Chapter 14 Quantitative Fluorescence Microscopy Using Photomultiplier Tubes and Imaging Detectors

Abstract

This chapter describes the quantitative fluorescence microscopy using photomultiplier tubes and imaging detectors. The most typical geometry for fluorescence microscopy is the epi-illumination geometry, where the exciting light is delivered to the sample through the objective lens. The main role of lenses in the illumination light path is to deliver the light to the limiting aperture and control its uniformity. Light rays emitted from the source are collected by a focusable lens or group of lenses called the collector lens. In the epifluorescence microscope, the objective functions as a condenser. This relationship yields a gain in brightness as a function of the fourth power of the numerical aperture of the objective. The choice of objective lens is critical for quantitative fluorescence microscopy. Each objective used must have high transmission efficiency and contribute no fluorescence from its internal parts. In most applications there are two additional criteria, high light collection efficiency and good resolution. The returning fluorescence signal in a conventional epifluorescence microscope must pass the beam splitter and any intermediated optics used by the manufacturer to control the internal light path.