Premium
Controlled microaspiration for high‐pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography
Author(s) -
TRIFFO W. J.,
PALSDOTTIR H.,
McDONALD K. L.,
LEE J. K.,
INMAN J. L.,
BISSELL M. J.,
RAPHAEL R. M.,
AUER M.
Publication year - 2008
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.2008.01986.x
Subject(s) - micromanipulator , ultrastructure , transmission electron microscopy , materials science , biomedical engineering , electron tomography , sample preparation , pipette , microscopy , electron microscope , biological specimen , biophysics , anatomy , nanotechnology , chemistry , computer science , optics , biology , chromatography , scanning transmission electron microscopy , artificial intelligence , physics , medicine
Summary 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 transmission electron microscopy. An established approach to surmount these difficulties is the use of cellulose microdialysis tubing to transport the sample. With an inner diameter of 200 μm, 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.