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Confocal Fluorescence Microscopy

Optical Imaging and Spectroscopy

  1. Oleg D. Lavrentovich

Published Online: 25 JUN 2012

DOI: 10.1002/0471266965.com127

Characterization of Materials

Characterization of Materials

How to Cite

Lavrentovich, O. D. 2012. Confocal Fluorescence Microscopy. Characterization of Materials. 1–15.

Author Information

  1. Kent State University, Kent, OH, USA

Publication History

  1. Published Online: 25 JUN 2012

Abstract

This article describes basic approaches to image materials by means of optical microscopy with fluorescence markers. The fluorescence microscopy is used to image materials in which the contrast cannot be achieved by light absorption, scattering, or birefringence. The fluorescence microscopy benefits greatly from a confocal mode of observation that eliminates signal coming from outside the small region of sample that is probed by a tightly focused laser beam. By scanning the focused laser beamthrough the sample, one obtains a 3D map of density of fluorescing molecules staining the sample. This technique is called confocal laser scanning microscopy (CLSM). By using fluorescent markers of anisometric shape and a polarized laser beam, one can produce 3D images of orientationally ordered materials such as liquid crystals. In this technique, called a fluorescent confocal polarizing microscopy (FCPM), the signal depends on the angle between the transition dipole of the dye and the polarization of probing light. Recent advances in the development of fluorescent markers that can be controllably switched between the bright state and the dark state lead to revolutionary development of far-field optical microscopy with resolution well below the diffraction limit.

Keywords:

  • fluorescence;
  • confocal laser scanning microscopy;
  • fluorescent confocal polarizing microscopy;
  • Nipkow disk