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Related Concept Videos

Cryo-electron Microscopy01:28

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Conventional electron microscopy (EM) involves dehydration, fixation, and staining of biological samples, which distorts the native state of biological molecules and results in several artifacts. Also, the high-energy electron beam damages the sample and makes it difficult to obtain high-resolution images. These issues can be addressed using cryo-EM, which uses frozen samples and gentler electron beams. The technique was developed by Jacques Dubochet, Joachim Frank, and Richard Henderson, for...
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A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen
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High subzero cryofixation: A technique for observing ice within tissues.

Larissa Lautner1, Nishaka William2, Jason P Acker3

  • 1Department of Surgery, University of Alberta, Edmonton, AB, T6G 2B7, Canada.

Cryobiology
|May 26, 2020
PubMed
Summary
This summary is machine-generated.

High-subzero cryofixation (HSC) allows ice observation in tissues at various temperatures without sample alteration. This method preserves ice grain integrity, offering a versatile alternative to traditional freeze substitution techniques for cryopreservation research.

Keywords:
CryofixationFreeze substitutionHigh sub-zero organ preservationIce recrystallizationIsothermal freeze fixationTissue fixation

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Area of Science:

  • Cryobiology
  • Histology
  • Materials Science

Background:

  • Existing methods for observing ice in tissues at sub-zero temperatures often require specific fixative compositions or temperature adjustments.
  • These limitations hinder consistent sample preparation across different storage temperatures.

Purpose of the Study:

  • To introduce and validate high-subzero cryofixation (HSC) as a versatile method for observing ice within tissues.
  • To demonstrate HSC's ability to maintain ice crystal integrity at various sub-zero temperatures without altering sample temperature.

Main Methods:

  • Rat liver sections were subjected to classical freeze-substitution (FS) or HSC protocols.
  • Samples were stored at -20°C for varying durations (1, 3, or 5 days) before processing.
  • Ice grain size in inner and outer tissue regions was quantified after paraffin embedding, slicing, and staining.

Main Results:

  • HSC fixative permeation was linear and mathematically modelable for predicting fixation duration.
  • Ice grain size in the inner regions of 5-day samples was consistent between HSC and FS (p=0.76).
  • FS processing resulted in larger ice grains in outer tissue regions compared to HSC (p=0.016), while HSC showed no difference between inner and outer regions (p=0.42).

Conclusions:

  • High-subzero cryofixation (HSC) is effective for observing ice in liver tissue stored at -20°C.
  • HSC offers a significant advantage over isothermal freeze fixation and freeze substitution by allowing fixation at various temperatures without sample or fixative modification.
  • The low melting point of the HSC fixative ensures consistent sample preparation across a range of sub-zero temperatures.