously thawed biological sample in a manner sufficient to prevent or at least reduce the formation of ice crystals generated by the freezing process, for instance, refreezing the sample under high pressure. In certain embodiments, the methods include re-freezing a biological sample in a manner sufficient to maintain the structural integrity of the biological sample. In certain embodiments, the methods include re-freezing a viable biological sample in a manner sufficient to maintain the viability of the biological sample. Additionally, once frozen the biological sample may be stored over a prolonged period of time and then thawed, or repeatedly frozen and thawed, while maintaining the structural integrity and/or viability of the biological sample.

In certain embodiments, the methods include freezing and/or thawing and/or refreezing and/or imaging and/or manipulating a sample or a previously thawed or frozen viable biological sample under pressure {e.g., high pressure) wherein the sample has not previously been chemically fixed, stained, embedded, or otherwise treated in a manner that destroys the viability of the biological sample.

The methods of the invention are useful for repeatedly freezing (e.g., cryofixing), thawing, imaging and/or manipulating a viable biological sample, e.g., under pressure, and in a manner sufficient to maintain the substantial structural integrity or viability of the sample. Once thawed or frozen the biological sample may be imaged and observed, for instance, via microscopy, e.g., optical microscopy, and/or manipulated by contacting it with physical probes or radiation or chemical reagents or molecular nanodevices, or the like. In this manner, a biological sample may be repeatedly frozen, imaged and/or manipulated, thawed, and imaged and/or manipulated over a prolonged period of time while maintaining the structural integrity and/or the viability of the biological sample. For instance, while in the frozen state, the sample may be contacted for arbitrarily long periods of time with photons, electrons, physical, chemical, molecular or other probes, allowing intricate, detailed and precise observation, imaging and/or manipulation of the sample. Also provided is an apparatus for performing the methods of the invention.

An apparatus of the invention is configured for freezing and/or thawing a sample, for instance, a biological sample, under pressure (e.g., high pressure). In certain embodiments, the apparatus is configured for being operated in conjunction with an apparatus for visualizing a frozen or thawed sample. In certain embodiments, the apparatus includes a chamber that includes an interior configured for holding a sample, a pressure modulator for modulating the pressure within the interior of the chamber and a temperature modulator for modulating the temperature of the interior of the chamber from a temperature that is below the freezing point of water to a temperature that is above the freezing point of water and vice-versa.

In certain embodiments, an apparatus of the invention is configured for both generating a high pressure within a chamber and for rapidly transferring heat into and out of the chamber and thereby thawing or heating or freezing or cooling a sample held therein. In certain embodiments, the apparatus includes a plurality of anvils, a pressure modulator, a pressure chamber and/or sample holding element, and a temperature modulator. In accordance with these embodiments, the anvils are configured for being compressed ,aπd thereby generating a high pressure within the pressure chamber. The pressure chamber may include a sample holding element, for instance, a gasket, which is configured for holding a sample and is adapted for withstanding the high pressure generated by the compression of the anvils. The pressure modulator, for instance, one or more levers, is configured for compressing the anvils so as to generate a high pressure and the temperature modulator, for instance, an applied gas or liquid, is configured for contacting the anvils and thereby modulating the temperature within the chamber.

In certain embodiments, one or more of the components of the chamber are configured in such a manner so as to allow the transmission of photons, electrons and the like, from the outside of the chamber to the interior of the chamber, as well as to allow transmission of photons, electrons and the like, from the interior of the chamber to the outside of the chamber, to facilitate the observation of a sample within the chamber. In certain embodiments, the apparatus includes, in addition to the chamber, an imaging element, for instance, one or more devices configured for and positioned to contact the sample with photons, electrons, or the like, while the sample is inside the chamber and thereby image the sample. In certain other embodiments, the apparatus includes an imaging element that is configured for and positioned to contact the sample with photons, electrons, or the like, while the sample is outside the chamber. In certain embodiments, the apparatus includes one or more elements configured for and positioned to contact the sample with physical, chemical, molecular or other probes while the sample is inside or outside of the chamber and thereby manipulate the sample. In certain embodiments, the apparatus includes devices configured to add or subtract material from the sample while the sample is frozen or unfrozen and while the sample is inside or outside the chamber.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates one embodiment of an apparatus of the invention. FIG. 2 illustrates another embodiment of an apparatus of the invention. FIG. 3 illustrates another embodiment of an apparatus of the invention. FIG. 4 illustrates the embodiment of an apparatus of FIG. 3 of the invention with photon source, objective and upper and lower cones of light indicated. FIG. 5 is an expanded cutaway view of the sample cell in FIG. 4. FIG. 6 is an oblique view of the delivery manifold component in FIG. 3. FIGS. 7 through 9 are cross section views of the predicted temperature distribution inside a sample cell of an apparatus of the invention, at various times during the cooling down of the sample.

FIGS. 10 through 12 are cross section views of the predicted temperature distribution inside a sample cell of an apparatus of the invention, at various times during the warming up of the sample.

FIG. 13 shows the predicted temperature at the center of the sample, during cooling down, inside an apparatus of the invention.

FIG. 14 shows the predicted temperature at the center of the sample, during warming up, inside an apparatus of the invention.

FIG. 15 is a slide of a tissue sample (control) that was neither pressurized nor frozen. Rat brain was kept in ice cold bicarbonated Hextend for 15 minutes, cut into 200μm thick slices, then stained with Molecular Probes L-3224 live/dead stain, and imaged with Zeiss Meta 510 Confocal Laser Scanning Microscope. The L-3224 Live/Dead stain stains the cytoplasm of living cells grey and the nuclei of dead cells black.

FIG. 16 is a slide of a tissue sample that was pressurized but was not frozen. Rat brain was kept in ice cold bicarbonated Hextend, pressurized to 2177 atm for 5 minutes, cut into 200//m thick slices, then stained with Molecular Probes L-3224 live/dead stain and imaged with Zeiss Meta 510 Confocal Laser Scanning Microscope.

FIG. 17 is a slide of a tissue sample that was frozen and thawed while pressurized. Rat brain was kept in ice cold bicarbonated Hextend, then pressurized to 2177 atm, frozen to -196° C over 5 minutes, held at this temperature and pressure for 5 minutes, then thawed over 15 minutes while held at 2177 atm pressure, then depressurized, cut into 200//m thick slices, stained with Molecular Probes L-3224 live/dead stain, and imaged with Zeiss Meta 510 Confocal LSM.

FIGS. 18 and 19 are slides of a tissue sample that was frozen quickly at 2100 atm pressure then thawed at 1 atm. Rat brain was cut into 200μm thick slices, then soaked in ice cold bicarbonated Hextend + 20% glycerol, then rapidly frozen under 2100 atm pressure to -196° C in a Bal-Tec HPM-010 machine, then thawed at 1 atm pressure by immersion in Hextend at 20° C, stained with Molecular Probes L-3224 live/dead stain, and imaged with Zeiss Meta 510 Confocal Laser Scanning Microscope.

DETAILED DESCRIPTION

Methods of freezing, thawing and imaging a biological sample, one or more times, in a manner sufficient to freeze, thaw, and/or image the biological sample, while maintaining the structural integrity and/or viability of the sample are provided. Aspects of embodiments of the methods include freezing, thawing and/or imaging a biological sample under pressure in a manner sufficient to maintain the structural integrity and/or viability of the sample. Also provided are devices and systems for use in practicing the methods. Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the stated ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference in their entirety as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. As summarized above, embodiments of the methods of the invention are directed to preparing and/or observing and/or imaging and/or manipulating a sample. An aspect of embodiments of the methods includes thawing and/or imaging a sample, for instance, a biological sample, in a manner sufficient to maintain the structural integrity of the biological sample. In certain embodiments, the biological sample is a viable biological sample and the methods of the invention include thawing and/or imaging the sample in a manner sufficient to maintain the viability of the biological sample. In certain embodiments, once thawed and/or imaged, the biological sample may be refrozen and/or imaged once frozen. For instance, the biological sample may be refrozen within a high pressure chamber and then manipulated and/or imaged (e.g., viewed) in a manner sufficient to maintain the structural integrity and/or viability of the sample.

Accordingly, in certain embodiments, the methods of the invention involve the high pressure thawing of a frozen sample. In certain embodiments, the methods of the invention involve the thawing of a frozen sample under pressure, for instance, high pressure, and then imaging of the sample once thawed. In certain embodiments, the methods of the invention also include the freezing or refreezing of a sample under pressure, for instance, high pressure, and imaging the sample once frozen. In one embodiment, the sample is imaged while under pressure, in a high pressure apparatus, such as the apparatus set forth herein below. In other embodiments, the sample is imaged outside of a high pressure apparatus once the sample has been frozen and/or thawed within a high pressure apparatus. By "thawing" is meant warming a sample so as to convert the frozen material therein to a non-frozen state. In certain embodiments, thawing of the sample is achieved by changing the temperature of the sample from a first temperature to a second temperature. In certain embodiments, the first temperature ranges from about 0 K to about 280 K, such as from about 40 K to about 273 K, including about 77 K to about 200 K and the second temperature ranges from about 250 K to about 350 K, such as from about 274 K to about 310 K, including about 288 K to about 298 K. Thawing of the sample may be acheived using any convenient protocol, e.g., via application of heat to the sample, where heat may be applied to the sample, for instance, by the specific protocols detailed below. Although the thawing can take place over a prolonged period of time, in certain embodiments, it occurs rapidly. For instance, in certain embodiments the thawing has a duration ranging from about 1 millisecond to about 1 second, from about 3 milliseconds to about 30 milliseconds, such as from about 5 milliseconds to about 20 milliseconds and including from about 7 to about 15 milliseconds, e.g., about 10 milliseconds.

As summarized above, thawing may occur under pressure, for instance, high pressure. By "high pressure" is meant a pressure in the range of about 200 atm to about 4000 atm, for instance, about 1800 atm to about 2800 atm, or about 2000 atm to about 2200 atm. Thawing can take place either with or without the addition of agents that suppress the formation of ice crystals. Ice crystal formation deterrent agents that may be used include, but are not limited to: glycerol, dimethyl sulfoxide, polyvinyl pyrridole, sucrose, and the like. Other ice crystal deterrent agents that may be used include antifreeze proteins and antifreeze glycoproteins and other freeze protecting compounds found naturally in certain cold tolerant organisms, such as certain arctic fishes and insects, and synthetic versions or derivatives or variants of such proteins and glycoproteins and other naturally occurring freeze protecting compounds found in organisms that live in cold environments. Combinations of such agents may also be used. Although thawing can take place with the addition of agents that suppress the formation of crystals during the thawing process, in certain embodiments, the thawing process occurs without the addition of such agents. In certain embodiments, the methods of the invention also include the freezing or refreezing of a sample under pressure, for instance, a high pressure. By "high pressure freezing" is meant a process of converting a material, such as a biological sample, into a frozen solid that is relativley free of crystalline structures (e.g., water crystals). By relatively free of crystalline structures is meant that the number and/or size of crystaline sturctures (e.g., water crystals) within the sample frozen under high pressure conditions is reduced as compared to the number and /or size of crystaline structures produced by freezing a similar sample while the sample is not under high pressure conditions, e.g., by at least about two fold, such as by at least about four fold and including by at least about ten fold.

In certain embodiments, a "high pressure frozen sample" of the invention is one wherein the liquid component of the sample has been converted to a solid form, under high pressure, with a reduced formation of ice crystals, as compared to a similar sample frozen at ambient pressure. Freezing occurs by the removal of heat from the sample, wherein a sample is cooled. In certain embodiments, freezing of a sample is achieved by. changing the temperature of the sample from a first temperature to a second temperature. In certain embodiments, the first temperature ranges from about 473 K to about 233 K, such as from about 350 K to about 252 K, such as from about 300 K to about 273 K, and the second temperature ranges from about 273 K to about 0 K, such as from about 225 K to about 77 K, while under pressure.

Freezing of the sample may be acheived using any convenient protocol, e.g., via application of a freezing (e.g., cryogenic) medium to the sample, where the freezing medium may be applied to the sample, for instance, by using the specific protocols detailed below. Although the freezing can take place over a prolonged period of time, in certain embodiments, it occurs rapidly, for instance, in a range of about 1 millisecond to about 1 second, from about 3 milliseconds to about 30 milliseconds, such as from about 7 milliseconds to about 15 milliseconds, e.g., about 10 milliseconds. Freezing can take place either with or without the addition of agents such as cryoprotectants (for instance, glycerol, dimethyl sulfoxide, polyvinyl pyrridole, sucrose, antifreeze proteins and glycoproteins and the like) that suppress the formation of crystals. Although freezing can take place with the addition of agents that suppress the formation of crystals, such agents can be toxic to or otherwise adversly effect the sample. In certain embodiments, the freezing process occurs without the addition of cryoprotectants or with cryoprotectants that do not adversly effect the sample.

In certain embodiments, freezing can take place with or without the addition of agents such as baroprotectants (for instance trimethyl amine oxide, TMAO) that suppress injury to biological systems due to exposure to high pressure. Although freezing can take place with the addition of agents that suppress injury due to high pressure, such agents can be toxic to or otherwise adversly effect the sample. In certain embodiments, the freezing process occurs without the addition of baroprotectants or with baroprotectants that do not adversely affect the sample.

The sample may be any sample the analysis and/or modification of which is desired. Any sample can be frozen, observed and/or imaged and/or manipulated, thawed, observed and/or imaged and/or manipulated one or more times in accordance with the methods of the invention. For instance, the methods are suitable for use with an environmental sample or a biological sample, for instance, an organ, tissue, or cell sample. In certain embodiments, a sample includes one or more cells, e.g., multiple cells. In certain embodiments, the cell sample includes one or more gamete cells, for instance, spermatozoa or ova. In certain embodiments, a sample includes multiple cells, stem cells, for instance, embryonic stem cells, or the like. In certain embodiments, a sample includes a multicellular organism.

The sample may be obtained from any suitable source in any manner sufficient to preserve the integrity of the sample, as is well known in the art. Where the sample is a biological sample it may be obtained from a suitable organ and/or tissue of interest. For instance, the sample may be a blood sample collected from a subject's veins via venipuncture, the sample may be an epidermal sample collected via skin grafting, the sample may be a tissue sample collected from some other organ (e.g., a liver, kidney, lungs, heart, brain or various other organs), the sample may be one or more ova, the sample may be one or more spermatozoa, the sample may be one or more embryos, the sample may be one or more embryonic or adult stem cells.

In certain embodiments, the sample is a viable biological sample. In certain embodiments, the methods of the invention are characterized in that they are performed in such a manner and under conditions that preserve or maintain the viability of a biological sample. By "viability" of a biological sample is meant that the biological sample and/or one or more of its components maintains its ability to function, divide, differentiate, grow or otherwise live. Where the sample is a biological sample, for instance, a cell or tissue sample or microorganism, the thawing, freezing, observing and/or imaging occur without substantially disrupting the structural integrity of the biological sample. Additionally, where the sample is a viable biological sample, the thawing, freezing, observing and/or imaging occur in a manner sufficient to maintain the viability of the sample. In certain embodiments, the methods of the invention are characterized in that they are performed in such a manner and under conditions that at least substantially preserve or maintain the structural integrity of the biological sample.

In certain embodiments, the sample may be one or more cells (e.g., a cell, a gamete cell, stem cell, or the like) that has been associated with a substrate, for instance, a glass, silicon or electronic chip. For instance, one or more cells may be arranged in a circuit configuration and may interact with other circuitry components to form a circuit that is capable of transferring a current or other signal from one point on the chip to another. Further, as is described in greater detail herein below, the one or more cells may be arranged in a circuit configuration and may interact with other circuitry components to form a circuit that is capable of transferring a current or other signal from one point on the chip to another.

Another aspect of the invention is an apparatus for both freezing (e.g., cooling) and thawing (e.g., heating) a sample under pressure, for instance, high pressure, in a manner sufficient to prevent or reduce in size and/or number the formation of ice crystals within the sample normally caused by a thawing or freezing process that is not performed under high pressure. Hence, in certain embodiments, an apparatus of the invention is characterized in that it is configured for both freezing and thawing a biological sample contained inside a high pressure chamber of the apparatus in a manner sufficient to maintain the structural integrity and/or viability of the biological sample, and for observing and/or imaging and/or manipulating the sample, or for being associated with an apparatus for observing and/or imaging and/or manipulating the sample. The_. apparatus may be configured for observing and/or imaging the sample before, after or during the freezing and/or thawing process while the sample is inside or outside of a chamber and/or inside or outside of the apparatus.

For instance, in certain embodiments, an apparatus of the invention allows observing and/or imaging and/or manipulating the sample while the sample is inside a sample containing element or high pressure chamber of the apparatus, e.g., the sample may be contained within a sample element that forms a high pressure chamber of the apparatus which is removed from the apparatus for observing and/or imaging and/or manipulation of the sample. In certain other embodiments, an apparatus of the invention may allow the sample to be removed temporarily from the sample containing element of the high pressure chamber of the apparatus for purposes of observing and/or imaging and/or manipulating the sample.

An apparatus of the invention includes a chamber, a pressure modulator and a temperature modulator and may include an element for observation and/or imaging and/or manipulation. The chamber includes an interior that is configured for holding a sample. For instance, where the pressure modulator includes two opposing surfaces (e.g. anvils) the chamber may be a cavity created between the two surfaces (e.g., anvils). The chamber may be formed from the interior of a sample holding element that is adapted for holding a sample, for instance, a biological sample, and configured for being associated between the two surfaces (e.g., anvils) of the pressure modulator. For instance, the sample holding element may be a gasket, foil, membrane, or the like. The sample holding element may be fabricated out of any material (e.g., metal) so long as it is capable of associating with the opposing surfaces of the pressure modulator in a manner sufficient to withstand a high pressure generated by the pressure modulator. In certain embodiments, the sample holding element is a hard metal foil associated between two opposing surfaces and adapted for both holding a sample and supporting the contact point of the two surfaces. In certain embodiments, the chamber may include a hydrostatic fluid.

The pressure modulator may be of any configuration so long as it is adapted for generating a pressure difference between the interior and the exterior of the chamber. Accordingly, the pressure modulator modulates the pressure of the interior of the chamber. In certain embodiments, the pressure modulator is a high pressure modulator capable of generating a high pressure within the chamber. By "high pressure" is meant a pressure in the range of about 200 atm to about 4000 atm, for instance, about 1800 atm to about of 2800 atm, or about 2000 atm to about 2200 atm. In certain embodiments, the pressure modulator is capable of generating a high pressure rapidly, for instance, in about 1 millisecond to about 30 milliseconds, such as from about 7 milliseconds to about 15 milliseconds, e.g., about 10 milliseconds. In certain embodiments, the pressure modulator may include two opposing surfaces and a force generating mechanism (e.g., a compression mechanism). For instance, the pressure modulator may include two opposing surfaces (e.g., anvils) that are configured to form a chamber and/or associate with a sample holding element in a manner so as to form a chamber and are additionally operatively connected to a force generating mechanism in a manner sufficient to allow the two opposing surfaces to be compressed one toward the other which thereby generates a high pressure within the chamber.

The force generating mechanism which is operatively connected to the two surfaces may include one or more lever arms, screws, hydraulic systems or the like that are configured for being tightened or pressurized and thereby compressing the two opposing surfaces toward one another. The operative connection may be such that it generates a substantially uniaxial force that is applied to the base of the opposing surfaces thereby compressing the surfaces together and consequently generating a high pressure within the chamber. As will be described in greater detail herein below, in certain embodiments, the two opposing surfaces of the pressure modulator may be anvils. By "anvil" is meant a hard, fixed surface that is operatively connected with a force generating mechanism and configured for .being compressed against a second hard, fixed surface and thereby generating a high pressure at the region of contact between the two surfaces. The anvils may be fabricated of any material capable of being compressed and withstanding the generation of a high pressure due to said compression without fracturing. The anvils may be fabricated from any suitable material, in certain embodiments, one or more of the anvils is a material with a high thermal conductivity. By high thermal conductivity material is meant a material with a thermal conductivity ranging between about 300 W m^"1 K^'1 to about 3000 W rrf¹ K^" ¹ or more, about 895 W m^"1 IC¹ to 2300 W rrf¹ K, or about 1000 W m^"1 K^'1 to 2000 W m^"1 K. For instance, the anvils may be diamonds, sapphires, or other precious or non-precious gem quality stones. Accordingly, a suitable device of the invention may be configured as a diamond anvil cell.

In certain embodiments, the anvils include at least one precious gemstone, for instance, a flawless diamond or other high gem quality stone, with between about 10 to about 20 facets, about 13 to 17 facets, or about 16 facets. The weight of a gemstone anvil may vary, but is typically about 1/8 to about 2 carats, about 1/4 to about 5/8 carat, or about 1/3 carat. The tip of the gemstone anvil, for instance, a diamond, may be cut, ground and/or polished so as to form a desired surface shape, for instance, a hexadecagonal surface.

The temperature modulator may be of any configuration so long as it is adapted for modulating the temperature of the interior and/or exterior of the chamber. By "modulating the temperature of the interior and/or exterior of the chamber" is meant that the temperature modulator is capable of changing the temperature of the interior or exterior of the chamber from a first temperature to a second temperature. Accordingly, the temperature modulator controls the temperature of the interior of the chamber and is configured for changing the temperature within the chamber along a broad range of temperatures. Generally, the temperature modulator is configured for modulating the temperature of the interior in a range that includes a temperature that is below the freezing point of water to a temperature that is above the freezing point of water. In certain embodiments, the temperature modulator is configured for modulating the temperature within the chamber over a range from about 0 K to about 473 K, about 40 K to about 350 K₁ or about 125 K to about 274 K. Hence, the temperature modulator is adapted for modulating the temperature within the chamber and thereby modulating the temperature of a contained sample. In certain embodiments, the temperature modulator is configured for rapidly heating or cooling the chamber, for instance in a about 1 millisecond to about 250 milliseconds, such as from about 2 milliseconds to about 150 milliseconds, such as from about 3 milliseconds to about 100 milliseconds, such as from about 5 milliseconds to about 50 milliseconds, such as from about 7 milliseconds to about 20 milliseconds, e.g., about 10 milliseconds.

In certain embodiments, the temperature modulator includes a heating element. The heating element may be any means capable of generating and causing the transference of a high temperature (i.e., heat) to the interior of the chamber. For instance, a heating element may include a fluid, such as a gas or liquid that contacts the pressure modulator and/or chamber and thereby warms it. In certain embodiments, the heating element includes a helium gas or water that is heated and contacted with one or more of the opposing surfaces, e.g., anvils, of the pressure modulator. In certain embodiments, the heating element is configured for contacting the pressure modulator with both a heated gas (e.g., a helium gas) and heated water. Accordingly^', in these embodiments, the heating element is configured for heating the exterior components of the apparatus (e.g., the pressure modulator, anvils, sample holding element, gasket, etc.) which in turn transfers heat to the inside of the chamber and thereby warms the sample. In certain embodiments, the heating element may add heat directly to the inside of the chamber, for instance by means of a resistive electrical element located inside the sample chamber. In certain embodiments, the heating element may add heat directly to the anvils, for instance by passing electrical current through anvils that are made of electrically conductive or semiconductive material, or by heating the anvils and/or the sample by means of magnetic inductive heating. In certain embodiments, the heating element may operate by irradiating the sample and/or the anvils with light or microwave energy or other electromagnetic energy which is absorbed by the material of the sample and/or the anvils. In certain embodiments, the heating element may operate by means of adiabatic magnetization of the anvils and/or the sample. Accordingly, in these embodiments, the heating element is configured for heating the interior or interior components of the apparatus (e.g., of the sample, anvils, sample holding element, gasket, etc.) In certain embodiments, the method of heating combines more than one of the methods described above (e.g. resistive heating and irradiation with electromagnetic energy). In certain embodiments, the temperature modulator includes a cooling element. The cooling element may be any means capable of withdrawing heat from the interior of the chamber. For instance, a cooling element may include a fluid, such as a gas or liquid that contacts the pressure modulator and/or chamber. In certain embodiments, the cooling element includes a cryogenic solution, for instance, liquid nitrogen that is contacted with one or more of the opposing surfaces, e.g., anvils, of the pressure modulator. Accordingly, in these embodiments the cooling element is configured for cooling the exterior components of the apparatus (e.g., the pressure modulator, sample holding element, etc.) that in turn cool the inside of the chamber and thereby freeze the sample, for instance, under high pressure. In certain embodiments, the cooling element is configured for cooling the interior of the interior components of the apparatus. For instance, the cooling element may remove heat from the sample or from the anvils by means of adiabatic demagnetization of the anvils, or of the sample, or of the gasket. In certain embodiments, the cooling element may operate by means of laser or optical cooling, as in the manner of the Los Alamos Solid State Optical Refrigerator. In this embodiment, the anvils may be made of glass doped with Ytterbium, or other suitable compounds. In certain embodiments, the method of cooling combines more than one of the methods described above (e.g. adiabatic demagnetization and optical cooling). In certain embodiments, the apparatus may include an imaging element. An imaging element may be any element that is capable of imaging a sample while it is either inside or outside of a chamber of the apparatus. Accordingly, the imaging element may be an element intimately associated with and/or integrated with the pressure chamber of the invention or the imaging element may be a stand alone element stationed within proximity to the pressure chamber. For instance, in certain embodiments, the apparatus includes, or is otherwise adapted to be associated with, a microscopic element, such as an optical microscope. !n certain embodiments, imaging includes forming a two- or three- dimensional image of a portion of a sample (e.g., a spatial image of the sample). In certain embodiments, imaging includes gathering spectral data with or without the forming of a two- or three-dimensional image of a portion of the sample. In certain embodiments, imaging includes merely observing a portion of the sample, with or without forming an image of the sample.

Accordingly, in certain embodiments, the chamber of the apparatus (or the sample containing element) may be positioned on the stage of a microscope, such that the sample inside the chamber or sample containing element is bombarded by photons or electrons or other radiation, and the resulting photons, electrons or other particles of radiation, after contacting the sample or passing through the sample chamber, pass out of the chamber and are collected and analyzed to form images of the sample or other data sets which record and/or describe one or more aspects of the structure and composition of the sample. Information so collected may contain 2- dimensional or 3-dimensional spatial information, spectral (wavelength or frequency) information, compositional (e.g. chemical) information, or any other information which describes the state of the sample.

In certain embodiments, the information describing the sample results from scattering of photons or other particles, or from absorption of photons or other particles, or from changing the polarization state of photons or other particles. In certain embodiments, the information describing the sample results from emission of photons or other particles from within the sample (e.g. from fluorescence or from stimulated emission). In certain embodiments, the information describing the sample results from emission caused by multiple photon absorption (e.g. two-photon microscopy). For instance, where a transparent material (e.g., diamond) is used to fabricate one or more of the opposing surfaces of the chamber (e.g., of a diamond anvil cell), the entire chamber (which may be inclusive of the opposing surfaces) may be mounted on the stage of a light microscope and the sample within the chamber (e.g., within the sample containing element) may be observed and/or imaged. Accordingly, the facets of the chamber (e.g., the diamond surfaces) may act as optical windows through which the sample may be observed and /or imaged.

Alternatively, the sample and/or sample containing element may be taken out of the chamber and placed directly on a light microscope stage for observation and/or imaging and/or manipulation. During any and/or all of these activities, the sample may be kept frozen at low temperatures, e.g., temperatures low enough to ensure no ice crystal formation takes place within the sample (e.g., at or below the glass transition temperature of the sample) and while maintained in a manner so as to a allow a large number of photons to contact the sample. Additionally, the apparatus may be configured to allow observing and/or imaging and/or manipulating the sample while in the thawed state within or outside the chamber.

An apparatus of the invention may have any configuration so long as it includes a chamber for holding a sample that can withstand a high pressure and includes both a means of generating a high pressure within the chamber and a means for rapidly transferring heat to and from the chamber (i.e., for thawing or freezing a sample). Accordingly, an apparatus of the invention can be fabricated from a wide variety of materials, as is known in the art, but should be fabricated out of materials that can withstand both high pressure and rapid changes in extreme temperatures. The general construction and operation of anvil-type high pressure chambers are well known in the art and disclosed in the following publications which are expressly incorporated in their entirety herein by reference.

The following references discuss the design, construction and use of high pressure chambers: Ruoff et al, "The Closing Diamond Anvil Optical Window in Multimegabar Research", J. Appl. Phys., 69 (9), 6413-6415, May 1 , 1991 ; Mao et al, "Optical Transitions in Diamond at Ultrahigh Pressures", Nature , vol. 351 , 721 et seq, Jun. 27, 1991 ; Phil M. Oger, lsabelle Daniel, Aude Picard, Development of a low-pressure diamond anvil cell and analytical tools to monitor microbial activities in situ under controlled P and T, Biochimica et Biophysica Acta v1764 p434-442 (2006); Isaac F. Silvera and Rinke J. Wijngaarden, Diamond anvil cell and cryostat for low-temperature optical studies, Review of Scientific Instruments v56 n1 p121- 124 (January 1985). R. Letoullec, J. P. Pinceaux and P. Loubeyre, The Membrane Diamond Anvil Cell: A New Device for Generating Continuous Pressure and Temperature Variations, High Pressure Research v1 p77-90 (1988); H. Tracy Hall, High Pressure Methods, in High Temperature Technology, p145-156, 335 & 336, McGraw-Hill, New York (1960); High Pressure Microscopic Cell PC400-MS, Teramecs Co., Ltd. Special Device Division, Kyoto, Japan (2006); Elena Mϋller, Detailed Investigations into the Propagation and Termination Kinetics of Bulk Homo- and Copolymerization of (Meth)Acrylates, doctoral dissertation, Mathematics and Science Faculty, Gottingen University (2005); Marcus Nowak, Harald Behrens and Wilhelm Johannes, A new type of high-temperature, high pressure cell for spectroscopic studies of hydrous silicate melts, American Mineralogist v81 p1507- 1512, (1996); K. Pressl, M. Kriechbaum, M. Steinhart and P. Laggner, High pressure cell for small- and wide-angle x-ray scattering, Review of Scientific Instruments v68 n12 p4588-4592 (December 1997); M. Steinhart, M. Kriechbaum, K. Pressl, H. Amenitsch, P. Laggner and S. Bemstorff, High-pressure instrument for small- and wide-angle x-ray scattering. II. Time-resolved experiments, Review of Scientific Instruments v70 n2 p1540-1545 (February 1999); N. Dahan, B. Barrau, G. Pinzutti, J. Moszkowski and G. Martinez, High-pressure design for optical measurements, Journal of Physics E: Scientific Instruments v15 n5 p587-590 (May 1982); Joachim D. Mϋller and Enrico Gratton, High-Pressure Fluorescence Correlation Spectroscopy, Biophysical Journal v85 p2711-2719 (2003). Field, The Properties of Diamond, Academic Press, New York City, N.Y. (1979); Manghnani, et al., High- Pressure Research and Mineral Physics, Terra Scientific Publishing Company, Tokyo, American Geophysical Union, Washington, D.C. (1987); Homan, "Higher Pressure in Science and Technology", Mat. Res. Soc. Symp. Proc, vol. 22, pp 2939, et seq., Elsevier Science Publishing Company (1984); Vodar, et al., High Pressure Science and Technology, Proceedings of the Vllth International AIRTAPT Conference, Le Creusot, France, JuI. 30-Aug. 3, 1979, Pergamon Press, New York, N.Y.; and Ruoff et al, "Synthetic Diamonds Produce Pressure of 125 GPa (1.25 Mbar)", J. Mater. Res., 2 (5), 614-617, September/October 1987. All of which are incorporated by reference in their entirety. In certain embodiments, the devices of the invention are adapted for both rapidly transferring heat to and from a chamber, for instance, a sample holding element under high pressure (i.e., for thawing and freezing a sample) and in a manner sufficient to maintain the structural integrity and/or viability of the sample. Specifically, an apparatus of the invention is configured for both supplying heat to and withdrawing heat from a sample, for instance, a biological sample, that is held within a pressure chamber (e.g., by a sample holding element). In certain embodiments, an apparatus of the invention is configured so as to both cryogenically cool and warm a sample sequentially, one or more times (e.g., repeatedly), in a controlled manner that allows for the precise control of the pressure and temperatures generated, as well as the time period during which those pressures and temperatures are generated.

To better understand an apparatus of the invention, a specific embodiment of a high pressure chamber in operative communication with two opposing anvils, a gasket sample holder and a temperature modulator is set forth herein below. Although the following description is set forth with reference to a particular embodiment of an apparatus of the invention for use in accordance with the methods of the invention, it is to be understood that an apparatus of the invention and its components can have a variety of configurations as will be understood by those of skill in the art. As can be seen with reference to Fig. 1 , in certain embodiments, an apparatus of the invention (100) contains a pressure modulator that includes both a force generating mechanism (e.g., a compression mechanism) (118) and two opposing elements configured as anvils (110a and 110b) (e.g., diamonds). The apparatus (100) further includes a sample holding element (116) (e.g., a gasket) with an interior that forms a chamber (not shown), which is configured for holding a sample. The apparatus (100) additionally includes a temperature modulator (130) configured for modulating the temperature of the chamber from a temperature that is below the freezing point of water to a temperature that is above the freezing point of water, or alternatively from a temperature that is above the freezing point of water to a temperature that is below the freezing point of water. The compression mechanism (118) includes two mount plates (120a and

120b) that are operatively joined by two screws (122a and 122b). The anvils (110a and 110b) include a base (111a and 111 b) and culets or tips (112a and 112b). The anvils (110a and 110b) are associated with the mount plates (120a and 120b) in such a manner that the base (111a and 111b) of each anvil contacts the mount plates (120a and 120b) and the tips (110a and 110b) of the anvils are parallel and face one another. The mount plates hold the anvils in a fixed position. Tips (112a and 112b) contact the two sides and the interior of the sample holding element (116). The juncture of the tips (112a and 112b) and the interior of the sample holding element (116) creates a chamber (not shown) within which a sample, for instance, a biological sample, can be held. Accordingly, within the created chamber an enclosed sample can be thawed and/or frozen in accordance with the methods of the invention, e.g., under pressure.

The pressure modulator is configured for modulating a pressure within the chamber. The compression mechanism (118) includes the mount plates (120a and 120b) which are associated with the bases (111a and 111 b) of anvils (110a and 110b) and is configured for compressing the tips (1 12a and 112b) of the anvils together. The force generated by the compression of the anvils (110a and 110b) together generates a pressure within the chamber. As shown, the two screws (122a and 122b) are configured for being tightened and thereby compressing the mount plates (120a and 120b) together. It is to be noted that although with respect to the illustrated embodiment, the compression mechanism is configured for being compressed by the tightening of one or more screws, this should in no way be construed as limiting the compression mechanism in that the desired compression could also be generated by one or more suitably configured lever arms. The temperature modulator (130) may include one or more of the following: a heating source (132), a cooling source (134), one or more delivery conduits (136) and/or one or more delivery mechanisms (138). The heating source (132) may be a fluid reservoir for containing and heating a fluid, such as a gas (e.g., helium) or liquid (e.g., water). The cooling source (134) may be a fluid reservoir for containing and super-cooling a fluid, such as a cryogenic fluid (e.g., liquid nitrogen). The heating or cooling source may further be connected to an electrical source. The cooling source may be a fluid reservoir for containing and cooling a fluid, such as a cryogenic fluid (e.g., liquid nitrogen).

The delivery conduit (136) is configured for delivering a heated or cooled fluid to the delivery mechanism (138). The delivery conduit may be connected to only the heating source, to only the cooling source, or to both. Accordingly, the delivery conduit may be one or a plurality of tubes, pipes, or the like. The delivery conduit may be fabricated from any material capable of transporting fluids and withstanding extreme temperatures. For instance, the delivery conduit may be fabricated from rubber, plastic, glass, metal or the like. The one or more delivery mechanisms (138) may be a manifold that is configured for receiving the heated or cooled fluid from the one or more delivery conduits and delivering the received fluid to the apparatus of the invention in a manner sufficient to heat or cool the other components of the device, for instance, the anvii(s) (110a and/or 110b) and/or the sample holding element (116). The delivery mechanism (e.g., manifold), as shown, may be configured for contacting one or more of the pressure modulators (e.g., one or more anvils thereof) and the sample holding element (116) with a heating or cooling fluid of the invention and thereby heating or cooling the sample chamber and its contents (e.g. a biological sample). Accordingly, within the sample chamber an enclosed sample (e.g. a biological sample) can be thawed and/or frozen in accordance with the methods of the invention (e.g. rapidly under controlled pressure and temperature conditions) and imaged within the sample chamber or imaged once removed from the sample chamber, as is described in greater detail below.

Although with respect to the illustrated embodiment, the temperature modulator is configured for heating or cooling a sample by contacting a chamber containing the sample and thereby heating or warming the sample, it is to be noted that other configurations for heating and/or cooling the sample may also be provided as is well known in the art and described above.

A feature of the temperature modulator is that it is configured for both heating and cooling the chamber one or more times (e.g., repeatedly). Because of the rapid heat transfer characteristics of the system (e.g., the rapid heat transfer characteristics of the opposing surfaces of the pressure modulator that interact to form the chamber), the temperature modulator is configured for modulating the temperature of the interior of the chamber and thereby heating or cooling a sample contained therein by heating or cooling the exterior of the chamber. For instance, the temperature modulator is configured for modulating the temperature of the interior of the chamber in a temperature range that is below the freezing point of water to a temperature that is above the freezing point of water. Specifically, the temperature modulator is configured for modulating the temperature within the chamber from about 0 K to about 473 K, about 40 K to about 350 K, or about 125 K to about 274 K. The rate of modulation may be from about 0.1 ° C/min to about 5000 ° C/ms, from about 1 ° C/min to about 200 ° C/ms, from about 10 ° C/min to about 100 ° C/ms, from about 5 ° C/min to about 40 ° C/ms, from about 10 ° C/min to about 1 ° C/msec. Accordingly, in certain embodiments, the thawing or freezing of a sample occurs at a rate ranging from about 0.1 ° C/min to about 5000 ° C/millisecond.

In another embodiment, the anvil includes at least one gem stone, for instance, a diamond, and a post, for instance, a metal post. In certain embodiments, the gem stone anvil (e.g., diamond) and the post interact with a sample containing element to produce a sample or pressure chamber. For example, as can be seen in reference to Fig. 2, in one embodiment, a sample chamber may include a disk (e.g., a metal disk, such as copper) (203) for containing a sample. The disk may contain a depression (201) in which the sample is placed. This disk may be placed between a single diamond anvil (202) and a metal post (208) to produce a pressure chamber in the depression (201). The diamond anvil (202) is contacted by a pressure plate (209) through mating surface (210) and is configured to contact the sample and cover the depression (201 ) in the disk (203) thereby enclosing the sample in the depression (201) in the disk. The sample may thereby be sealed inside the depression (201) with a pressure tight seal by applying uniaxial compressive forces to surfaces (207) and (206) of the pressure plate (209) and the post (208).

In accordance with this embodiment, the post may contain a hole (205). This hole may further contain a fluid. For instance, a liquid or a gas (e.g. helium) may be contained within the hole (205) of the post (208). The bottom surface (204) of the disk (203) may be positioned to cover the hole (205) in the post (208). The sample chamber may be sealed against the bottom surface of the diamond anvil (202) by applying a moderate uniaxial compressive force to surfaces (206) and (207), which application of these compressive forces also creates a pressure tight seal between the bottom surface (204) of the disk (203) and the fluid volume of the hole (205). Pressure may be applied to the sample by means of a fluid (e.g. helium gas) in the hole in the post. For example, when pressure is applied via the fluid in the hole (205), the pressure is transmitted to the bottom (204) of the metal disk (203) containing the sample. The pressure may then deform the bottom (204) of the metal disk and pressurize the sample in the sample chamber (201 ). After pressurizing the sample in the sample chamber, the temperature modulating fluid (e.g. helium gas and/or water) may be applied by means of a fluid delivery manifold (211 ) to the outside of the diamond anvil (202), the metal disk (203) and/or the metal post (208). In addition, heat may also be added to the sample in the sample chamber (201) by irradiating the sample chamber with light (212) or other electromagnetic radiation from a source (213), which may pass through the diamond anvil (202) into the sample chamber (201 ).

Another embodiment of an apparatus of the invention is set forth below with respect to Figs. 3 and 4. As can be seen in reference to Fig. 3, in certain embodiments, an apparatus of the invention (300) contains a pressure modulator that includes both a force generating mechanism (e.g., a compression mechanism, not shown) and two opposing elements configured as anvils (303 and 304) (e.g., diamond disks). In this embodiment, each of the diamond anvils has an overall diameter of approximately 6 mm and a thickness of approximately 1.8 mm. In certain embodiments, the diamonds of the diamond anvils are of sufficient clarity and cut that they function as windows, capable of engaging a sample holding element, as well as allowing the transmission of photons through one or more of the various facets of the diamond so as to allow visualization of a sample contained within the sample holding element. As can be seen in reference to Fig. 4, in this embodiment, photons in the bottom cone of light (316) emerge from the photon source (318), enter the sample chamber (301) by passing through facet (305), contact or pass through the sample contained in the chamber (301 ), leave the sample chamber and pass through facet (306), and enter the objective (319) of the observing system via the upper cone of light (317). The apparatus (300) further includes a sample holding element (302) (e.g., a copper gasket). In this embodiment, the sample holding element (302) is a gasket in the shape of a washer approximately 200 μm thick with an internal diameter of approximately 4 mm and an external diameter of approximately 8 mm. The two diamond anvils (303 and 304) engage the sample holding element In a manner sufficient to enclose a sample in the center of the gasket (302) thereby forming a sample chamber (301 ).

The pressure modulator may further include one or more pressure plates. For instance, in certain embodiments the compression mechanism of the pressure modulator may be configured to apply uniaxial perpendicular compressive forces to the outer surfaces (312 and 313) of two circular metal alloy (e.g. tungsten carbide) pressure plates (308 and 309) such that the applied forces are transmitted to the anvils (303 and 304) via mating surfaces (314 and 315). The applied forces push the anvils together, thereby compressing the sample and the gasket (302) surrounding the sample, and thereby modulating the pressure inside the sample chamber (301). The pressure plates hold the anvils in position relative to the gasket (302), with the anvil bases contacting the sample parallel and facing one another.

The bottom pressure plate (309) may contain a spherical bearing (310) with a spherical bearing surface (311 ) that allows the bottom diamond anvil (304) to automatically position itself parallel to the top diamond anvil (303) as forces are applied to surfaces (312 and 313) of the pressure plates. A thin layer of soft metal (e.g. lead foil) may be inserted at the mating surfaces (314 and 115) between the pressure plates (308 and 309) and the anvils (303 and 304), to ensure that the forces applied to the anvils by the pressure plates are applied evenly.

The apparatus (300) additionally includes a temperature modulator (not shown) and a delivery conduit that is configured for delivering a heated or cooled fluid to the delivery manifold (307), as described above.

Fig. 5 is a cutaway diagram showing a representative sample cell of Fig. 3. The sample chamber (501 ) is enclosed between the diamond windows (503 and 504), and surrounded on the edge by gasket (502). Mating surfaces (505 and 506) transmit force to the diamond windows, and light may be passed through diamond window surfaces (507 and 508) to allow illumination and observation of the sample chamber.

Fig. 6 is a diagram showing a representative delivery manifold (307 of Fig. 3) located between the pressure plates and surrounding the sample cell. Referring to Fig. 3, the manifold (307) may be of any shape or size, for instance, square, hexagonal, circular or the like, but the thickness of the manifold is such that, when compressive force is applied to surfaces (312 and 313) to compress the anvils (303 and 304), the pressure plates (308 and 309) do not interfere with or contact the manifold (307). In the present embodiment the manifold is circular. The fluid manifold (307) may be made of any suitable material (e.g. metal or glass) through which temperature modulating fluids may be passed, to modulate the temperature of the sample cell. The manifold may contain a number of passages (e.g., 1 , 2, 3, 4, 5, 10, 15, 20 or more).

Referring to Fig. 6, in this embodiment, the manifold contains six tubular passages (606) for the application of a temperature modulating fluid, and six tubular passages (607) for removal of a temperature modulating fluid from the central space containing the sample cell. In this embodiment, fluid delivery manifold also contains six each tubular passages (604 and 605) for passing temperature modulating fluids through the manifold without contacting these fluids to the sample cell, e.g., for the purposes of precooling or prewarming of the fluid manifold itself. The fluid manifold may also be operatively connected to a reservoir for containing the temperature modulating fluid. Temperature sensing devices (e.g. thermocouples, not shown) may be mounted at appropriate places in the manifold, to monitor the temperature near the sample cell. In the center of the fluid manifold is the gasket (602) and the sample chamber volume (603).

Additionally, the assembly of any of Figs.1 , 2 or 3 may be mounted, or be configured to be mounted, on the stage of an optical system (e.g. a microscope) such that the optical system may illuminate and/or observe and/or image the sample by passing light or other radiation through one or more of the diamond windows. Portions of the optical system (e.g. a microscope) may also be used to illuminate the sample with laser beams or other radiation sources, to allow optical or other manipulation of the sample while the sample is inside the sample chamber.

In operation, a sample (e.g. a biological sample) is placed in the sample chamber and the upper diamond window, mounted in its pressure plate, is placed over the sample so as to seal the sample inside the sample chamber. Pressure is then applied to the sample by applying force to the pressure plates, compressing the sample and the gasket between the diamond windows. Before and/or while pressure is being applied, the fluid delivery manifold may be precooled or prewarmed by passing a cryogenic fluid (e.g. liquid nitrogen) or a warming fluid (e.g., heated water or helium gas) through the manifold precooling/prewarming passages. Once the pressure reaches a desired value, and the manifold has been precooled or prewarmed, a cryogenic fluid or a warming fluid may be applied to the sample cell by passing such fluid through the application and removal passages of the manifold. Contact of the cryogenic or warming fluid to the sample cell rapidly cools or warms the cell and freezes (e.g. vitrifies) or thaws the sample contained within it. In the frozen (e.g. vitrified) or thawed state, the sample may be observed and/or imaged and/or manipulated, while in the chamber, using an optical system (e.g. a light or optical microscope) or by other optical or microscopic means (e.g., via infra-red or x- ray or electron microscopy), for arbitrarily long periods of time, for instance, to a allow a large number of photons, electrons, or the like, to contact the sample.

Thus, in certain embodiments, the methods of the invention allow for the detailed ultra-structural observation and imaging of a viable sample (e.g., via optical microscopic means, such as structured light microscopy) so as to generate a super resolution (e.g. better than Rayleigh criterion) image of the sample while preserving the viability and/or structural integrity of the sample. In certain embodiments, this may be achieved by increasing the time period over which the light (e.g., photons) is contacted with the sample and thereby increasing the number of photons which contact the sample and thereby increasing the maximum attainable overall signal-to- noise ratio and/or spatial resolution and/or spectral resolution of the data, without increasing the photon intensity or irradiance.

In certain other embodiments, an apparatus of the invention is characterized in that it is configured for both freezing and/or thawing a biological sample inside a chamber of a high pressure modulator in a manner sufficient to maintain the structural integrity and/or viability of the biological sample, and for removing the sample from the high pressure modulator for imaging and/or manipulating the sample, while the sample is outside of the high pressure modulator, and then returning the sample to the high pressure modulator for refreezing or rethawing after observing and/or imaging and/or manipulating the sample.

For instance, an apparatus of the invention may include a transfer element, such as a robotic arm which can transfer the sample while frozen or thawed and still contained within the chamber or sample containing element (e.g., an annular enclosing gasket) from the location of the high pressure modulator or chamber, to the viewing stage of an optical microscope. In this embodiment, the microscope viewing stage may be kept at cryogenic or warming temperatures so that the sample remains frozen (e.g., at 77 K) or thawed (e.g., at ambient temperatures) while it is observed by means of the microscope, and the portion of the robot arm which contacts the sample may also be kept at cryogenic or warmed (e.g. ambient) temperatures so that the sample remains frozen or thawed during the transfer. In certain embodiments, the sample may be returned to the chamber after viewing and/or manipulation (if desired) by the same or a different transfer element (e.g., a different robotic arm mechanism).

In certain embodiments, the sample and the opposing surfaces of the pressure modulator (e.g., the diamond anvils) may be separated with the aid of chemical parting substances (e.g. lecithin or 1-hexadecene) coating the surfaces of the diamond anvils which contact the sample, as is well known in the art. In certain embodiments, the sample chamber may be opened by lowering the bottom pressure plate and enclosing gasket, which causes the bottom anvil and the enclosing gasket, containing the sample, to part contact with the upper anvil. When the bottom portion of the apparatus containing the bottom anvil and the sample and gasket have cleared the top portion, a robotic arm may be engaged to contact and grasp the gasket and the sample contained within it and then move the sample onto the cryogenic microscope stage for viewing or manipulation. In certain embodiments, the bottom anvil is also carried to the cryogenic microscope stage along with the gasket and sample. In certain embodiments, the sample and gasket and lower anvil remain stationary, and the microscope objective is moved into place over the sample, after the top portion of the apparatus is removed.

In another embodiment, the apparatus is configured so that the sample may be removed from the enclosing gasket for viewing. In this configuration, the inner surface of the gasket may be shaped in the form of a truncated cone, so that the enclosed sample may be more easily removed from the gasket by lifting the sample in the direction of the big end of the cone, while the gasket is lifted in the opposite direction. In this embodiment, the inner surface of the gasket may be coated with chemical parting substances as mentioned above. In embodiments in which it is desired to return the sample to the pressure chamber after observation or modification, the chamber may be filled with a cryogenic liquid (e.g. liquid nitrogen) or a warming fluid before it is closed, to fill up any spaces in the chamber not occupied by the sample, so that hydrostatic pressure may be reestablished after the chamber is closed. In certain embodiments, filling the chamber this way may be accomplished by closing the chamber under cryogenic liquid or warming fluid.

After observation, imaging and/or manipulation are completed, the sample, in the sample cell, may be rewarmed or re-frozen by applying pressure (if not already pressurized) to the chamber and, while pressurized, applying rewarming or cooling fluids, in appropriate sequence and timing, to the sample cell. These rewarming or cooling fluids may be applied to the sample cell by passing them through the application and removal passages in the prewarmed/precooled fluid manifolds.

In one embodiment, the tissue to be observed and/or imaged and/or analyzed and/or manipulated is from an organ (e.g., a brain) and the tissue of interest (e.g., neural tissue) is excised from that organ in a manner sufficient to preserve the viability of the sample. Accordingly, the organ (e.g., brain) from which the tissue (e.g., neural tissue) is to be harvested may first be put into a state of cold but not frozen suspended animation (e.g., at a temperature between 273 K and 283 K) and then carefully sliced in a manner to reduce damage to the tissue sections collected, as is well known in the art. The sliced sections may be from about 10 μm to about 300,000 μm, such as from about 20 μm to about 1000 μm, e.g., from about 200 μm to about 400 μm thick. The tissue (e.g., neuronal cells) collected, along with hydrostatic fluid (e.g. Hextend or Ringer's lactate solution) if desired, may then be placed into a chamber of a device of the invention. For instance, a chamber formed between the two opposing surfaces of the pressure modulator or a chamber formed from the interior of a suitable sample holder (e.g., a gasket which is then placed between the two opposing surfaces of the pressure modulator).

The sample is placed in between the two opposing surfaces of the pressure modulator, (e.g., within a sample holding element of the chamber) the opposing surfaces are aligned and manipulated so as to generate a pressure within the chamber, for instance, a high pressure. The high pressure may be generated by manipulating the force generating mechanism (e.g., suitably configured lever arm(s) or screw(s) or hydraulic system) of the pressure modulator in a manner sufficient to cause the tips of the two opposing surfaces to move toward, contact, and be compressed against one another and/or the sample and/or gasket, which thereby generates a high pressure within the chamber.

A high pressure is generated within the chamber and the temperature modulator is then be engaged to apply a cooling fluid to the exterior of the chamber (e.g., to the opposing surfaces of the pressure modulator and/or the exterior of the sample holder) in a manner sufficient to cause the freezing of the sample with minimal to no ice crystal formation within the sample (e.g., both within and between the cells of the sample). The freezing of the sample may take place rapidly, as described above, and in a manner such that the sub-cellular structures and their positioning remains substantially unaffected (e.g., by ice crystal formation) and the cell to cell alignment within the tissue remains substantially intact. In certain embodiments, the freezing of a biological sample takes place in a manner such that the chemical, biochemical and molecular processes within the biological sample cease.

After the sample has been frozen in a manner sufficient to fix (e.g. immobilize) the sample without compromising the structural integrity of the majority of the components of the sample and/or with preserving its viability, the sample may then be manipulated and/or imaged (e.g., observed and/or analyzed) in any of a number of ways over a short or long period of time, for instance, while the sample remains frozen during the manipulating and/or imaging and/or analyzing.

Accordingly, in the cryogenically fixed state, the observation, imaging and/or analysis of the sample may be performed via optical microscopy over a prolonged period of time in a manner sufficient to allow a large number of photons to be contacted with and/or passed through the sample and thereby to produce one or more high signal-to-noise ratio, high resolution images or data sets of the sample. In the cryogenically fixed state, photons may be made to contact or pass through the sample over arbitrarily long periods of time, ranging from one minute to three minutes, or from 10 seconds to 30 minutes, or from 1 second to 24 hours, or from 100 milliseconds to 30 days, or from 10 milliseconds to 1 year, or for any arbitrarily long period of time. For instance, the image acquisition process herein described may be used to obtain detailed structural information about the sample, the cells of the sample or the various components within the cells, such as the location, orientation and composition of sub-cellular structures of the cells of the sample as well as the cell to cell structure of the overall tissue.

The sample may be analyzed in any of a number of ways. In one embodiment, once frozen the sample may be observed and otherwise analyzed via a sample observation element, for instance, a microscope, such as a light microscope (e.g., for optical microscopy) or other microscopic means (e.g., via infra- red or x-ray microscopy). For instance, where a transparent material (e.g., a diamond) is used to fabricate one or more of the opposing surfaces of the pressure modulator (e.g., which may be configured as a diamond anvil cell), the entire chamber (which may be inclusive of the opposing surfaces) may be mounted on the stage of a light microscope and the sample within the chamber (e.g., within the diamond anvil cell) may be observed. Wherein the facets of the chamber (e.g., the diamond) surface act as an optical window through which the sample may be observed. Alternatively, the sample may be taken out of the chamber and placed directly on a light microscope stage for observation. Accordingly, the methods of the invention allow for the acquisition of detailed, high resolution images (e.g., optical microscopic images) of the collected sample (e.g., a cellular, tissue or multicellular organism sample). Because the sample is frozen (e.g., cryogenically fixed) without the production of ice crystal artifacts, ultra- structural details of the sample, the cells within the sample and/or the components within the cell(s) may be observed, imaged and otherwise analyzed with little to no interference due to artifacts. The observation, imaging and/or analysis may be performed while the sample remains frozen (e.g., while the chemical, biochemical and molecular processes of the cell are ceased) or while the sample is thawed.

In this manner, the observation, imaging and/or analyzing may be performed via optical microscopy over a prolonged period of time in a manner sufficient to allow a large number of photons to be contacted with and/or passed through the sample and thereby to produce high resolution data of the sample. For instance, the image acquisition process herein described may be used to obtain detailed information about the sample, the cells of the sample or the various components within the cells, such as the location, orientation and sub-cellular structures of the cells of the sample as well as the cell to cell structure of the overall tissue.

Contacting a large number of photons with the sample over a prolonged period of time both reduces photon noise and reduces overall light (e.g., photon) intensity or irrradiance, which is an important factor when visualizing (e.g., imaging) a sample in such a manner so as to maintain the viability of the sample. If the intensity or irradiance of the light is too great, structural and cellular perturbation increases and the viability of the sample may be compromised. Thus, the methods of the invention allow for the ultra-structural observation of a viable sample (e.g., via optical microscopic means, such as structured light microscopy) so as to generate a super resolution image of the sample while preserving the viability of the sample. This is achieved by increasing the time period over which the light {e.g., photons) are contacted with the sample and thereby increasing overall resolution without increasing intensity or irradiance.

Where multiple samples are collected from a single organ or organism, multiple images of the samples may be collected (e.g., via a suitable detector), stored and analyzed, for instance, via a computer means. Such multiple images may be used to gain detailed knowledge of the organ or organism from which the samples were collected. For instance, a complete detailed image (e.g., a three dimensional digital image) of an organ (e.g., a brain) and its structure(s) may be obtained, stored, examined, reproduced and otherwise analyzed to give detailed information of the organ, how it works, and how the individual cells (e.g., neurons) interact or associate with one another within the organ. For example, the data obtained can be used to generate a computer simulation of the structure and/or function of the organ (e.g. a brain) or its individual cells (e.g. neurons and other brain cells). The sample may be manipulated or perturbed in a number of ways while in the frozen state, including: physical, chemical, electrical, optical, molecular, and nanotechnological perturbation. While in the frozen state, cells may be added to or removed from the sample, or subcellular components may be added to or removed from the cells of the sample. The sample may be manipulated and/or imaged either while still in the sample chamber or after having been removed from the sample chamber, as described above.

A frozen and/or manipulated and/or imaged and/or analyzed sample may be thawed in a manner sufficient to maintain the structural integrity and/or viability of the sample. Accordingly, to thaw a frozen sample, the sample is placed within a chamber of the apparatus (if not already therein). For instance, the sample may be placed within a sample holder (e.g., a gasket, washer or the like) and the sample holder may then be placed between the opposing surfaces of the pressure modulator. The opposing surfaces of the pressure modulator (e.g., anvils) are then aligned and brought together so as to enclose the chamber. Once the chamber is enclosed between the two opposing surfaces the pressure modulator is manipulated so as to generate a pressure within the chamber, for instance, a high pressure. The high pressure may be generated by manipulating the force generating mechanism (e.g., a suitably configured lever arm(s) or screw(s)) of the pressure modulator in a manner sufficient to cause the two opposing surfaces to move toward one another thereby generating a high pressure within the chamber. After a high pressure is generated within the chamber, the temperature modulator may then be engaged to apply a heating fluid to the exterior of the chamber (e.g., to the opposing surfaces of the pressure modulator and/or the exterior of the sample holder) in a manner sufficient to cause the thawing of the sample with minimal to no ice crystal formation within the sample (e.g., both within and between the cells of the sample). The thawing of the sample may take place rapidly, as described above, and in a manner that the sub-cellular structures and their positioning remains relatively unaffected (e e.g., due to the melting of fluid components and/or recrystallization of ice within and between the cells of the sample) and the cell to cell alignment within the tissue remains intact. Once thawed the cells of the sample maintain their viability and continue their typical cellular processes.

After the sample has been thawed in a manner sufficient to maintain the structural integrity of the majority of the components of the sample and/or to preserve its viability, the sample may then be analyzed via optical microscopy as described above, or otherwise manipulated. For instance, where the sample is a viable biological sample, once thawed, the viable sample or one or more of the viable cells of the sample may be manipulated or perturbed in a number of ways well known in the art, including: physical, chemical, electrical, optical, molecular, and nanotechnological perturbation. Individual or multiple cells may be added or removed from the sample. Subcellular components of cells may be modified, or added to or removed from the cells of the sample. After manipulation the sample may then be frozen and observed in the manner described above, i.e., in a manner sufficient to maintain the structural integrity and the viability of the sample, and observed and/or imaged and/or manipulated while in the frozen state. In this way, one or more biological (e.g., cellular) process may be observed and/or imaged in a living tissue or viable cell over time and over one or more (e.g., several) cycles of freezing and thawing, wherein the tissues or cell is manipulated in some manner, frozen, observed, thawed, re- manipulated in some manner, re-frozen, observed, etc.

Accordingly, because when the sample is in a frozen state, the cellular structures are immobilized and cellular processes of the viable cells of the sample are arrested, the effects of a previously applied perturbation can be observed and imaged in great detail and with high spatial and/or spectral resolution, because such observation or imaging may be accomplished over arbitrarily long periods of time, allowing high signal-to-noise ratios to be attained in the data sets, as described above, as described above. In one embodiment, the manipulated sample is frozen to produce a frozen viable sample following the manipulation, which may then be observed and re-thawed under pressure and in a manner sufficient to maintain the structural integrity of the sample.

Although, the above has been described with respect to determining the functioning of an organ this should not be construed as limiting the scope of the invention in any way as modifications to the above description may be made without diverging from the invention. For instance, the above methods may be used to characterize the contents and interactions between the various components of a cell or other sub-cellular structure (e.g., nucleus, chromosomes, etc.), or between the components of a portion of an organ (e.g. a brain or other neural tissue).

Hence, using the methods disclosed herein, one is capable of observing a tissue or a eel), manipulating the tissue or cell, freezing the tissue or cell so as to cryogenically fix (e.g., arrest) the tissue or cell and observing the cellular changes that have taken place after the manipulation and before the cryofixation (e.g., via light microscopy, as described above) and then thawing the tissue and cell while maintaining is structural integrity and vitality. This process may be performed once or performed repeatedly over several cycles of freezing and thawing. Using the methods disclosed herein, one is capable of manipulating a tissue or cell in the frozen state, and observing the changes that have taken place after the manipulation while still in the frozen state, and after subsequent thawing. The methods described herein may be used to observe the time evolution of one or more cells or cellular processes. Accordingly, individual cellular structures and identifiable chemical components (e.g., components labeled with an observable dye that does not compromise the viability of the cell) can be observed using super- resolution imaging techniques (e.g. structured light microscopy) and followed over time and over several cycles of manipulation (e.g., perturbing), freezing, manipulation and/or observation, and thawing. It is to be noted that although the observation methods disclosed herein have been described with respect to observing a frozen sample, the thawed sample may also be observed as part of the experimental process. Repeated high resolution imaging of the same cellular structures, combined with the ability to perturb those structures as desired, may allow greater understanding of the relationship of structure to function in biological samples and/or systems.

Additionally, the methods described herein are also useful for storing a viable biological sample that may or may not be observed but maintains its structural integrity and viability so as to be thawed at a later time. By use of the methods disclosed herein, a biological sample may be frozen and stored and later thawed while maintaining its structural integrity and/or viability. The methods of the invention are useful for reversibly and repeatedly cryofixing a viable biological sample under high pressure, and once thawed or frozen, the biological sample may be imaged, for instance, by optical microscopy (including: light, infrared and/or x-ray microscopy). In this manner, a biological sample may be repeatedly frozen, thawed, imaged and/or stored over a prolonged period of time while maintaining the structural integrity and viability of the biological sample.

For instance, the methods herein described are useful in maintaining and storing living cells contained on silicon chips. Mammalian cells, such as nervous system cells (e.g., neurons) may be associated with a silicon microchip so as to form a biological circuit. For example, a combination biological and electronic circuit may be formed, as is well known in the art. In certain embodiments, one or more cells (e.g., a cell, a neuron, a gamete cell, stem cell, or the like) may be associated with a substrate, for instance, a glass, silicon or electronic chip so as to form a biological circuit. For instance, in certain embodiments, the one or more cells may be arranged in a circuit configuration and may interact with other circuitry components to form a biological circuit.

The one or more cells may be arranged in a circuit configuration and may interact with other circuitry components to form a circuit that is capable of transferring a current or other signal from one point on the chip to another. In another example, the one or more cells may be arranged in a way to allow the cells to be stimulated and/or observed by optical, electronic or other means. In one embodiment, the microchip substrate is configured for being contained within a chamber of the high pressure apparatus and is capable of being moved into and out of the chamber and/or for being associated with a stage of an imaging device for imaging the components (e.g., the biological components) of the microchip (e.g., the associated biological cells).

The association of a biological cell with a substrate so as to form a microchip that contains biological components is well known in the art and disclosed in following references, which are hereby incorporated by reference in their entirety for their teaching on the production and use of biochips. The Neurally Controlled Animat: Biological Brains Acting with Simulated Bodies. Thomas B. DeMarse, Daniel A. Wagenaar, Axel W. Blau and Steve M. Potter Autonomous Robots v.11 n.3 p.305-310 (November 2001 ). Noninvasive neuroelectronic interfacing with synaptically connected snail neurons immobilized on a semiconductor chip Gϋnther Zeck and Peter Fromherz PNAS | August 28, 2001 , vol. 98 no. 18 p. 10457-10462. Engineering a biospecific communication pathway between cells and electrodes Joel H. Collier and Milan Mrksich PNAS | February 14, 2006, vol. 103 no. 7 p. 2021-2025. Closing the Loop: Stimulation Feedback Systems for Embodied MEA Cultures S. M. Potter, D.A. Wagenaarand T.B. DeMarse. In: Advances in Network Electrophysiology Using Multi-Electrode Arrays, M.Taketani and M. Baudry (Eds.), Springer (2005). For instance, cortical neurons from a suitable organism may be dissociated and cultured on a surface containing a grid of electrodes (multi-electrode arrays, or MEAs) capable of both recording and stimulating neural activity.

Such microchips containing biological circuits may be useful in studying the behavior of neurons, analyzing the information processing functions of particular neurons or samples of neural tissue, as detectors for environmental pathogens or toxins, drug screening systems, as chemical sensors (artificial noses), for development of medical devices, such as neural prostheses, for the generation of organic computers using living neurons, and for other applications. Accordingly, the methods of the invention are useful for imaging, analyzing and/or manipulating the neuron containing biochips once they have been fabricated, or during their fabrication as part of the fabrication process. Additionally, the methods herein disclosed are useful for cryostoring neurons before their employment in such a biochip and/or for cryostoring the neuron containing biochips once they have been fabricated. Hence, the methods herein disclosed are useful in both studying the effects of and implementing cryobiological storage of biochips containing neurons or other cells.

In another aspect, the present invention is directed to a computer program that may be utilized to carry out the above steps. The device of the invention may include mechanisms to open and close the sample chamber, place the sample into and remove the sample from the chamber, control the application of forces applied to the pressure plates, monitor and control the application of the cooling and warming fluids, and operate various devices (e.g., the robotic arm and/or imaging apparatus) to manipulate and/or observe the sample either inside or outside the chamber. One or more of the steps including: the placement of a sample into a chamber, the alignment of the opposing surfaces of the pressure modulator, the enclosing of the chamber, the generation of a force, the modulation of the pressure within the chamber, the modulation of the temperature of the chamber, the placement of the sample and/or chamber components on an observation stage, and/or the observing (e.g., imaging and storing) of the sample, in accordance with the invention, may all be done automatically under computer control, that is, with the aid of a computer. The computer may be driven by software specific to the methods described herein. Examples of software or computer programs used in assisting and conducting the present methods may be written in any convent language, e.g. Visual BASIC, FORTRAN and C++ (PASCAL, PERL or assembly language). It should be understood that the above computer information and the software used herein are by way of example and not limitation.

Programming according to the present invention, i.e., programming that allows one to carry out the methods of the invention, as described above, can be recorded on computer readable media, e.g., any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM and DVD; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. In certain embodiments, a processor of the subject invention may be in operable linkage, i.e., part of or networked to, the aforementioned apparatus, and capable of directing its activities. A processor may be pre-programmed, e.g., provided to a user already programmed for performing certain functions, or may be programmed by a user. Thus, in certain embodiments, the programming is further characterized in that it provides a user interface, where the user interface presents to a user the option of selecting among one or more different, including multiple different, rules for individually controlling the steps of the methods herein disclosed. A processor may be remotely programmed by "communicating" programming information to the processor, i.e., transmitting the data representing that information as electrical signals over a suitable communication channel (for example, a private or public network). Any convenient telecommunications means may be employed for transmitting the programming, e.g., facsimile, modem, Internet, LAN, WAN or other network means, wireless communication, etc. EXAMPLES ^•

Modeling in Software the Cooling and Warming of a Sample

High pressure freezing is used by electron microscopists freeze biological samples with the formation of few or no ice crystals, and/or ice crystals of greatly reduced size, thus preserving the fine structure of those samples for later imaging. Success in freezing without ice crystal formation, for aqueous samples thicker than about 10 to 20 microns, may depend on the timed application of sufficiently high pressure and sufficiently rapid cooling to the biological sample being frozen. Use of chemical cryoprotectants may relax these conditions to some degree, but in certain embodiments, little or no cryop rote eta nt need be used, for instance, in circumstances where the use of cryoprotectant chemicals may be toxic to viable biological samples. Thus, the present methods and devices may be configured for reversibly thawing and freezing viable biological samples, without sacrificing viability and without the use of cryoprotectant chemicals. In certain embodiments, a device of the invention may be configured to thaw a biological sample under high pressure with the same rapidity that the sample was frozen.

The heat transfer characteristics of a device of this patent were modeled, using COMSOL Multiphysics version 3.2b software. The materials of the device were alloy steel, copper, water, and diamond. Images of the temperature distribution at all relevant points in the device, for various times after commencement of warming or cooling, were generated by the software.

FIGS. 7 through 9 show the predicted temperature distribution in the sample cell of a device of the invention during cooling. The figures are side view cross sections, and the sample cell has axially symmetric geometry. The sample cell also has bilaterally symmetric geometry, about the plane perpendicular to the device axis passing through the center of the sample cell. Accordingly, FIGS. 7 through 9 show only one quarter (the upper right quadrant) of the complete cross section. Therefore in the figures the leftmost edge is the centerline axis of the device, and the bottom edge cuts through the center of the sample and the copper gasket that holds the sample. The diamond anvil cooling plate is 1.2 mm thick by 6 mm in diameter, and is represented in the figures by a rectangle 1.2 mm high by 3 mm wide. The alloy steel post holding the diamond anvil cooling plate is represented by the rectangular region immediately above the diamond anvil cooling plate. Initial temperature conditions for the model at time t = 0 are 275 K throughout the volume of the device, and 77 K at the outer surface of the device. The temperature scale is shown by the vertical grey scale bar at the right, with light grey representing 75 K and dark grey representing 275 K. Times shown are 1 millisecond, 10 milliseconds, and 100 milliseconds.

Figs. 10 through 12 show the predicted temperature distribution in the sample cell of a device of the invention during warming. Device geometry and time intervals are the same as in Figs. 7 through 9. At time t = 0, the entire volume of the device is at 77 K, with the outside surface of the device warmed to 305 K.

Also generated were predicted temperature versus time curves for the point most central in the sample, which is the point in the sample most slowly cooled or warmed. Fig. 13 shows the predicted temperature in the center of the sample, for the case of cooling down in a device of this application. Fig. 14 shows the predicted temperature in the center of the sample, for the case of warming up in a device of this patent. Both figures show temperature versus time for times t = 0 to t = 3 seconds.

Comparing these images and the cooling and warming curves shows that the cooling rates achieved by the device of this patent are comparable to those obtained by electron microscopists in preparing their samples. Furthermore, the time to warm (e.g. thaw) the sample, using the device of this patent, is substantially equivalent to the cooling times of the device of this patent. This is what we want to achieve in this particular embodiment of a device of this patent. The images and curves presented indicate the temperature versus time for points in the sample during cooling and warming, for instance, in the center of the sample. Points near the center of the sample will coo! and warm most slowly, compared to points near the edges of the sample. The methods and devices of the invention are configured to rapidly cool and/or warm the entire sample with little or no ice crystal formation and while maintaining the structural integrity and/or viability of the sample. Experimental Evaluation of the Survival of HP Frozen Rat Brain Tissue

The survival of rat brain tissue when exposed to low temperatures and high pressures was evaluated. In one procedure, a young female Sprague-Dawley rat, 273 grams, was anesthetized with 0.35ml KAX (a mixture of ketamine, xylazine and acepromazine) intraperitoneally, weighed and cannulated in the femoral artery. The femoral vessel was opened and the animal was perfused with 45ml of ice cold bicarbonated Hextend, until the hematocrit was reduced to less than one. The brain was then removed and sectioned into quarters and the four quarters treated as follows: 1 ) control, held for 15 minutes at 2⁰C; 2) pressurized to 2177atm for 5 minutes while at 2°C, then depressurized; 3) frozen to -196⁰C at ambient {1 atm) pressure for 5 minutes and then thawed; and 4) pressurized to 2177atm (221 MPa), then frozen to -196°C over 5 minutes, held at this temperature and pressure for 5 minutes, then thawed to 2°C over 15 minutes, then depressurized. Brain tissue in each case was kept in ice-cold bicarbonated Hextend saturated with BioBlend®¹³, a gas mixture consisting of 95% oxygen and 5% carbon dioxide.

After these steps, the brain tissue was cut into 200//m thick coronal slices using a VIBRATOME®. While being sliced, the tissue was held at 2° C while it was bathed in artificial cerebrospinal fluid (ACSF) bubbled with BioBlend. After slicing, the tissue samples were allowed to rest for about 45 minutes at room temperature, while bathed in ACSF bubbled with BioBlend. Slices were then stained by immersion for 30 minutes in room temperature BioBlend-bubbled Hextend to which the two-component Molecular Probes L-3224 Live/Dead stain was added. The L- 3224 Live/Dead stain stains the cytoplasm of living cells grey and the nuclei of dead cells black. Concentration of the calcein-AM component was 4.5 μM and concentration of the ethidium homodimer component was 6.7 //M.

Immediately after staining, the tissue was imaged using a Zeiss Meta-510 Confocal Laser Scanning Microscope (LSM). The microscope was fitted with a 4OX water immersion objective and the illumination source was a 488 nm argon-ion laser. Three-dimensional stacks 236μm high consisting of 50 2-D images measuring 650μm x 650μm were acquired. The resulting 3-D image data sets were imported into the Zeiss LSM image browser program and individual 2-D slices were selected as representative images of the 3-D data sets. This live/dead viability assay showed that, when compared to the control, the pressure only tissue showed comparable viability, and the pressure frozen tissue showed much less viability. Figs. 15 through 17 illustrate the results of the different treatment of the sections. Fig. 15 illustrates the treatment of the control sample. As can be seen with reference to Fig. 15, the sample shows very few black spots interspersed on a strongly grey colored cytoplasm matrix. Ropey structures which are the intact capillaries of the vascular bed can be seen threading through the image. As can be seen with reference to Fig. 16, the tissue sample treated with pressure only shows less intense grey color, but still very few black spots. The capillaries are clearly visible. Accordingly, the application of 2177 atm pressure to ice cold brain tissue for 5 minutes does not compromise tissue viability, when compared to control tissue samples (see Fig. 15). Images of brain tissue frozen to -196° C at ambient pressure were not obtained because samples frozen without the application of high pressure were so delicate and friable that it was impossible to slice them on the Vibratome without causing the tissue to fall apart.

As can be seen with reference to Fig. 17, the pressure frozen and thawed sample shows much less grey cytoplasm background, many more black spots and no visible capillary structures.

In another procedure, the survival of rat brain tissue when thawed after rapid high pressure freezing was evaluated. In this procedure a young female Sprague- Dawley rat of 243 g was anesthetized, cannulated and perfused with ice cold bicarbonated Hextend until the hematocrit was reduced to less than one. The brain was then removed and sliced into 200/vm thick coronal slices. The slices were then soaked in bicarbonated Hextend to which glycerol had been added to a concentration of 20% by volume. Slices were then quickly pressure frozen at 2100 atm in a Bal-Tec HPM-010 high pressure freezing machine and stored in liquid nitrogen for several days. Cooling from room temperature to -196° C took about 10 milliseconds in the Bal-Tec machine. After storage in liquid nitrogen for several days, slices were rewarmed at 1 atm pressure by immersing them for about 45 minutes in Hextend at 20° C bubbled with BioBlend. Slices were then immediately stained with L-3224 Live/Dead stain and imaged with the Zeiss Meta 510 Confoca! Laser Scanning Microscope (LSM) fitted with a 2OX air objective. Illumination was with the 488 nm argon-ion laser. Three dimensional stacks 42μm high consisting of 20 2-D images measuring 460μm x 460μm (512 x 512 pixels) were acquired. The resulting 3-D image data sets were imported into the Zeiss LSM image browser program and individual 2-D slices were selected as representative images of the 3-D data sets. See FIGS. 18 and 19. As seen in comparisons between Fig. 15 (control) and Figs. 18 and 19, brain tissue slices prepared, pressurized, frozen and thawed in this way showed good viability although structure was altered due to the formation of ice crystals while thawing. Structures resembling the capillaries are visible and viability of the tissue is comparable to that of control samples. In a further procedure, the survival of rat brain tissue when thawed under high pressure is evaluated. In this procedure a young female Sprague-Dawley rat of 243 g is anesthetized, cannulated and perfused with ice cold bicarbonated Hextend® until the hematocrit is reduced to less than one, as described above. The brain is then removed and sliced into 200//m thick coronal slices. The slices are then soaked in bicarbonated Hextend to which glycerol is added to a concentration of 20% by volume. Slices are then quickly pressure frozen at 2100 atm in a high pressure freezing and thawing apparatus of the invention and stored in liquid ^' nitrogen for several days.

After storage the slices are quickly pressure thawed at 2100 atm and the temperature of the sample is changed from -196° C to room temperature within about 100 msec in an apparatus of the invention. The slices are then immediately stained with L-3224 Live/Dead stain and imaged with the Zeiss Meta 510 Confocal Laser Scanning Microscope (LSM) fitted with a 2OX air objective. Illumination is with the 488 nm argon-ion laser. Three dimensional stacks 42//m high consisting of 20 2- D images measuring 460/ym x 460μm (512 x 512 pixels) are acquired. The resulting 3-D image data sets are imported into the Zeiss LSM image browser program and individual 2-D slices are selected as representative images of the 3-D data sets. Image data sets show that the tissue is intact and strong enough to be easily sliced into 200μm thick slices. Structures resembling the capillaries are visible and viability of the tissue is comparable to that of control samples. It is evident from the above discussion that the subject invention provides an important breakthrough in the preparing biological samples without destroying the structural integrity and/or viability of the sample. Specifically, the subject invention allows one to thaw and freeze a viable biological sample one or more times in a manner sufficient to maintain the structural integrity and viability of the sample. Accordingly, the subject invention represents a significant contribution to the art.

All publications and patents cited in this specification are herein incorporated by reference, in their entirety, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

References

The following references discuss the design, construction and operation of high pressure diamond anvil cells: Field, The Properties of Diamond, Academic Press, New York City, N.Y. (1979); Manghnani, et al., High-Pressure Research and Mineral Physics, Terra Scientific Publishing Company, Tokyo, American Geophysical Union, Washington, D.C. (1987); Homan, "Higher Pressure in Science and Technology", Mat. Res. Soc. Symp. Proc, vol. 22, pp 2939, et seq., Elsevier Science Publishing Company (1984); Vodar, et al., High Pressure Science and Technology, Proceedings of the Vllth International AIRTAPT Conference, Le Creusot, France, JuI. 30-Aug. 3, 1979, Pergamon Press, New York, N.Y.; and Ruoff et al, "Synthetic Diamonds Produce Pressure of 125 GPa (1.25 Mbar)", J. Mater. Res., 2 (5), 614-617, September/October 1987

The following references discuss the design, construction and operation of high pressure freezing machines, for the purposes of cryofixing biological samples as a step in preparing samples for imaging in the electron microscope:

D. Studer, W. Graber, A. Al-Amoundi and P. Eggli, A new approach for cryofixation by high-pressure freezing, Journal of Microscopy v203 part 3 p285-294 (September 2001); Hans Moor, Cryotechniques in Electron Microscopy p176-191 (1987); E. Shimoni and M. Muller, On optimizing high-pressure freezing: from heat transfer theory to a new microbiopsy device, Journal of Microscopy v192 part 3 p236-247 (December 1998); D. Studer, W. Graber, A. Al-Amoundi and P. Eggli, A new approach for cryofixation by high-pressure freezing, Journal of Microscopy v203 part 3 p285-294 (September 2001); D. Studer, S. Zhao, W. Graber, P. Eggli and M. Frotscher, High Pressure Freezing of Brain Tissue Slices, Microscopy and Microanalysis v12 supplement 2 (2006).

Claims

WHAT IS CLAIMED IS:

1. A method, comprising: thawing a frozen biological sample under pressure in a manner sufficient to maintain structural integrity of said biological sample.

2. The method of Claim 1 , wherein said biological sample comprises multiple cells.

3. The method according to Claim 2, wherein said biological sample comprises a tissue sample.

4. The method according to Claim 1 , wherein said biological sample comprises a multicellular organism.

5. The method of Claim 1 , wherein said biological sample is viable.

6. The method of Claim 5, wherein said thawing occurs in a manner sufficient to maintain viability of said sample.

7. The method of Claim 1 , wherein said pressure ranges from about 200 atm to about 4000 atm.

8. The method of Claim 1 , wherein said thawing has a duration ranging from about 1 millisecond to about 1 second.

9. The method of Claim 1 , wherein said thawing comprises warming said sample to a temperature ranging from about 288 ° K to about 310⁰ K.

10. The method of Claim 1 , wherein said thawing occurs at a rate ranging from about 0.1 ° C/min to about 200 ° C/ms.

11. The method of Claim 1 , further comprising imaging said thawed sample.

12. The method of any of the previous claims, further comprising manipulating said sample after said sample is thawed.

13. The method of Claim 12, wherein said manipulation is selected from the group consisting of a physical, chemical, optical, molecular, and a nanotechnological manipulation.

14. The method of Claim 13, wherein said manipulating comprises physically manipulating said sample.

15. The method of Claim 13, wherein said manipulating comprises chemically manipulating said sample.

16. The method of Claim 15, further comprising imaging said sample after said manipulation.

17. The method of Claim 16, further comprising freezing said sample in a manner so as to maintain the viability of the sample to produce a frozen viable sample following said manipulating.

18. The method of Claim 17, further comprising observing said frozen viable sample.

19. The method of Claim 18, further comprising manipulating said frozen viable sample.

20. The method of Claims 12 or 19, wherein said manipulation comprises removing an element of the sample or adding one or more elements to the sample.

21. The method of Claim 18, further comprising, following said observing, thawing said frozen sample under pressure in a manner sufficient to maintain the structural integrity of said sample.

22. The method of Claim 11 , wherein said imaging is performed by an optical microscope.

23. An apparatus comprising: a chamber having an interior configured to hold a sample; a pressure modulator for modulating the pressure of said interior; and a temperature modulator for modulating the temperature of said interior from a temperature that is below the freezing point of water to a temperature that is above the freezing point of water.

24. The apparatus of Claim 23, further comprising an imaging element for imaging said sample.

25. The apparatus of Claim 26, wherein said imaging element comprises an optical microscope.

26. The apparatus of Claim 23, wherein said imaging element is configured for imaging said sample while the sample is inside of said chamber.

27. The apparatus of Claim 26, wherein said imaging element is configured for imaging said sample once the sample is removed from said chamber.

28. The apparatus of Claim 24, wherein said imaging element is associated with said chamber.

29. The apparatus of Claim 24, wherein said imaging element is configured for receiving said chamber.

30. The apparatus of Claim 29, further comprising a transfer element for transferring said chamber from said pressure modulator to said imaging element.

31. The apparatus of Claim 30, wherein said transfer element comprises a robotic arm.

32. The apparatus of Claim 25, wherein said chamber is configured for being mounted on a stage of an optical system.

33. The apparatus of Claim 23, wherein said chamber is configured to hold a biological sample.

34. The apparatus of Claim 23, wherein said chamber comprises: two or more anvils configured for generating a high pressure; and a sample holder for holding a sample.

35. The apparatus of Claim 34, wherein at least one of said anvils is fabricated from a high thermal conductivity material.

36. The apparatus of Claim 35, wherein said high thermal conductivity material has a thermal conductivity ranging from about 300 W m^"1 K^"1 to about 2300 W m^"1 1C¹.

37. The apparatus of Claim 35, wherein said high thermal conductivity material has a thermal conductivity ranging from about 895 W m^'1 K^"1 to about 2300 W m^"1 K^'

38. The apparatus of Claim 35, wherein said high thermal conductivity material is chosen from the group consisting of diamond, sapphire, ruby and emerald.

39. The apparatus of Claim 23, wherein said temperature modulator comprises a heating element.

40. The apparatus of Claim 39, wherein said heating element comprises a heated fluid.

41. The apparatus of Claim 40, wherein said heated fluid is a gas

42. The apparatus of Claim 40, wherein said heated fluid is a liquid.

43. The apparatus of Claim 39, wherein said heating element contacts said heated fluid with a chamber of said apparatus.

44. The apparatus of Claim 39, wherein said heating element is capable of generating a temperature within the chamber between about 288 ° K to about 298 ° K in about 1 millisecond to about 20 milliseconds.

45. The apparatus of Claim 23, wherein said temperature modulator further comprises a cooling element.

46. The apparatus of Claim 45, wherein said cooling element comprises a liquid.

47. The apparatus of Claim 45, wherein said cooling element is capable of reducing the temperature within said chamber to between about 0 ° K to about 273 ° K within about 1 millisecond to about 20 milliseconds.

48. The apparatus of Claim 23, further comprising a compression mechanism for moving said anvils so as to generate a high pressure within the sample holder.

49. The apparatus of Claim 48, wherein said compression mechanism is capable of generating a pressure of about 200 atm to about 4000 atm within the chamber.

50. The apparatus of Claim 49, wherein said pressure is capable of being generated within about 1 millisecond to about 20 milliseconds.

51. The apparatus of Claim 23, wherein said chamber is configured to hold a biological sample and to allow the transmission of photons or electrons into and out of said chamber.