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High-Pressure Frozen Mouse Lung: Cryo Scanning Electron Microscopy

Published online by Cambridge University Press:  02 July 2020

Jacob Bastacky ,
Paul Walther ,
Jon Goerke ,
Eve Clausnitzer ,
Charles Lee  and
Martin Müller
Jacob Bastacky
Affiliation:
Life Sciences Division, Lawrence Berkeley Laboratory, Berkeley, California94720 Children's Hospital Oakland Research Institute, 747 52nd St., Oakland, California94609
Paul Walther
Affiliation:
Sektion Elektronenmikroskopie, University of Ulm, Germany Laboratorium für Elektronenmikroskopie 1, Institut für Zellbiologie, ETH, Zürich, Switzerland
Jon Goerke
Affiliation:
CVRI and Department of Physiology, University of California, San Francisco, California94143
Eve Clausnitzer
Affiliation:
Children's Hospital Oakland Research Institute, 747 52nd St., Oakland, California94609
Charles Lee
Affiliation:
Life Sciences Division, Lawrence Berkeley Laboratory, Berkeley, California94720 Children's Hospital Oakland Research Institute, 747 52nd St., Oakland, California94609
Martin Müller
Affiliation:
Laboratorium für Elektronenmikroskopie 1, Institut für Zellbiologie, ETH, Zürich, Switzerland
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Extract

High-pressure facilitates freezing of biological specimens: pressurizing to 2000 bar before cooling retards growth and reduces size of ice crystals in the specimen. Although the lung is 80% air and is generally difficult to freeze, we report here good cellular ultrastructure in high-pressure frozen lung when: 1) the lung is allowed to collapse (empty of air) prior to sampling, or 2) air-filled lung is allowed to compress during pressurization in the high-pressure freezer prior to freezing or 3) airspaces are filled with relatively incompressible fluorocarbon liquid.

Small Balb mice (28-30g) were anaesthetized and ventilated via endotracheal tube. The chest was opened and a 2mm diameter disk of lung at the thin anterior margin of the right lung was clamped between two aluminum tophats (200μm wells), sealing in air. In other experiments, the lung was allowed to collapse to 0 cm water transpulmonary pressure prior to clamping. In the third series of experiments, air was gradually replaced with fluorocarbon liquid (FC5312, 3M, Minneapolis MN) and the lung inflated with liquid to functional residual capacity. The top hat assembly containing the sealed lung was transferred to the high-pressure freezer (HPM 010, Bal-Tec, Balzers, Lichtenstein) where they were pressurized and frozen.

Type
Cryotechniques, Immunocytochemistry, and Electron Microscopy II. Cells and Tissues
Information
Microscopy and Microanalysis , Volume 5 , Issue S2: Proceedings: Microscopy & Microanalysis '99, Microscopy Society of America 57th Annual Meeting, Microbeam Analysis Society 33rd Annual Meeting, Portland, Oregon August 1-5, 1999 , August 1999 , pp. 440 - 441
Copyright
Copyright © Microscopy Society of America

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References

1 Studer, D., Michel, M., and Müller, M.. Scanning Microscopy. Supplement (1989). 3:253-69.Google Scholar

2 Bastacky, J., et al.Microscopy Research and Technique (1995). 32:457–458.CrossRefGoogle Scholar

3 Walther, P. and Müller, M.. Scanning (1997). 19:343–348.CrossRefGoogle ScholarPubMed

4 Supported by NIH HL52161, HL24075, HL60288, TRDRP 4RT-0382, CF 96PO, and the Swiss National Science Foundation; with thanks to K. McDonald, Electron Microscope Laboratory, University of California, Berkeley.Google Scholar