Luminescence Lifetime Imaging Microscopy: The Lemasters laboratory has developed a time-resolved imaging of long lifetime luminescence technique 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 the pinhole is selectively shifted in the lagging direction with respect to the rasting laser spot short lifetime luminescence disappeares much more rapidly than the long life time luminescence. Figure 1 shows a schemactic of the principle of this technique. Figure 2 shows acquired images of europium (Eu), a red emitting probe with short lifetime green-fluorescing microspheres and/or fluorescein and rhodamine in solution. Using 720-nm two-photon excitation and a pinhole diameter of one Airy unit, short lifetime fluorescence of fluorescein, rhodamine and the green microspheres disappeared much more rapidly than the long life time phosphorescence of Eu microspheres as the pinhole was repositioned in the lagging direction. By contrast, repositioning of the pinhole in the leading and orthogonal directions caused equal loss of short and long lifetime luminescence. This simple adaptation is the basis for quantitative 3-dimensional luminescence lifetime imaging microscopy. We have named this technique as luminescence lifetime imaging microscopy by confocal pinhole shifting (LLIM-CPS).
Figure 1. Principle of phosphorescence lifetime imaging microscopy by confocal pinhole shifting. When the detection pinhole is aligned to the crossover of the rasting laser beam (a), short lifetime luminescence passes the pinhole to the photodetector. When the pinhole is shifted in the lagging direction with respect to the rasting laser spot (b), short lifetime luminescence is rejected but delayed long lifetime luminescence is collected instead.
Figure 2. Confocal images of long lifetime europium microspheres and short lifetime green microspheres, rhodamine and fluorescein. In A, 1-µm europium microspheres were imaged with 1-µm green microspheres. In B, europium microspheres were imaged with rhodamine (400 µM) in solution. In C, europium microspheres were imaged with fluorescein (400 µM) in solution. The center column shows images obtained with the pinholes aligned to the laser spot. The left column shows images obtained with pinholes shifted in the lagging direction relative the rasting laser spot by 1 Airy unit. The right column shows images obtained with pinholes shifted by 1 Airy unit in the leading direction.