Pinhole shifting lifetime imaging microscopy

Venkat K. Ramshesh; John James Lemasters

doi:10.1117/1.3027503

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.

Citation Download Citation

Venkat K. Ramshesh, Venkat K. Ramshesh, John James Lemasters, John James Lemasters, } "Pinhole shifting lifetime imaging microscopy," Journal of Biomedical Optics 13(6), 064001 (1 November 2008). https://doi.org/10.1117/1.3027503 . Submission:

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