Confocal sample preparation

Scanning force microscopy (SFM) has been widely used for visualization of biomedical objects because of combination of extreme resolution, simplicity of sample preparation and ability to operate under physiological conditions. Nowadays SFM is increasingly applied to investigate the ultrastructure of biomedical samples embedded in epoxy resin [1]. In the present work, we are focusing on application of SFM, confocal laser scanning microscopy and ultramicrotomy to the K562 leukemic cells study. 

Bom out of the need to overcome an imaging problem in the 1950s, confocal microscopes today allow researchers to go beyond simple imaging and ask biochemical questions. This chapter provides background information on the development of modern confocal microscopes, their uses and applications. Sample preparation and observation are also discussed. Information is also provided about more advanced applications such as FRAP, FRET and 2-photon imaging. The requirements for setting up a confocal laboratory and the instrumentation needs are also discussed. 

Key words Confocal microscopy, Sample preparation, DAPI, FRAP, FRET, 2-Photon imaging... 

Shifting from confocal microscopy to electron microscopy requires a completely different method of sample preparation and sectioning, which translates into more time and more money. A first step in preparation is light microscopic immunocytochemistry to determine the correct antibody dilutions, the proper detergent, and the needed blocking agents. Then, as needed, move to electron microscopic immunocytochemistry with the same sample. 

Figure 19. Confocal micrograph of an MFT sample that was unfrozen and observed using the confocal optical technique. The orientation or structuring of the clay component is identical to that observed with the cryogenic sample preparation for the electron microscope (Figure 18). Scale bar, 100 pms. Photograph courtesy of V. A. Munoz.

In the present chapter, we discuss the principles and techniques commonly used for observing biological surface structures, including optical microscopy (light microscopy, laser scanning confocal microscopy), electron microscopy (scanning electron microscopy, transmission electron microscopy), and scanning probe microscopy. We describe and contrast the sample preparation of each technique. Quantitative data analysis as well as the limitations of each technique is also addressed. 

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