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Erschienen in:
Modern TCSPC Electronics: Principles and Acquisition Modes Kapitel lesen Erstes Kapitel lesen

2015 | OriginalPaper | Buchkapitel

MFD-PIE and PIE-FI: Ways to Extract More Information with TCSPC

verfasst von : Anders Barth, Lena Voith von Voithenberg, Don C. Lamb

Erschienen in: Advanced Photon Counting

Verlag: Springer International Publishing

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Abstract

Pulsed interleaved excitation (PIE) is the method of fast alternation of pulsed lasers for quasi-simultaneous observation of fluorophores with different spectral properties. PIE was originally introduced to enable artifact-free fluorescence cross-correlation measurements, while first experiments with alternating laser excitation (ALEX) used the dual excitation of donor and acceptor for single-pair Förster resonance energy transfer (spFRET). In this article, we will review the benefit of PIE for spFRET experiments with multiparameter fluorescence detection (MFD). The direct probing of the acceptor fluorophore in PIE increases the robustness of the quantitative MFD analysis and extends it to even more parameters.
Recently, PIE has been combined with commonly used fluorescence fluctuation imaging techniques such as raster image correlation spectroscopy (RICS) and the number and brightness analysis (N&B). We highlight how PIE improves these methods, and how artifacts in the analysis can be avoided. Similar to PIE-FCS, quantitative cross-correlation raster image correlation spectroscopy (ccRICS) is greatly simplified. Additionally, the lifetime information can be used to further increase the contrast and sensitivity of the method with raster lifetime image correlation spectroscopy (RLICS).

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Vorheriges Kapitel Lifetime-Weighted FCS and 2D FLCS: Advanced Application of Time-Tagged TCSPC
Nächstes Kapitel Photon Antibunching in Single Molecule Fluorescence Spectroscopy
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Metadaten
Titel
MFD-PIE and PIE-FI: Ways to Extract More Information with TCSPC
verfasst von
Anders Barth
Lena Voith von Voithenberg
Don C. Lamb
Copyright-Jahr
2015
Buch
Advanced Photon Counting

Print ISBN: 978-3-319-15635-4

Electronic ISBN: 978-3-319-15636-1

Copyright-Jahr: 2015

DOI
https://doi.org/10.1007/4243_2014_66

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