EP0136014A2
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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: 1984-08-08
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Granted: —
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Expires: 2004-08-08
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 loss 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
EP
Data Source
google_patents
Last Synchronized
2026-10-23 16:27
CPC Classifications
G; G01; G01N; G01N1/00; G01N1/28; G01N1/42
Claims Analysis
Total Claims: 18
Claim 1
Independent Claim
91 words
A method for cryopreparing a biological tissue sample for ultrastructural analysis characterized in that: (a) a biological tissue sample is vitrified; (b) the atmosphere surrounding said sample is depressurized; (c) said vitrified tissue sample is brought to equilibrium at a temperature of less than -140° C; (d) said vitrified tissue sample is dehydrated while said sample is maintained in a state of equilibrium; (e) said dehydrated tissue sample is infiltrated with a degassed resin; and (f) said resin is polymerized in said infiltrated tissue sample to form an embedded tissue sample.
Claim 2
Independent Claim
91 words
A method for cryopreparing a biological tissue sample for ultrastructural analysis characterized in that: (a) a biological tissue sample is vitrified; (b) the atmosphere surrounding said sample is depressurized; (c) said vitrified tissue sample is brought to equilibrium at a temperature of less than -140° C; (d) said vitrified tissue sample is dehydrated while said sample is maintained in a state of equilibrium; (e) said dehydrated tissue sample is infiltrated with a degassed resin; and (f) said resin is polymerized in said infiltrated tissue sample to form an embedded tissue sample.
Claim 3
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 4
Dependent Claim
19 words
The method of claim 1 wherein the equilibrium of said vitrified tissue sample is indicated by a constant temperature.
Claim 5
Dependent Claim
13 words
The method of claim 1 wherein said dehydration is by means of sublimation.
Claim 6
Dependent Claim
19 words
The method of claim 4 wherein said dehydration is enhanced by the addition of energy from a secondary source.
Claim 7
Independent Claim
138 words
A method for cryopreparing a biological tissue sample for ultrastructural analysis characterized in that: (a) a biological tissue sample is vitrified by reducing the temperature of said sample to -140° C or below; (b) the atmosphere surrounding said sample is depressurized to less than 3 x 10 -9 Torr by drawing a vacuum, said vacuum to be drawn in less than 300 seconds; (c) said vitrified tissue sample is brought to equilibrium at a temperature of from -140° C to -196° C; (d) said vitrified tissue sample is dehydrated, while said sample is maintained in a state of equilibrium, by sublimation; (e) a contrast enhancing material is added to said sample; (f) said dehydrated tissue sample is infiltrated with a degassed resin; and (g) said resin is polymerized in said infiltrated tissue sample to form an embedded sample.
Claim 8
Independent Claim
138 words
A method for cryopreparing a biological tissue sample for ultrastructural analysis characterized in that: (a) a biological tissue sample is vitrified by reducing the temperature of said sample to -140° C or below; (b) the atmosphere surrounding said sample is depressurized to less than 3 x 10 -9 Torr by drawing a vacuum, said vacuum to be drawn in less than 300 seconds; (c) said vitrified tissue sample is brought to equilibrium at a temperature of from -140° C to -196° C; (d) said vitrified tissue sample is dehydrated, while said sample is maintained in a state of equilibrium, by sublimation; (e) a contrast enhancing material is added to said sample; (f) said dehydrated tissue sample is infiltrated with a degassed resin; and (g) said resin is polymerized in said infiltrated tissue sample to form an embedded sample.
Claim 9
Independent Claim
154 words
A method for cryopreparing a biological tissue sample for ultrastructural analysis characterized in that: (a) a biological tissue sample is vitrified by reducing the temperature of said sample to -140° C or below by the use of a liquid nitrogen bath; (b) the atmosphere surrounding said sample is depressurized to less than 3 x 10-9 Torr in less than 300 seconds by drawing a vacuum on said sample; (c) said vitrified tissue sample is brought to equilibrium at a temperature of from -140° C to -196° C; (d) said vitrified tissue sample is dehydrated, while said sample is maintained in a state of equilibrium, by sublimation and by the use of a secondary source of energy; (e) osmium tetraoxide is added to enhance the contrast of said tissue sample; (f) said dehydrated tissue sample is infiltrated with a degassed resin; and (g) said resin is polymerized in said infiltrated tissue sample by adding heat.
Claim 10
Independent Claim
154 words
A method for cryopreparing a biological tissue sample for ultrastructural analysis characterized in that: (a) a biological tissue sample is vitrified by reducing the temperature of said sample to -140° C or below by the use of a liquid nitrogen bath; (b) the atmosphere surrounding said sample is depressurized to less than 3 x 10-9 Torr in less than 300 seconds by drawing a vacuum on said sample; (c) said vitrified tissue sample is brought to equilibrium at a temperature of from -140° C to -196° C; (d) said vitrified tissue sample is dehydrated, while said sample is maintained in a state of equilibrium, by sublimation and by the use of a secondary source of energy; (e) osmium tetraoxide is added to enhance the contrast of said tissue sample; (f) said dehydrated tissue sample is infiltrated with a degassed resin; and (g) said resin is polymerized in said infiltrated tissue sample by adding heat.
Claim 11
Dependent Claim
71 words
Apparatus for cryopreparing a biological tissue sample for ultrastructural analysis characterized in that it includes: (a) means for vitrifying a biological tissue sample; (b) means for depressurizing the atmosphere of a biological tissue sample; (c) means for dehydrating a vitrified tissue sample while said sample is maintained in a state of equilibrium; (d) means for infiltrating a dehydrated tissue sample with a degassed resin; and (e) means for polymerizing said resin.
Claim 12
Dependent Claim
71 words
Apparatus for cryopreparing a biological tissue sample for ultrastructural analysis characterized in that it includes: (a) means for vitrifying a biological tissue sample; (b) means for depressurizing the atmosphere of a biological tissue sample; (c) means for dehydrating a vitrified tissue sample while said sample is maintained in a state of equilibrium; (d) means for infiltrating a dehydrated tissue sample with a degassed resin; and (e) means for polymerizing said resin.
Claim 13
Dependent Claim
19 words
The apparatus of claim 8 wherein the equilibrium of said vitrified tissue sample is indicated by a constant temperature.
Claim 14
Dependent Claim
13 words
The apparatus of claim 8 wherein said dehydration is by means of sublimation.
Claim 15
Dependent Claim
76 words
Specimen holding apparatus for the cryopreparation of biological samples characterized in that it includes: (a) a specimen chamber adapted to retain one or more tissue samples; (b) a support assembly attached to said specimen chamber, said support assembly insulating said specimen chamber from energy means; and (c) spacing means adapted to isolate tissue samples from one another while said tissue samples are in said specimen chamber, said spacing means being removably engaged by said specimen chamber.
Claim 16
Dependent Claim
76 words
Specimen holding apparatus for the cryopreparation of biological samples characterized in that it includes: (a) a specimen chamber adapted to retain one or more tissue samples; (b) a support assembly attached to said specimen chamber, said support assembly insulating said specimen chamber from energy means; and (c) spacing means adapted to isolate tissue samples from one another while said tissue samples are in said specimen chamber, said spacing means being removably engaged by said specimen chamber.
Claim 17
Dependent Claim
124 words
Specimen holding apparatus for the cryopreparation of biological samples characterized in that it includes: (a) a specimen chamber adapted to retain one or more tissue samples, said chamber including a plurality of apertures to permit the flow of moisture and energy to said tissue samples; (b) a support assembly attached to said specimen chamber, said support assembly including a tubular member attached to said specimen chamber, said tubular member being fittably received by a suport housing; and (c) spacing means adapted to isolate tissue samples from one another while said tissue samples are in said specimen chamber, said spacing means being removably engaged by said specimen chamber and wherein said spacing means comprise a plurality of planar flanges pivotally attached to a retaining pair.
Claim 18
Dependent Claim
124 words
Specimen holding apparatus for the cryopreparation of biological samples characterized in that it includes: (a) a specimen chamber adapted to retain one or more tissue samples, said chamber including a plurality of apertures to permit the flow of moisture and energy to said tissue samples; (b) a support assembly attached to said specimen chamber, said support assembly including a tubular member attached to said specimen chamber, said tubular member being fittably received by a suport housing; and (c) spacing means adapted to isolate tissue samples from one another while said tissue samples are in said specimen chamber, said spacing means being removably engaged by said specimen chamber and wherein said spacing means comprise a plurality of planar flanges pivotally attached to a retaining pair.
References Cited (Backward Citations)
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