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Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 21, Iss. 10 — May. 20, 2013
  • pp: 11769–11782

High speed multispectral fluorescence lifetime imaging

Farzad Fereidouni, Keimpe Reitsma, and Hans C. Gerritsen  »View Author Affiliations


Optics Express, Vol. 21, Issue 10, pp. 11769-11782 (2013)
http://dx.doi.org/10.1364/OE.21.011769


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Abstract

We report a spectrally resolved fluorescence lifetime imaging system based on time gated single photon detection with a fixed gate width of 200 ps and 7 spectral channels. Time gated systems can operate at high count rates but usually have large gate widths and sample only part of the fluorescence decay curve. In the system presented in this work, the fluorescence signal is sampled using a high speed transceiver. An error analysis is carried out to characterize the performance of both lifetime and spectral detection. The effect of gate width and spectral channel width on the accuracy of estimated lifetimes and spectral widths is described. The performance of the whole instrument is evaluated at count rates of up to 12 MHz. Accurate fluorescence lifetimes (error < 2%) are recorded at count rates as high as 5 MHz. This is limited by the PMT performance, not by the electronics. Analysis of the large spectral lifetime image sets is challenging and time-consuming. Here, we demonstrate the use of lifetime and spectral phasors for analyzing images of fibroblast cells with 2 different labeled components. The phasor approach provides a fast and intuitive way of analyzing the results of spectrally resolved fluorescence lifetime imaging experiments.

© 2013 OSA

OCIS Codes
(030.5260) Coherence and statistical optics : Photon counting
(110.2960) Imaging systems : Image analysis
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(170.6920) Medical optics and biotechnology : Time-resolved imaging
(110.4234) Imaging systems : Multispectral and hyperspectral imaging

ToC Category:
Imaging Systems

History
Original Manuscript: March 15, 2013
Revised Manuscript: April 25, 2013
Manuscript Accepted: April 26, 2013
Published: May 7, 2013

Virtual Issues
Vol. 8, Iss. 6 Virtual Journal for Biomedical Optics

Citation
Farzad Fereidouni, Keimpe Reitsma, and Hans C. Gerritsen, "High speed multispectral fluorescence lifetime imaging," Opt. Express 21, 11769-11782 (2013)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-21-10-11769


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