Pinhole shifting lifetime imaging microscopy.

Journal of Biomedical Optics
Venkat K Ramshesh, John J Lemasters

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 (Eu(3+)), 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 Eu(3+) 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 micros was estimated for the Eu(3+) microspheres, consistent wi...Continue Reading

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Citations

Oct 23, 2013·Inorganic Chemistry·Megha RajendranLawrence W Miller
Sep 9, 2010·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Nivriti Gahlaut, Lawrence W Miller
Jul 23, 2015·Biophysical Journal·Megha Rajendran, Lawrence W Miller
Jan 12, 2010·Photochemical & Photobiological Sciences : Official Journal of the European Photochemistry Association and the European Society for Photobiology·Michael P CooganDavid Lloyd
Dec 31, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Sebastiano Di PietroRanieri Bizzarri

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