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Title: Microaspiration for high-pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography

Abstract

High-pressure freezing is the preferred method to prepare thick biological specimens for ultrastructural studies. However, the advantages obtained by this method often prove unattainable for samples that are difficult to handle during the freezing and substitution protocols. Delicate and sparse samples are difficult to manipulate and maintain intact throughout the sequence of freezing, infiltration, embedding, and final orientation for sectioning and subsequent TEM imaging. An established approach to surmount these difficulties is the use of cellulose microdialysis tubing to transport the sample. With an inner diameter of 200 micrometers, the tubing protects small and fragile samples within the thickness constraints of high-pressure freezing, and the tube ends can be sealed to avoid loss of sample. Importantly, the transparency of the tubing allows optical study of the specimen at different steps in the process. Here, we describe the use of a micromanipulator and microinjection apparatus to handle and position delicate specimens within the tubing. We report two biologically significant examples that benefit from this approach, 3D cultures of mammary epithelial cells and cochlear outer hair cells. We illustrate the potential for correlative light and electron microscopy as well as electron tomography.

Authors:
; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
Life Sciences Division
OSTI Identifier:
936096
Report Number(s):
LBNL-748E
TRN: US200818%%570
DOE Contract Number:  
DE-AC02-05CH11231
Resource Type:
Journal Article
Journal Name:
Journal of Microscopy
Additional Journal Information:
Journal Volume: 230; Related Information: Journal Publication Date: 05/2008
Country of Publication:
United States
Language:
English
Subject:
59; CELLULOSE; ELECTRON MICROSCOPY; ELECTRONS; FREEZING; ORIENTATION; PRESERVATION; THICKNESS; TOMOGRAPHY; TRANSPORT; high-pressure freezing TEM electron tomography

Citation Formats

Auer, Manfred, Triffo, W J, Palsdottir, H, McDonald, K L, Inman, J L, Bissell, M J, Raphael, R M, Auer, M, and Lee, J K. Microaspiration for high-pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography. United States: N. p., 2008. Web.
Auer, Manfred, Triffo, W J, Palsdottir, H, McDonald, K L, Inman, J L, Bissell, M J, Raphael, R M, Auer, M, & Lee, J K. Microaspiration for high-pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography. United States.
Auer, Manfred, Triffo, W J, Palsdottir, H, McDonald, K L, Inman, J L, Bissell, M J, Raphael, R M, Auer, M, and Lee, J K. Wed . "Microaspiration for high-pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography". United States. https://www.osti.gov/servlets/purl/936096.
@article{osti_936096,
title = {Microaspiration for high-pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography},
author = {Auer, Manfred and Triffo, W J and Palsdottir, H and McDonald, K L and Inman, J L and Bissell, M J and Raphael, R M and Auer, M and Lee, J K},
abstractNote = {High-pressure freezing is the preferred method to prepare thick biological specimens for ultrastructural studies. However, the advantages obtained by this method often prove unattainable for samples that are difficult to handle during the freezing and substitution protocols. Delicate and sparse samples are difficult to manipulate and maintain intact throughout the sequence of freezing, infiltration, embedding, and final orientation for sectioning and subsequent TEM imaging. An established approach to surmount these difficulties is the use of cellulose microdialysis tubing to transport the sample. With an inner diameter of 200 micrometers, the tubing protects small and fragile samples within the thickness constraints of high-pressure freezing, and the tube ends can be sealed to avoid loss of sample. Importantly, the transparency of the tubing allows optical study of the specimen at different steps in the process. Here, we describe the use of a micromanipulator and microinjection apparatus to handle and position delicate specimens within the tubing. We report two biologically significant examples that benefit from this approach, 3D cultures of mammary epithelial cells and cochlear outer hair cells. We illustrate the potential for correlative light and electron microscopy as well as electron tomography.},
doi = {},
journal = {Journal of Microscopy},
number = ,
volume = 230,
place = {United States},
year = {2008},
month = {2}
}