Cite as: Cold Spring Harb. Protoc.; 2006; doi:10.1101/pdb.ip16

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information_panelInformation Panel

An Experimental Setup for Frequency Domain FLIM

Peter J. Verveer, Oliver Rocks, Ailsa G. Harpur, and Philippe I.H. Bastiaens

This information panel was adapted from "Imaging Protein Interactions by FRET Microscopy," Chapter 32, in Protein-Protein Interactions (ed. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

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The following are key elements of a frequency domain fluorescence lifetime imaging system: (1) a standing wave acousto-optic modulator (SW-AOM) that modulates the intensity of the excitation light source at high frequency and (2) a frequency mixing device, such as an image intensifier, to perform phase-sensitive detection of the fluorescence emission. Figure 1 shows a single-frequency FLIM configuration. The instrument is based around a vibrationally isolated, inverted microscope (e.g., Zeiss Axiovert 135 TV). The basic light source requirement is an argon laser with sufficient output power (>100 mW per line). The 457-nm, 488.0-nm, and 514.5-nm lines are ideal for excitation . . . [Full Text of this Article]

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