Imaging of cytoskeletal elements by low‐temperature high‐resolution scanning electron microscopy

Abstract

reasons have been the low resolving power of the Actin Elaments and microtubules, both in situ and in vitro, were imaged using high-resolution scanning electron microscopy (HRSEM) at low temperature. For visualization of cytoskeletal elements in situ, fibroblasts were first extracted and fixed; for cytoskeletal elements in vitro, purified proteins were polymerized and fixed. Both types of specimen were then subjected to plunge freezing, controlled freeze-drying, cryo-sputter coating with a thin chromium layer, cryo-transferring and cryo-observation in an FESEM. The three-dimensional architecture of the cytoskeleton was well preserved, permitting examination of the structural relationships among cytoskeletal elements. Actin Elaments and microtubules were identified by their characteristic helical features. Two periodicities of actin filaments, the short pitch of the left-handed helix measured at 5-5 nm and the 37-nm-long pitch helix, were revealed.Individual protofilaments were seen in microtubules as well as the characteristic 4-nm repeat of tubulin subunits along the protofilament. Clathrin cages were also observed. This technique provides a powerful approach for direct imaging of macromolecular structures with high contrast and high signal-to-noise ratio at a resolution of 2-3 nm.--

The median body of Giardia lamblia: an ultrastructural study

“…Several previous reports have used whole mounts of cytoskeletons for high resolution electron microscopy (Bell, 1995;Bell et al, 1988;Braet et al, 1996;Chen et al, 1995;Lindroth et al, 1988;Ris, 1985). There are several problems associated with the preparation of whole mounts, in which detergent extraction has been made, and the researcher must be aware for effects of critical point drying, and type of fixatives used on the morphology of Triton-resistant cytoskeletons when observed by SEM and TEM Lindroth et al, 1988).…”

“…There are several problems associated with the preparation of whole mounts, in which detergent extraction has been made, and the researcher must be aware for effects of critical point drying, and type of fixatives used on the morphology of Triton-resistant cytoskeletons when observed by SEM and TEM Lindroth et al, 1988). In the last years, a number of studies using these techniques called the attention to the possible distortions that can arise during CPD procedure and how to avoid them (Chen et al, 1995;Ris, 1985). In order to increase SEM signal yield, enhance contrast and reduce charging problems, coating of the specimens with a metal layer is necessary (Pawley, 1990).…”

“…In order to increase SEM signal yield, enhance contrast and reduce charging problems, coating of the specimens with a metal layer is necessary (Pawley, 1990). Other important factors in high resolution SEM are contamination, beam damage, and mechanical stability (Chen et al, 1995). The effects of CPD and freeze-drying on the morphology of microtubules by scanning and transmission electron microscopy were presented by some authors , showing that cytoskeletons attached to Formvar films suffer less structural damage than cells attached to glass, because the Formvar film absorbs some of the stress associated with shrinkage during drying.…”

Field emission scanning electron microscopy of plant cells

“…Early attempts at examining intracellular structure, including the use of freeze-fracturing techniques, by SEM were generally constrained by the low resolution of conventional instruments (Haggis and Phipps-Todd 1977). However, the development of high-resolution FESEM, with resolution approaching that of the transmission electron microscope (TEM), has opened up a whole new area of investigation of biological samples involving imaging with both low and high accelerating voltages, and cryo-microscopy (for references, see Chen et al 1995). Application of FESEM to important questions in animal cell biology is becoming widespread (Allen and Goldberg 1993,Allen et al 1997, Ris 1997; however, papers dealing with problems in plant cell biology are still relatively few (Vesk et al 1996, Fowke et al 1999.…”

Proceedings of SCANNING 96 Monterey, California, USA

“…Knowing the structure of fibronectin fibrils is important to understanding how the conformation of fibronectin fibrils can be regulated and why this affects cell behavior. In this study, we use high-resolution cryo-scanning electron microscopy (cryo-HRSEM), (Chen et al 1994 to examine the supramolecular organization of fibronectin fibrils formed in cultures and formed in a cell-free system (Mosher and Johnson 1983). 200 Scanning Vol.…”