Apparatus and method for cryopreparing biological tissue for ultrastructural analysis
First Claim
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1. Apparatus for cryopreparing vitrified biological tissue comprising:
- (a) a sample holder for retaining vitrified biological tissue;
(b) means for maintaining the sample holder at a cryogenic temperature;
(c) ultra-high vacuum means for depressurizing the atmosphere in the sample holder; and
(d) means for dehydrating said vitrified biological tissue while maintaining said tissue at a cryogenic temperature of less than -80°
C.
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Abstract
This invention relates to apparatus for the cryopreparation of biological tissue samples for ultrastructural analysis. The use of the apparatus comprises vitrifying a biological tissue sample under cryogenic temperature conditions and ultra low vacuum conditions. The depressurized, vitrified tissue sample is brought to equilibrium in a sample holder at a temperature of less than -140° C. The tissue sample is then dehydrated while maintained in a state of thermal equilibrium. After reaching equilibrium the tissue sample is optionally infiltrated with a degassed resin followed by a polymerization of the resin to form an embedded tissue sample.
75 Citations
43 Claims
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1. Apparatus for cryopreparing vitrified biological tissue comprising:
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(a) a sample holder for retaining vitrified biological tissue; (b) means for maintaining the sample holder at a cryogenic temperature; (c) ultra-high vacuum means for depressurizing the atmosphere in the sample holder; and (d) means for dehydrating said vitrified biological tissue while maintaining said tissue at a cryogenic temperature of less than -80°
C. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. Apparatus for cryopreparing biological tissue comprising:
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(a) apparatus for rapid supercooling of biological tissue, said apparatus including a metal rod adapted to transform said biological tissue to the vitreous phase, contact with said metal rod maintaining said tissue at a temperature of less than -123°
C.;(b) a sample holder assembly adapted to receive a sample holder containing said vitrified biological tissue, said sample chamber assembly being movable to facililate insertion into and withdrawal from a cryogenic bath; (c) an ultra high vacuum pump assembly for depressurizing said sample chamber assembly to from 1×
108 Torr to 1×
10-10 Torr, said ultra high vacuum pump assembly being removably attached to said sample chamber assembly; and(d) a cryogenic bath for maintaining the temperature of said sample chamber assembly at -123°
C. or below, said cryogenic bath being adapted to receive at least a portion of said sample chamber assembly. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 41, 42, 43)
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32. Apparatus for cryopreparing biological tissue comprising:
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(a) apparatus for rapid supercooling of biological tissue, said apparatus including a copper rod adapted to transform said biological tissue to the vitreous phase, said copper rod maintaining said tissue at a temperature of less than -140°
C.;(b) a sample holder designed to hold said vitrified tissue; (c) a sample chamber assembly adapted to receive said sample holder containing said vitrified biological tissue, said sample chamber assembly being movable to facilitate said insertion into and withdrawal from a cryogenic bath; (d) a low vacuum mechanical pump connected to said sample chamber assembly for depressurizing said sample chamber assembly to 1×
10-3 Torr;(e) an ultra high vacuum pump assembly for depressurizing said sample chamber assembly to from 1×
10-8 Torr to 1×
10-10 Torr, said ultra high vacuum pump assembly being removably attached to said sample chamber assembly;(f) a cryogenic bath for maintaining the temperature of said sample chamber assembly at -140°
C. or below, said cryogenic bath being adapted to receive at least a portion of said sample chamber assembly; and(g) a control panel electrically connected to said sample chamber assembly to monitor the vacuum, temperature and residual gas in said sample chamber assembly. - View Dependent Claims (33, 34, 35, 36, 37, 38, 39)
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40. Apparatus for cryopreparing biological tissue comprising:
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(a) apparatus for rapid supercooling of biological tissue, said apparatus including a solid copper rod adapted to transform said biological tissue to the vitreous phase, said solid copper rod maintaining said tissue at a temperature of less than -140°
C.;(b) a sample holder designed to hold said vitrified tissue, said sample holder being connected to radiant heating means; (c) a sample chamber assembly adapted to receive said sample holder containing said vitrified biological tissue, said sample chamber assembly being movable to facilitate insertion into and withdrawal from a cryogenic bath, said sample chamber assembly further including a resin embedding chamber and sample staining means; (d) a low vacuum mechanical pump connected to said sample chamber assembly for depressurizing said sample chamber assembly to 1×
10-3 Torr;(e) a turbomolecular pump for depressurizing said sample chamber assembly to from 1×
10-8 Torr to 1×
10-10 Torr, said turbomolecular pump being removably attached to said sample chamber assembly;(f) a gate valve interposed between said turbomolecular pump and said sample chamber assembly, said gate valve being sealed; (g) a bakeout jacket encasing said turbomolecular pump; (h) a cryogenic bath for maintaining the temperature of said sample chamber assembly at -140°
C. or below, said cryogenic bath being adapted to receive at least a portion of said sample chamber assembly; and(i) a control panel connected to said sample chamber assembly to monitor the vacuum, temperature and residual gas in said sample chamber assembly.
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Specification