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.
Cardiac optical mapping currently provides 2-D maps of transmembrane voltage-sensitive fluorescence localized near the tissue surface. Methods for interrogation at different depths are required for studies of arrhythmias and the effects of defibrillation shocks in 3-D cardiac tissue. We model the effects of coloading with a dye that absorbs excitation or fluorescence light on the radius and depth of the interrogated region with specific illumination and collection techniques. Results indicate radii and depths of interrogation are larger for transillumination versus epi-illumination, an effect that is more pronounced for broad-field excitation versus laser scanner. Coloading with a fluorescence absorber lessens interrogated depth for epi-illumination and increases it for transillumination, which is confirmed with measurements using transillumination of heart tissue slices. Coloading with an absorber of excitation light consistently decreases the interrogated depths. Transillumination and coloading also decrease the intensities of collected fluorescence. Thus, localization can be modified with wavelength-specific absorbers at the expense of a reduction in fluorescence intensity.
Limited optical interrogation depth prevents studies of arrhythmias in 3D tissue. Our experiments with single photon excitation and computer simulations with single and two-photon excitation show that the addition of a fluorescence absorber can modify the region of tissue interrogated with transillumination. Experiments were performed with a pair of 0.1 cm thick rabbit cardiac slices. The slices were excited with 488 nm single photon laser excitation while light from opposite side was collected for transillumination. One slice was stained with di-4-ANEPPS while the other was not. In different measurements, both slices were also stained with the red light absorbing dye, blue 1. In our models fluorescent photons were launched from specific regions of the tissue to mimic staining of different layers with di-4-ANEPPS. In additional simulations the fluorescence absorption coefficient was increased 3-fold. In our experiments with the di-4-ANEPPS slice facing away from the laser, measured light intensity was 68% of the value found with di-4-ANEPPS slice facing the laser while it was 21% in the model. This reduction in intensity from the deeper layer became less pronounced after addition of the red absorber. Then with di-4-ANEPPS slice facing away from the laser the measured light intensity was 81% of the value found with di-4-ANEPPS slice facing the laser in experiments while it was 34% in the model. This indicates deepening of the interrogated region by addition of red absorbing dye. In computer simulations using two-photon excitation at 1064 nm, increasing the fluorescence absorption further deepend the interrogated region compared with one photon excitation at 488 nm.
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