Epifluorescence Microscopy

Research output: Chapter in Book/Report/Conference proceedingChapter


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.

Original languageEnglish (US)
Title of host publicationPrinciples of Light Microscopy
Subtitle of host publicationFrom Basic to Advanced
PublisherSpringer International Publishing
Number of pages19
ISBN (Electronic)9783031044779
ISBN (Print)9783031044762
StatePublished - Jan 1 2022


  • CCD cameras
  • CMOS cameras
  • Filters
  • Fluorescence microscopy
  • Fluorophores
  • Light engines
  • Photobleaching
  • Widefield microscopy

ASJC Scopus subject areas

  • General Medicine
  • General Agricultural and Biological Sciences


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