Combined video fluorescence and 3D electron microscopy.
Three-dimensional structure of cells and organelles examined with the power of resolution of electron microscopy (EM) including EM tomography represents the average view of cell processes. More precise and detailed analysis is limited by the significant variations in the structure of cells in temporal dynamics of cellular events. Therefore EM cannot identify rare and fast events that could be extremely important for understanding molecular mechanisms underlying these cellular processes. Observation of living cells under EM is still impossible. In contrast, observations of cellular dynamics with the help of laser scanning confocal microscopes or light digitalized microscopes became an indispensable tool of cell biology. However, the resolution of even confocal microscope is limited to the half of the light wave. Therefore, in studies of dynamic cellular processes, it would be ideal to be able to combine the capability of in vivo fluorescence video microscopy with the EM. This chapter describes this technique with details and useful tricks, including the way to localize the same cell after its transfection with a protein fused with a fluorescent tag, examination under the microscope in living condition, fixation, immunolabeling, embedding, serial sectioning from the first section, and observation under EM. We also illustrate here the kinds of questions that the CVLEM approach was designed to address, as well as the particular know-how that is important for the successful application of this technique.
Mironov
Alexander A
AA
Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro, Chieti, Italy.
Polishchuk
Roman S
RS
Beznoussenko
Galina V
GV
D000818:Animals; D002468:Cell Physiological Phenomena; D006046:Gold / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D007150:Immunohistochemistry; D053768:Metal Nanoparticles; D008854:Microscopy, Electron / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D018715:Microscopy, Video / Q000295:instrumentation / Q000379:methods*; D008867:Microtomy / Q000379:methods; D008954:Models, Biological; D016707:Tissue Fixation / Q000379:methods
D006046:Gold; D006735:Horseradish Peroxidase