Methods in Cellular Imaging
Other Advanced Microscopy Methods for Cellular Imaging
We have seen in the previous chapters that fluorescence microscopy methods have become the standard approach for cellular imaging and studying the functional activity of cells with high temporal and spatial resolution. The microscopic techniques listed in this section are used for different kinds of biological investigations. Multicolor fluorescence should become routine, especially with the development of novel fluorescent reagents with reduced photobleaching and large and variable Stokes shifts. Chapter 20 describes recent technological improvements to image biological specimens by spectral methods with microscopic spatial resolution. Spectral imaging could improve molecular–biological procedures by automatic detection such as the mapping of brain oxygenation and the targeting of cellular components such as actin, endosomes, mitochondria and much more in a mouse 3T3 fibroblast cells. Total internal reflection fluorescence (TIRF) microscopy provides a means to selectively excite in an aqueous or cellular environment without exciting fluorescence from regions farther from the surface (Chapter 21). Theory and optical configurations of TIRF are discussed in this chapter. TIRF can be used for many biological applications such as to observe the position, extent, composition, and motion of cell substrate contact regions, single molecule studies, measurements of the kinetic rates of binding of extra-cellular and intracellular proteins to cell surface receptors and artificial membranes and much more.
A laser trap is a beam of laser light brought to a diffraction-limited focus through a microscope objective. At the focal point, a translucent particle or a living cell may be captured, held, and manipulated. Chapter 22 clearly summarizes the knowledge of laser traps, indicates when the technique is appropriate for certain applications, and points out common problems and their solutions. The physics and fundamental limitations of laser traps in cell biology are also discussed.
The bioluminescence method is widely used for monitoring gene expression in living cells and tissues (Chapter 23). Bioluminescence provides additional advantages in the ability to couple enzyme reactions to luciferase. This chapter outlines several bioluminescent reporter genes currently used in live cells and mammalian nervous systems. Also, it explains the instrumentation involved for bioluminescence imaging. Chapter 24 describes the instrumentation involved in atomic force microscopy (AFM) imaging and the details of cellular and molecular level applications. AFM provides higher resolution on the atomic scale than other microscopic techniques described in this book for cellular imaging.