title: Pinhole shifting lifetime imaging microscopy.
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 with independent measurements. This simple adaptation is the basis for quantitative 3-D lifetime imaging microscopy.
authors.affiliation: Medical University of South Carolina, Center for Cell Death, Injury and Regeneration and Hollings Cancer Center, Charleston, South Carolina 29425, USA.
authors.forename: Venkat K
authors.initials: VK
authors.lastname: Ramshesh
authors.forename: John J
authors.initials: JJ
authors.lastname: Lemasters
mesh_terms: D017076:Computer-Aided Design; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007089:Image Enhancement / Q000295:instrumentation* / Q000379:methods; D008163:Luminescent Measurements / Q000295:instrumentation* / Q000379:methods; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods; D019047:Phantoms, Imaging; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013382:Subtraction Technique / Q000295:instrumentation*