Research Papers

Pinhole shifting lifetime imaging microscopy

[+] Author Affiliations
Venkat K. Ramshesh

Medical University of South Carolina, Center for Cell Death, Injury and Regeneration and Hollings Cancer Center, Charleston, South Carolina 29425

John J. Lemasters

Medical University of South Carolina, Center for Cell Death, Injury and Regenerationand Departments of Pharmaceutical Sciences and Biochemistry & Molecular Biology and Hollings Cancer Center, Charleston, South Carolina 29425

J. Biomed. Opt. 13(6), 064001 (December 08, 2008). doi:10.1117/1.3027503
History: Received January 30, 2008; Revised August 12, 2008; Accepted August 15, 2008; Published December 08, 2008
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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 270μs was estimated for the Eu3+ microspheres, consistent with independent measurements. This simple adaptation is the basis for quantitative 3-D lifetime imaging microscopy.

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© 2008 Society of Photo-Optical Instrumentation Engineers

Citation

Venkat K. Ramshesh and John J. Lemasters
"Pinhole shifting lifetime imaging microscopy", J. Biomed. Opt. 13(6), 064001 (December 08, 2008). ; http://dx.doi.org/10.1117/1.3027503


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