Preparing whole mounts of biological specimens for imaging macromolecular structures by light and electron microscopy

Abstract

In this article we critically review the procedures involved in preparing whole mounts of biological samples for microscopic observation, particularly at high resolution. We discuss practical methods for optimizing specimen preservation to achieve the two principal goals of biological specimen preparation: (a) preserving biological structures as close to their living configuration as possible, and (b) rendering them visible with the desired imaging method. Drawing on examples taken from our own work, and using fluorescence light microscopy and scanning electron microscopy to study the cytoskeleton of vertebrate cells in culture, we show that obtaining optimal results is often a compromise between maximum preservation and the clear visualization of structures. We have found that fixing cells with bifunctional protein crosslinking reagents, in combination with detergent extraction, allows internal cell structures to be preserved, labeled with specific probes, and visualized with microscopic methods. We also review and discuss the relative merits of different procedures for fixing (chemical fixation and cryofixation), drying (air-drying, critical point-drying, and freeze-drying) and coating biological specimens with metals to facilitate visualization in the electron microscope. © 1997 John Wiley & Sons, Inc. Int. J. Imaging Syst Technol, 8, 225–239, 1997