US4865871A
Expired / Lapsed
US
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Method for cryopreparing biological tissue
Assignee: The University of Texas System
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Filing: 1988-07-11
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Granted: 1989-09-12
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Expires: 2008-07-11
Abstract
Abstract This invention relates to apparatus for the cryopreparation of biological tissue samples. The method comprises treating a biological tissue sample with a cryoprotectant which raises the glass transition temperature range of the sample followed by vitrification under cryogenic temperature conditions and ultra low vacuum conditions. The depressurized, vitrified tissue sample is brought to equilibrium in a sample holder. The tissue sample is then dehydrated while maintained in a state of thermal nonequilibrium.
Inventors
- 👤 Stephen A. Livesey
- 👤 John G. Linner
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-22 23:44
CPC Classifications
G; G01; G01N; G01N1/00; G01N1/28; G01N1/42; A; A01; A01N; A01N1/00; A01N1/10; A01N1/12; A01N1/122; A01N1/125; A01N1/126; A01N1/16; A01N1/165; F; F26; F26B; F26B5/00; F26B5/04; F26B5/06
Claims Analysis
Total Claims: 16
Claim 1
Independent Claim
123 words
A method for cryopreparing a biological tissue sample comprising: treating a biological tissue sample with a cryoprotectant; vitrifying the tissue sample at a rate and under conditions so as to preclude the formation of significant resolvable ice crystals; equilibrating said vitrified tissue sample at temperature and pressure conditions below the glass transition range of said sample adding energy to said equilibrated tissue sample at a rate sufficient to cause vitrified water in said sample to change physical state directly to the vapor phase without substantially disturbing said equilibrium or completing the induced transition; and removing water from said tissue sample by molecular distillation by the addition of energy until substantially no vaporized water can be detected in the atmosphere surrounding said tissue sample.
Claim 2
Independent Claim
123 words
A method for cryopreparing a biological tissue sample comprising: treating a biological tissue sample with a cryoprotectant; vitrifying the tissue sample at a rate and under conditions so as to preclude the formation of significant resolvable ice crystals; equilibrating said vitrified tissue sample at temperature and pressure conditions below the glass transition range of said sample adding energy to said equilibrated tissue sample at a rate sufficient to cause vitrified water in said sample to change physical state directly to the vapor phase without substantially disturbing said equilibrium or completing the induced transition; and removing water from said tissue sample by molecular distillation by the addition of energy until substantially no vaporized water can be detected in the atmosphere surrounding said tissue sample.
Claim 3
Dependent Claim
24 words
A method of preparing a biological tissue sample as defined in claim 1 wherein the cryoprotectant increases the glass transition characteristics of the sample.
Claim 4
Dependent Claim
37 words
A method of cryopreparing a biological tissue sample as defined in claim 2 wherein the cryoprotectant is selected from the group consisting of DMSO, dextran, sucrose, 1,2-propanediol, glycerol, sorbitol, fructose, trehalose, hydroxyethyl starch, propylene glycol, and polyvinylpyrolidine.
Claim 5
Dependent Claim
18 words
A method for cryopreparing a biological tissue sample as defined in claim 3 wherein the cryoprotectant comprises trehalose.
Claim 6
Dependent Claim
23 words
A method for cryopreparing a biological tissue sample as defined in claim 1 wherein the vitrification step comprises rapidly supercooling said tissue sample.
Claim 7
Dependent Claim
27 words
A method for cryopreparing a biological tissue sample as defined in claim 5 wherein the vitrifcation step comprises contacting the tissue sample with a supercooled metal block.
Claim 8
Independent Claim
126 words
A method for cryopreparing a biological tissue sample for ultrastructural analysis comprising: treating a biological tissue sample with a cryoprotectant which increases the glass transition temperature range of the sample; vitrifying the tissue sample under conditions so as to preclude the formation of significant resolvable ice crystals; bringing the vitrified tissue sample to equilibrium at a temperature below the glass transition temperature range; adding energy to said equilibrated tissue sample at a rate sufficient to cause vitrified water in said sample to change physical state directly to the vapor phase without substantially disturbing said equilibrium; and removing water from said tissue sample by molecular distillation by the addition of energy until substantially no vaporized water can be detected in the atmosphere surrounding said biological tissue sample.
Claim 9
Independent Claim
126 words
A method for cryopreparing a biological tissue sample for ultrastructural analysis comprising: treating a biological tissue sample with a cryoprotectant which increases the glass transition temperature range of the sample; vitrifying the tissue sample under conditions so as to preclude the formation of significant resolvable ice crystals; bringing the vitrified tissue sample to equilibrium at a temperature below the glass transition temperature range; adding energy to said equilibrated tissue sample at a rate sufficient to cause vitrified water in said sample to change physical state directly to the vapor phase without substantially disturbing said equilibrium; and removing water from said tissue sample by molecular distillation by the addition of energy until substantially no vaporized water can be detected in the atmosphere surrounding said biological tissue sample.
Claim 10
Dependent Claim
24 words
A method of preparing a biological tissue sample as defined in claim 7 wherein the cryoprotectant increases the glass transition temperature of the sample.
Claim 11
Dependent Claim
37 words
A method of cryopreparing a biological tissue sample as defined in claim 8 wherein the cryoprotectant is selected from the group consisting of DMSO, dextran, sucrose, 1,2-propanediol, glycerol, sorbitol, fructose, trehalose, hydroxyethyl starch, propylene glycol, and polyvinylpyrolidine.
Claim 12
Dependent Claim
18 words
A method for cryopreparing a biological tissue sample as defined in claim 9 wherein the cryoprotectant comprises trehalose.
Claim 13
Dependent Claim
23 words
A method for cryopreparing a biological tissue sample as defined in claim 7 wherein the vitrification step comprises rapidly supercooling the tissue sample.
Claim 14
Dependent Claim
27 words
A method for cryopreparing a biological tissue sample as defined in claim 11 wherein the vitrification step comprises contacting the tissue sample with a supercooled metal block.
Claim 15
Dependent Claim
21 words
A method of preparing a biological tissue sample as defined in claim 7 further comprising coating said sample with osmium vapors.
Claim 16
Dependent Claim
34 words
A method of preparing a biological tissue sample as defined in claim 7 further comprising infiltrating said sample with a degassed resin followed by polymerization of the resin to form an embedded tissue sample.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1992-09-11 | 1993-03-13 | 1993-09-12 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-09-11 | 1997-03-13 | 1997-09-12 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-09-11 | 2001-03-13 | 2001-09-12 | $7700.00 | Window Closed / Lapsed |