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Journal of Biomedical Optics

Pinhole shifting lifetime imaging microscopy
Author(s): Venkat K. Ramshesh; John James Lemasters
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Paper Abstract

Lifetime imaging microscopy is a powerful tool to probe biological phenomena independent of luminescence intensity and fluorophore concentration. We describe time-resolved imaging of long-lifetime luminescence with an unmodified commercial laser scanning confocal/multiphoton microscope. The principle of the measurement is displacement of the detection pinhole to collect delayed luminescence from a position lagging the rasting laser beam. As proof of principle, luminescence from microspheres containing europium (Eu3+), a red emitting probe, was compared to that of short-lifetime green-fluorescing microspheres and/or fluorescein and rhodamine in solution. Using 720-nm two-photon excitation and a pinhole diameter of 1 Airy unit, the short-lifetime fluorescence of fluorescein, rhodamine and green microspheres disappeared much more rapidly than the long-lifetime phosphorescence of Eu3+ microspheres as the pinhole was repositioned in the lagging direction. In contrast, repositioning of the pinhole in the leading and orthogonal directions caused equal loss of short- and long-lifetime luminescence. From measurements at different lag pinhole positions, a lifetime of 270 μs was estimated for the Eu3+ microspheres, consistent with independent measurements. This simple adaptation is the basis for quantitative 3-D lifetime imaging microscopy.

Paper Details

Date Published: 1 November 2008
PDF: 10 pages
J. Biomed. Opt. 13(6) 064001 doi: 10.1117/1.3027503
Published in: Journal of Biomedical Optics Volume 13, Issue 6
Show Author Affiliations
Venkat K. Ramshesh, Medical Univ. of South Carolina (United States)
John James Lemasters, Medical Univ. of South Carolina (United States)

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