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Pengfei Zhang, Azhar Zam, Edward Pugh, Robert J Zawadzki; In vivo low light level mouse retinal SLO imaging: limits for backscattered intensity and autofluorescense detection. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4116. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate feasibility of a custom mouse scanning light ophthalmoscope (SLO) for low light level (below 1µW) mouse retinal imaging. Low light imaging is desired to reduce potential deleterious effects of light-tissue interactions, including heating and photochemical reactions (e.g., reactive oxygen species; retinoid byproducts from pigment bleaching).
A custom widefield (~50 deg VA) high-resolution retinal imaging SLO with a 0 Dpt contact lens (affixed to the SLO optics) was used to simultaneously acquire both backscattered intensity as well as autofluorescense signal from mouse retina. Different scanning speeds and light levels were tested to find the minimum light power that gives good quality images. 469 nm light from a Fianium sc400 Supercontinuum laser source was used for imaging and a 503 nm long-pass filter was used to select retinal autofluorescense. The SLO detection system consisted of a Hamamatsu PMT (H7422-20) for reflected light and H7422-40 for fluorescense and Femto preamplifiers (107 gain), and the sampling rate is 500kHz. Custom Labview acquisition software allowed selection of the sampling region (ROI) and frame rate. Pigmented (C57BL/6) and albino (BALB/cJ) mice from Jackson Laboratory were used as subjects. During imaging, mice were anesthetized with 2-3% inhalational isoflurane anesthetic. Pupils were dilated and cyclopleged with Tropicamide and phenylephrine.
Example images of mouse retina (BALB/cJ, 13 mo) acquired simultaneously in reflectance and fluorescence channels during a single imaging session with different light levels are presented (Fig. 1). The data were acquired with pixel sample time of 6 μs and sampling density of 256 x 256 pixels, resulting in frame rate of ~2Hz. Control gain voltage of the PMTs was adjusted to match the dynamic range of the DAQ during imaging.
Low light level mouse retinal imaging is essential to allow study of physiological processes of the retina in vivo without adversely affecting retinal structure and function by the probing beam itself. Our data suggest that low light level SLO is feasible for mouse retinal imaging. In particular, light levels below 1µW can be used during mouse positioning, keeping light exposure to minimum.
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