Title: Implementation and Application of Pulsed Interleaved Excitation for Dual-Color FCS and RICS
Authors: Hendrix, Jelle
Lamb, Don C #
Issue Date: 2014
Publisher: Humana Press, Inc.
Series Title: Methods in Molecular Biology vol:1076
Host Document: Fluorescence Spectroscopy and Microscopy: Methods and Protocols pages:653-682
Abstract: Pulsed interleaved excitation (PIE) employs pulsed laser sources that are interleaved such that differentially colored fluorophores can be measured or imaged quasi-simultaneously in the absence of spectral crosstalk. PIE improves the robustness and reduces data analysis complexity of many fluorescence techniques, such as fluorescence cross-correlation spectroscopy (FCCS) and raster image cross-correlation spectroscopy (ccRICS), two methods used for quantitative investigation of molecular interactions in vitro and in living cells. However, as PIE is most often used for fluorescence fluctuation spectroscopy and burst analysis experiments and utilizes time-correlated single-photon counting detection and advanced optoelectronics, it has remained a technique that is mostly used by specialized single-molecule research groups. This protocols chapter provides an accessible overview of PIE for anyone considering implementing the method on a homebuilt or commercial microscope. We give details on the instrumentation, data collection and analysis software, on how to properly set up and align a PIE microscope, and finally, on how to perform proper dual-color FCS and RICS experiments.
ISSN: 1064-3745
Publication status: published
KU Leuven publication type: IHb
Appears in Collections:Non-KU Leuven Association publications
# (joint) last author

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