Bridging fluorescence microscopy and electron microscopy

Abstract

Development of new fluorescent probes and fluorescence microscopes has led to new ways to study cell biology. With the emergence of specialized microscopy units at most universities and research centers, the use of these techniques is well within reach for a broad research community. A major breakthrough in fluorescence microscopy in biology is the ability to follow specific targets on or in living cells, revealing dynamic localization and/or function of target molecules. One of the inherent limitations of fluorescence microscopy is the resolution. Several efforts are undertaken to overcome this limit. The traditional and most well-known way to achieve higher resolution imaging is by electron microscopy. Moreover, electron microscopy reveals organelles, membranes, macromolecules, and thus aids in the understanding of cellular complexity and localization of molecules of interest in relation to other structures. With the new probe development, a solid bridge between fluorescence microscopy and electron microscopy is being built, even leading to correlative imaging. This connection provides several benefits, both scientifically as well as practically. Here, I summarize recent developments in bridging microscopy.

Fig. 1

Labeling proteins for both light microscopic and electron microscopic analysis. Flowchart indicating numerous possibilities for correlative microscopy. See text and (Sosinsky et al. 2007) for details. Reproduced from (Sosinsky et al. 2007). Copyright: AP/Elsevier

Fig. 2

Pulse-chase in the microscope. Four amino-acid substitutions in the gap junction protein conexin43 create a binding site for tetracysteines. This minor protein modification allows determination of gap junction turnover. Sequence of labeling: proteins in transfected cells are labeled with FlAsH (green); excess label is washed away. After a 6-h chase, the new protein is labeled with ReAsH (red), revealing that gap junctions grow at the edge of the plaque. This finding is consistent with another study, where FRAP was used to study the gap junction turnover (see Gaietta et al. ; Lauf et al. for further details). [Image: BNGG at the National Center for Microscopy and Imaging Research (NCMIR), San Diego, CA, USA]

Fig. 3

Detection of proteins by Quantum dots. Top Composite of six cultured fibroblasts (false-colored) from six different dishes immuno-labeled for a-tubulin and detected with Quantum dots analyzed by confocal microscopy: QD525 (blue), QD565 (green), QD585 (yellow), QD605 (orange), QD655 (red) and QD705 (purple). Bottom EM-analysis of a similar experiment. Note the specific labeling of microtubules, but not of the actin fibers. [Image: T.J. Deerinck and BNGG at NCMIR]

Fig. 4

Golgi twins revealed by LM/EM. Main picture Proliferating population of HeLa cells, of which some cells express Golgi-targeted GFP-tetracysteine (green) fixed and stained for the Golgi protein Giantin (blue) and microtubules (green). Note the different morphologies of the Golgi apparatus depending on cell cycle phase. Inset Sequence of time lapse of the same marker (GFP: green; tetracysteine-labeled with ReAsH red, depicted on the transmission image). The cells were fixed when the Golgi twins were formed and subsequently processed and analyzed by electron microscopy (bottom-right). See text and (Gaietta et al. 2006) for further details. [Image: G.M. Gaietta, T.J. Deerinck and BNGG at NCMIR]