Abstract
A nonlinear optical (NLO) microscopy system integrating stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) was developed to image fresh mouse retinas. The morphological and functional details of various retinal layers were revealed by the endogenous NLO signals. Particularly, high resolution label-free imaging of retinal neurons and nerve fibers in the ganglion cell and nerve fiber layers was achieved by capturing endogenous SRS and TPEF signals. In addition, the spectral and temporal analysis of TPEF images allowed visualization of different fluorescent components in the retinal pigment epithelium (RPE). Fluorophores with short TPEF lifetime, such as A2E, can be differentiated from other long-lifetime components in the RPE. The NLO imaging method would provide important information for investigation of retinal ganglion cell degeneration and holds the potential to study the biochemical processes of visual cycle in the RPE.
Highlights
The retina is the light-sensitive tissue at the back of eye
Based on the previous findings that most of the two-photon excited fluorescence (TPEF) in the inner retina was contributed by endogenous NADH fluorescence, the excitation wavelength was set at 740 nm, which was optimal to excite NADH TPEF signal [12]
We demonstrate the application of multimodal label-free nonlinear optical (NLO) microscopy to extract the morphological and biochemical details in different retinal layers
Summary
The retina is the light-sensitive tissue at the back of eye. Its morphological and functional alterations are associated with a variety of ocular diseases, leading to a special interest in the study of its fine layered structures [1]. The innermost layers of the retina are the nerve fiber layer (NFL) and the ganglion cell layer (GCL). The retinal nerve fibers comprise the axons of retinal ganglion cells (RGCs). They relay electrical signals generated from the outer retina to the central nervous system. The inner nuclear layer (INL) contains a variety of neuronal cells including bipolar cells, amacrine cells and horizontal cells that function as a bridge connecting the photoreceptors in the outer retina and the RGCs in the inner retina. Similar to the IPL, the outer plexiform layer (OPL) is composed of a network of neuronal synapses connecting the bipolar cells and photoreceptors. A number of retinal diseases, for example, agerelated macular degeneration (AMD), retinitis pigmentosa, Best’s disease, Stargardt’s disease and Leber’s congenital amaurosis are associated with abnormalities in the RPE [4,5,6,7,8]
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