Fluorescence Microscopy

Abstract

To harness the power of fluorescence for biological microscopic imaging, a number of additional components must be introduced to the standard light microscope. The most important modifications are (1) a strong illumination system that provides suitable wavelengths for exciting a specific fluorochrome, (2) some mechanism to limit the illumination beam to specific wavelengths so that only the fluorochrome of choice is excited, and (3) a means to image only the light emitted from the fluorochrome so that the excitation light (and other stray wavelengths) does not degrade the image. In this chapter, we will discuss a basic setup that meets these criteria. Subsequent chapters will expand on this theme and discuss additional modifications required for laser scanning and spinning disk confocal fluorescence. Additionally, in confocal imaging the signal generated by the sample is often more limited than in standard wide-field fluorescence. For this reason, understanding what limits the signal projected by the specimen and how to minimize signal loss is even more critical in confocal microscopy. In this chapter we review how to optimize instrument setup to minimize signal loss.