Laser-based measurements in cell biology.
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.
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ABSTRACT: Establishing the temporal and concentration profiles of neurotransmitters during synaptic release is an essential step towards understanding the basic properties of inter-neuronal communication in the central nervous system. A variety of ingenious attempts has been made to gain insights into this process, but the general inaccessibility of central synapses, intrinsic limitations of the techniques used, and natural variety of different synaptic environments have hindered a comprehensive description of this fundamental phenomenon. Here, we describe a number of experimental and theoretical findings that has been instrumental for advancing our knowledge of various features of neurotransmitter release, as well as newly developed tools that could overcome some limits of traditional pharmacological approaches and bring new impetus to the description of the complex mechanisms of synaptic transmission.Molecular Neurobiology 10/2009; 40(3):289-306. · 5.74 Impact Factor