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James Feeks, Qiang Yang, Jennifer J Hunter; Cellular-scale fluorescence lifetime imaging of the retina in living mice. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2208.
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© 2017 Association for Research in Vision and Ophthalmology.
Fluorescence lifetime imaging microscopy has revolutionized biological imaging due to its ability to measure the rate of fluorescence decay, which is an intrinsic property of the fluorophore that is modified by its environment. Previous studies in ophthalmoscopy with low resolution and poor axial sectioning have taken advantage of this property, but a technique is needed which can longitudinally quantify changes in retinal micro-environment at cellular-scale in vivo. Here, we have assessed the feasibility of cellular-scale fluorescence lifetime imaging ophthalmoscopy (FLIO) using a two-photon adaptive optics scanning light ophthalmoscope (TPAOSLO).
A TPAOSLO was designed and constructed for the mouse eye. A polygon scanner replaced the resonance scanner to ensure linearity of the scanned beam. For FLIO, a single photon counting detector and time-correlated single photon counting (TCSPC) module (Becker&Hickl) were added to the fluorescence detection pathway. Thy1-EGFP mice were imaged in vivo to visualize enhanced green fluorescent protein (EGFP) in sparsely labeled ganglion cells. C57BL/6J and Thy1-EGFP mice were injected with sodium fluorescein to image vasculature. Images were acquired over 3 minutes using 6 mW of 910 nm light (70 fs, 80 MHz). Fluorescence lifetime decays were fit with exponential decay curves.
EGFP labeled ganglion cell somas and dendrites were resolved using the TCSPC technique. The fluorescence lifetime was bi-exponential (τ1 = 1.49 ± 0.27 ns (mean ± SD) and τ2 = 2.90 ± 0.19 ns), consistent with previous reports of EGFP lifetime. Fluorescein could be clearly identified in the vasculature and had a measured lifetime of 3.21 ± 0.06 ns. EGFP-labeled ganglion cells could be identified in an image dominated by fluorescein fluorescence due to the difference in lifetime.
We have demonstrated two-photon FLIO at a cellular scale in the living mouse eye. This method allows differentiation of spectrally-overlapping fluorophores and repeated longitudinal measurements of fluorescent markers. By using two-photon excitation, this technique is translatable to the primate eye, wherein the ocular transmission window inhibits single-photon excitation of key retinal fluorophores such as NADH and retinol. The use of high resolution FLIO could provide information about the micro-environment of the living retina, and may yield additional insight into the dynamics of disease progression.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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