Conductive fixation for electron microscopy

Patent metadata

Field	Value
Publication number	US-12224154-B2
Application number	US-201916961328-A
Country	US
Kind code	B2
Filing date	Jan 10, 2019
Priority date	Jan 10, 2018
Publication date	Feb 11, 2025
Grant date	Feb 11, 2025

Abstract

Disclosed are compositions and methods for the conductive fixation of organic material, including biological samples. The compositions and methods described herein can address the problems of charging and sample damage caused by electron beam-sample interactions within an electron microscope.

First claim

What is claimed is:

1. A method of preparing a biological sample for microanalysis, the method comprising:
(i) contacting the biological sample with a freeze substitution solution comprising a lithium salt under conditions effective to permit permeation of the lithium salt into the biological sample;
(ii) contacting the biological sample with a first polymerizable resin under conditions effective to permit permeation of the polymerizable resin into the biological sample;
(iii) disposing the biological sample in a volume of a second polymerizable resin; and
(iv) heating the so-prepared biological sample to a temperature effective to cure the first polymerizable resin and the second polymerizable resin, thereby forming a 3-dimensional conductive sample block;
wherein the lithium salt is chosen from lithium trifluoromethanesulfonate, lithium carbonate, lithium hydroxide, lithium citrate, lithium nitrate, lithium benzoate, lithium formate, and combinations thereof.

2. The method of claim 1 , wherein the method further comprises contacting the biological sample with a fixation fluid prior to step (i).

3. The method of claim 2 , wherein the fixation fluid comprises an aldehyde.

4. The method of claim 3 , wherein the fixation fluid comprises an aqueous solution comprising from 2% to 8% by weight aldehyde, based on the total weight of the fixation fluid.

5. The method of claim 1 , wherein the lithium salt exhibits a solubility in water of at least 10 g/100 mL at 20° C.

6. The method of claim 1 , wherein the lithium salt comprises from 0.1% to 10% by weight of the freeze substitution solution.

7. The method of claim 1 , wherein the freeze substitution solution further comprises an oxidizing fixative, and
wherein the oxidizing fixative comprises from 0.5% to 5% by weight of the freeze substitution solution.

8. The method of claim 1 , wherein the freeze substitution solution further comprises a negative stain, and
wherein the negative stain comprises from 0.5% to 8% by weight of the freeze substitution solution.

9. The method of claim 1 , wherein the freeze substitution solution further comprises a mordant,
and wherein the mordant comprises from 0.01% to 1% by weight of the freeze substitution solution, based on the total weight of the freeze substitution solution.

10. The method of claim 1 , wherein contacting step (i) comprises:
immersing the biological sample in the freeze substitution solution at a first temperature below 0° C. for a first duration; and
immersing the sample in the freeze substitution solution at a second temperature above 0° C. for a second duration.

11. The method of claim 10 , wherein contacting step (i) further comprises immersing the biological sample in the freeze substitution solution at a third temperature below 0° C. for a third duration before immersing the sample in the freeze substitution solution at the second temperature above 0° C. for the second duration.

12. The method of claim 11 , wherein contacting step (i) further comprising immersing the biological sample in the freeze substitution solution at a fourth temperature above 0° C. for a fourth duration.

13. The method of claim 10 , wherein the first temperature is from −70° C. to −80° C. the second temperature is from 0° C. to 10° C., the first duration is from 8 hours to 24 hours, and the second duration is from 8 hours to 24 hours.

14. The method of claim 11 , wherein the third temperature is from −10° C. to −40° C., and wherein the third duration is from 2 hours to 12 hours.

15. The method of claim 12 , wherein the fourth temperature is from 15° C. to 30° C., and wherein the fourth duration is from 2 hours to 12 hours.

16. The method of claim 1 , wherein the first and second polymerizable resin comprises an epoxy resin.

17. The method of claim 1 , wherein the second polymerizable resin comprises an accelerator.

18. The method of claim 1 , step (iv) comprises heating the so-prepared biological sample to a temperature of at least 40° C.

19. The method of claim 1 , wherein the biological sample comprises a tissue sample.

20. A method of preparing a biological sample for microanalysis, the method comprising:
(i) contacting the biological sample with a freeze substitution solution comprising a lithium salt under conditions effective to permit permeation of the lithium salt into the biological sample;
(ii) contacting the biological sample with a first polymerizable resin under conditions effective to permit permeation of the polymerizable resin into the biological sample;
(iii) disposing the biological sample in a volume of a second polymerizable resin; and
(iv) heating the so-prepared biological sample to a temperature effective to cure the first polymerizable resin and the second polymerizable resin, thereby forming a 3-dimensional conductive sample block;
wherein the lithium salt consists of lithium trifluoromethanesulfonate, lithium carbonate, lithium hydroxide, lithium citrate, lithium nitrate, lithium benzoate, lithium formate, or any combination thereof.

Assignees

• Univ Kansas

Inventors

• Rosa-Molinar Eduardo

Classifications

• G01Q30/02

  Non-SPM analysing devices, e.g. SEM [Scanning Electron Microscope], spectrometer or optical microscope · CPC title

• G01N2001/305

  Fixative compositions · CPC title

• G01N2001/302

  Stain compositions · CPC title

• G01N1/42

  Low-temperature sample treatment, e.g. cryofixation · CPC title

• G01N1/30

  Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis} · CPC title