ABSTRACT
This chapter is devoted to techniques that rely on light absorption and subsequent fluorescence emission to generate images in which color variations create contrast. The chapter begins with a brief synopsis of the reasons that fluorescence microscopy dominates the life sciences. This is followed by a discussion of the quantum mechanical origins of fluorescence, absorption and emission bands, and the Stokes shift. Additional follow-up topics include (1) methods of staining samples so they are rendered fluorescent, and (2) optical components, like filters and dichromatic mirrors, that play a key role in the fluorescence microscope. The later sections of the chapter discuss the most basic form of fluorescence microscopy, termed widefield, and its primary weakness, which is image-degrading blur that arises due to the collection of both in-focus and out-of-focus light. The chapter concludes with a discussion of two prominent methods of reducing image blur: confocal microscopy, and deconvolution of blur-contaminated images.
