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US4510169A
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Method and apparatus for cryopreparing biological tissue for ultrastructural analysis
Assignee: The University of Texas System
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Filing: 1983-08-23
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Granted: 1985-04-09
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Expires: 2003-08-23
Abstract
Abstract This invention relates to a method for the cryopreparation of biological tissue samples for ultrastructural analysis. The process comprises vitrifying a biological tissue sample under severely rapid conditions and then depressurized. The depressurized, vitrified tissue sample is brought to equilibrium at a temperature of less than -140° C. The tissue sample is then dehydrated while maintained in a state of thermal equilibrium. After reaching equilibrium the tissue sample is infiltrated with a degassed resin and followed by a polymerization of the resin to form an embedded tissue sample. This invention additionally relates to a sample holder apparatus which has unique use characteristics in the process of this invention.
Inventors
- 👤 John G. Linner
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 12:33
CPC Classifications
G; G01; G01N; G01N1/00; G01N1/28; G01N1/42
Claims Analysis
Total Claims: 34
Claim 1
Independent Claim
82 words
A method for cryopreparing a biological tissue sample for ultrastructural analysis comprising: (a) vitrifying a biological tissue sample; (b) depressurizing the atmosphere surrounding said sample; (c) bringing said vitrified tissue sample to equilibrium at a temperature of less than -140° C.; (d) dehydrating said vitrified tissue sample while said sample is maintained in a state of equilibrium; (e) infiltrating said dehydrated tissue sample with a degassed resin; (f) polymerizing said resin in said infiltrated tissue sample to form an embedded tissue sample.
Claim 2
Independent Claim
82 words
A method for cryopreparing a biological tissue sample for ultrastructural analysis comprising: (a) vitrifying a biological tissue sample; (b) depressurizing the atmosphere surrounding said sample; (c) bringing said vitrified tissue sample to equilibrium at a temperature of less than -140° C.; (d) dehydrating said vitrified tissue sample while said sample is maintained in a state of equilibrium; (e) infiltrating said dehydrated tissue sample with a degassed resin; (f) polymerizing said resin in said infiltrated tissue sample to form an embedded tissue sample.
Claim 3
Dependent Claim
17 words
The method of claim 1 wherein said vitrifying is to a temperature of -140° C. or below.
Claim 4
Dependent Claim
14 words
The method of claim 2 wherein said vitrifying is by means of liquid nitrogen.
Claim 5
Dependent Claim
15 words
The method of claim 3 wherein said vitrifying is completed in less than one second.
Claim 6
Dependent Claim
22 words
The method of claim 1 wherein said depressurizing is by means of drawing a vacuum on the atmosphere surrounding said tissue sample.
Claim 7
Dependent Claim
14 words
The method of claim 5 wherein said vacuum is at least 3×10 -9 Torr.
Claim 8
Dependent Claim
15 words
The method of claim 6 wherein said vacuum is created in less than 300 minutes.
Claim 9
Dependent Claim
19 words
The method of claim 1 wherein the equilibrium of said vitrified tissue sample is indicated by a constant temperature.
Claim 10
Dependent Claim
16 words
The method of claim 8 wherein said constant temperature is between -140° C. to -196° C.
Claim 11
Dependent Claim
13 words
The method of claim 1 wherein said dehydration is by means of sublimation.
Claim 12
Dependent Claim
19 words
The method of claim 10 wherein said dehydration is enhanced by the addition of energy from a secondary source.
Claim 13
Dependent Claim
14 words
The method of claim 11 wherein said secondary source of energy is heat energy.
Claim 14
Dependent Claim
14 words
The method of claim 11 wherein said secondary source of energy is radiant energy.
Claim 15
Dependent Claim
16 words
The method of claim 13 wherein said radiant energy is by means of nuclear magnetic resonance.
Claim 16
Dependent Claim
20 words
The method of claim 11 wherein said radiant energy is infrared produced by a laser and transmitted by fiber optics.
Claim 17
Dependent Claim
14 words
The method of claim 1 further comprising the addition of a contrast enhancing material.
Claim 18
Dependent Claim
13 words
The method of claim 16 wherein said contrast enhancing material is osmium tetraoxide.
Claim 19
Dependent Claim
11 words
The method of claim 1 wherein said polymerization is by heat.
Claim 20
Dependent Claim
14 words
The method of claim 1 wherein said polymerization is by means of ultraviolet light.
Claim 21
Independent Claim
131 words
A method for cryopreparing a bilogical tissue sample for ultrastructural analysis comprising: (a) vitrifying a biological tissue sample by reducing the temperature of said sample to -140° C. or below; (b) depressurizing the atmosphere surrounding said sample to less than 3×10 -9 Torr by drawing a vacuum, said vacuum to be drawn in less bhan 300 minutes; (c) bringing said vitrified tissue sample to equilibrium at a temperature of from -140° C. to -196° C.; (d) dehydrating said virified tissue sample, while said sample is maintained in a state of equilibrium, by sublimation or adiabatic molecular distillation; (e) adding a contrast enhancing material to said sample; (f) infiltrating said hehydrated tissue sample with a degassed resin; and (g) polymerizing said resin in said infiltrated tissue sample to form an embedded sample.
Claim 22
Independent Claim
131 words
A method for cryopreparing a bilogical tissue sample for ultrastructural analysis comprising: (a) vitrifying a biological tissue sample by reducing the temperature of said sample to -140° C. or below; (b) depressurizing the atmosphere surrounding said sample to less than 3×10 -9 Torr by drawing a vacuum, said vacuum to be drawn in less bhan 300 minutes; (c) bringing said vitrified tissue sample to equilibrium at a temperature of from -140° C. to -196° C.; (d) dehydrating said virified tissue sample, while said sample is maintained in a state of equilibrium, by sublimation or adiabatic molecular distillation; (e) adding a contrast enhancing material to said sample; (f) infiltrating said hehydrated tissue sample with a degassed resin; and (g) polymerizing said resin in said infiltrated tissue sample to form an embedded sample.
Claim 23
Dependent Claim
14 words
The method of claim 20 wherein said vitrifying is by means of liquid nitrogen.
Claim 24
Dependent Claim
15 words
The method of claim 21 wherein said vitrifying is completed in less than one second.
Claim 25
Dependent Claim
19 words
The method of claim 20 wherein said dehydration is enhanced by the addition of energy from a secondary source.
Claim 26
Dependent Claim
14 words
The method of claim 23 wherein said secondary source of energy is heat energy.
Claim 27
Dependent Claim
14 words
The method of claim 23 wherein said secondary source of energy is radiant energy.
Claim 28
Dependent Claim
16 words
The method of claim 23 wherein said radiant energy is by means of nuclear magnetic resonance.
Claim 29
Dependent Claim
20 words
The method of claim 23 wherein said radiant energy is infrared produced by a laser and transmitted by fiber optics.
Claim 30
Dependent Claim
13 words
The method of claim 20 wherein said contrast enhancing material is osmium tetraoxide.
Claim 31
Dependent Claim
11 words
The method of claim 20 wherein said polymerization is by heat.
Claim 32
Dependent Claim
14 words
The method of claim 20 wherein said polymerization is by means of ultraviolet light.
Claim 33
Independent Claim
148 words
A method for cryopreparing a biological tissue sample for ultrastructural analysis comprising: (a) vitrifying a biological tissue sample by reducing the temperature of said sample to -140° C. or below by the use of a liquid nitrogen bath; (b) depressurizing the atmosphere surrounding said sample to less than 3×10 -9 Torr in less than 300 minutes by drawing a vacuum on said sample; (c) bringing said vitrified tissue sample to equilibrium at a temperature of from -140° C. to -196° C.; (d) dehydrating said vitrified tissue sample, while said sample is maintained in a state of equilibrium, by sublimation or adiabatic moledular distillation and by the use of a secondary source of energy; (e) adding osmium tetraoxide to enhance the contrast of said tissue sample; (f) infiltrating said dehydrated tissue sample with a degassed resin; and (g) polymerizing said resin in said infiltrated tissue sample by adding heat.
Claim 34
Independent Claim
148 words
A method for cryopreparing a biological tissue sample for ultrastructural analysis comprising: (a) vitrifying a biological tissue sample by reducing the temperature of said sample to -140° C. or below by the use of a liquid nitrogen bath; (b) depressurizing the atmosphere surrounding said sample to less than 3×10 -9 Torr in less than 300 minutes by drawing a vacuum on said sample; (c) bringing said vitrified tissue sample to equilibrium at a temperature of from -140° C. to -196° C.; (d) dehydrating said vitrified tissue sample, while said sample is maintained in a state of equilibrium, by sublimation or adiabatic moledular distillation and by the use of a secondary source of energy; (e) adding osmium tetraoxide to enhance the contrast of said tissue sample; (f) infiltrating said dehydrated tissue sample with a degassed resin; and (g) polymerizing said resin in said infiltrated tissue sample by adding heat.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1988-04-08 | 1988-10-08 | 1989-04-09 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1992-04-08 | 1992-10-08 | 1993-04-09 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1996-04-08 | 1996-10-08 | 1997-04-09 | $7700.00 | Window Closed / Lapsed |