Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina

Abstract

Adaptive optics scanning laser ophthalmoscopy (AO-SLO) has recently been used to achieve exquisite subcellular resolution imaging of the mouse retina. Wavefront sensing-based AO typically restricts the field of view to a few degrees of visual angle. As a consequence the relationship between AO-SLO data and larger scale retinal structures and cellular patterns can be difficult to assess. The retinal vasculature affords a largescale 3D map on which cells and structures can be located during in vivo imaging. Phase-variance OCT (pv-OCT) can efficiently image the vasculature with near-infrared light in a label-free manner, allowing 3D vascular reconstruction with high precision. We combined widefield pv-OCT and SLO imaging with AO-SLO reflection and fluorescence imaging to localize two types of fluorescent cells within the retinal layers: GFPexpressing microglia, the resident macrophages of the retina, and GFPexpressing cone photoreceptor cells. We describe in detail a reflective afocal AO-SLO retinal imaging system designed for high resolution retinal imaging in mice. The optical performance of this instrument is compared to other state-of-the-art AO-based mouse retinal imaging systems. The spatial and temporal resolution of the new AO instrumentation was characterized with angiography of retinal capillaries, including blood-flow velocity analysis. Depth-resolved AO-SLO fluorescent images of microglia and cone photoreceptors are visualized in parallel with 469 nm and 663 nm reflectance images of the microvasculature and other structures. Additional applications of the new instrumentation are discussed.

Whole-body positional manipulators for ocular imaging of anaesthetised mice and rats: a do-it-yourself guide

“…[20][21][22] The high resolution of third-generation spectral-domain OCT devices renders in vivo retinal imaging in mice and rats possible, gaining an increasing importance in ophthalmological and neurological preclinical research. [23][24][25][26][27][28][29][30][31][32][33] The obtained results are in good accordance with histological sections of the animals' retinae. 34 The application of OCT technology in rodent models, however, is still challenging, mainly because of the small size of the rodents' eyes.…”

In vivowide-field multispectral scanning laser ophthalmoscopy–optical coherence tomography mouse retinal imager: longitudinal imaging of ganglion cells, microglia, and Müller glia, and mapping of the mouse retinal and choroidal vasculature

“…Clearly, further studies with fluorescent reporters expressed in other cells types and possibly images of microglia with higher resolution AO-based imaging systems will be needed to determine the nature of this pathology. 49 This result nonetheless provides an example of the potential power of en face intensity projections of the different retinal layers for fast phenotypic multimodal screening of experimental animals. Notably, these scattering structures are not readily seen in Bscans, because they do not have a distinctive cross-sectional profile.…”

In vivo optophysiology reveals that G-protein activation triggers osmotic swelling and increased light scattering of rod photoreceptors

“…Mouse rod outer segments (OS) are in effect genetically manipulable, femtoliter test tubes whose protein constituents (15,16), as well as their biochemical reactions (17)(18)(19) and light-stimulated translocations (20)(21)(22), are well known and have been measured primarily with ex vivo methods. Advances in imaging technology have now made it possible to achieve subcellular-resolution imaging of the mouse retina in vivo (23)(24)(25)(26). We recently developed a multimodal ocular imaging system for mice that combines confocal laser scanning ophthalmoscopy (SLO) and OCT (27) and used this system to quantify photoactivation (bleaching) of rhodopsin in vivo (28).…”