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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 15, Iss. 20 — Oct. 1, 2007
  • pp: 12548–12561

Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging

S. Kumar, C. Dunsby, P. A. A. De Beule, D. M. Owen, U. Anand, P. M. P. Lanigan, R. K. P. Benninger, D. M. Davis, M. A. A. Neil, P. Anand, C. Benham, A. Naylor, and P. M. W. French  »View Author Affiliations

Optics Express, Vol. 15, Issue 20, pp. 12548-12561 (2007)

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We report a multifocal multiphoton time-correlated single photon counting (TCSPC) fluorescence lifetime imaging (FLIM) microscope system that uses a 16 channel multi-anode PMT detector. Multiphoton excitation minimizes out-of-focus photobleaching, multifocal excitation reduces non-linear in-plane photobleaching effects and TCSPC electronics provide photon-efficient detection of the fluorescence decay profile. TCSPC detection is less prone to bleaching- and movement-induced artefacts compared to wide-field time-gated or frequency-domain FLIM. This microscope is therefore capable of acquiring 3-D FLIM images at significantly increased speeds compared to single beam multiphoton microscopy and we demonstrate this with live cells expressing a GFP tagged protein. We also apply this system to time-lapse FLIM of NAD(P)H autofluorescence in single live cells and report measurements on the change in the fluorescence decay profile following the application of a known metabolic inhibitor.

© 2007 Optical Society of America

OCIS Codes
(110.0180) Imaging systems : Microscopy
(180.2520) Microscopy : Fluorescence microscopy

ToC Category:

Original Manuscript: August 9, 2007
Revised Manuscript: September 6, 2007
Manuscript Accepted: September 11, 2007
Published: September 17, 2007

Virtual Issues
Vol. 2, Iss. 11 Virtual Journal for Biomedical Optics

S. Kumar, C. Dunsby, P. A. A. De Beule, D. M. Owen, U. Anand, P. M. P. Lanigan, R. K. P. Benninger, D. M. Davis, M. A. A. Neil, P. Anand, C. Benham, A. Naylor, and P. M. W. French, "Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging," Opt. Express 15, 12548-12561 (2007)

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