Epifluorescence Microscopy

In previous chapters, we have seen that the interaction of light with matter produces one or a combination of the following phenomena: transmission, absorption, reflection, scattering and diffraction, refraction and polarization, phase change and fluorescence emission [1]. Each one of these effects can be used to generate contrast and hence create an image. In this chapter, we will discuss the light-matter interaction that leads to the absorption of a photon and the subsequent emission of a photon with lower energy: Fluorescence. We will explore its principles, advantages over classic bright field techniques, limitations and some of its main applications in life and material sciences. By providing technical analysis as well as a step-by-step protocol, the reader will be able to understand the concept of fluorescence microscopy, get an introduction to labelling techniques, understand the components of a fluorescence microscope and learn how to design and set up experiments with the optimal compromise between Acquisition Speed, Signal-to-Noise Ratio and Resolution. The chapter will be divided into four sections: theoretical aspects of fluorescence microscopy, microscope setup, sample preparation and key applications of widefield fluorescence microscopy.