Abstract

In this chapter, we review the imaging techniques and methods of molecular interrogation made possible by integrating laser light sources with microscopy. We discuss the advantages of exciting fluorescence by laser illumination and review commonly used laser-based imaging techniques such as confocal, multiphoton, and total internal reflection microcopy. We also discuss emerging imaging modalities based on intrinsic properties of biological macromolecules such as second harmonic generation imaging and coherent anti-Raman resonance spectroscopy. Super resolution techniques are presented that exceed the theoretical diffraction-limited resolution of a microscope objective. This chapter also focuses on laser-based techniques that can report biophysical parameters of fluorescently labeled molecules within living cells. Photobleaching techniques, fluorescence lifetime imaging, and fluorescence correlation methods can measure kinetic rates, molecular diffusion, protein-protein interactions, and concentration of a fluorophore-bound molecule. This chapter provides an introduction to the field of laser-based microscopy enabling readers to determine how best to match their research questions to the current suite of techniques. © 2007 Elsevier Inc. All rights reserved.