Multimodal photoacoustic ophthalmoscopy in mouse - PubMed (original) (raw)

Multimodal photoacoustic ophthalmoscopy in mouse

Wei Song et al. J Biophotonics. 2013 Jun.

Abstract

Photoacoustic ophthalmoscopy (PAOM) is a novel imaging technology that measures optical absorption in the retina. The capability of PAOM can be further enhanced if it could image mouse eyes, because mouse models are widely used for various retinal diseases. The challenges in achieving high-quality imaging of mouse retina, however, come from the much smaller eyeball size. Here, we report an optimized imaging system, which integrates PAOM, spectral-domain optical coherence tomography (SD-OCT), and autofluorescence-scanning laser ophthalmoscopy (AF-SLO), for mouse eyes. Its multimodal capability was demonstrated by imaging transgenic Nrl-GFP mice that express green fluorescent protein (GFP) in photoreceptors. SD-OCT provided guidance of optical alignment for PAOM and AF-SLO, and complementary contrast with high depth-resolution retinal cross sections. PAOM visualized the retinal vasculature and retinal pigment epithelium melanin, and AF-SLO measured GFP-expressing in retinal photoreceptors. The in vivo imaging results were verified by histology and confocal microscopy.

Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Figures

Figure 1

Figure 1

Schematic diagram of the multimodal retinal imaging system. SLED: superluminescent laser diode; Ref: OCT reference arm; 2×2: 50:50 optical fiber coupler; SPEC: spectrometer; BS: beam splitter; DM: dichroic mirror; HM; hot mirror; GM: 2D galvanometer mirrors; AMP: amplifier; UT: ultrasonic transducer; PD: photodiode; APD: avalanche photodetector.

Figure 2

Figure 2

In vivo multimodal retinal imaging. (a) and (d) are en face SD-OCT images; (b) and (e) are PAOM fundus images; (c) and (f) are AF-SLO fundus images. The images on the left are acquired from a transgenic Nrl-GFP mouse and the images on the right are acquired from a control mouse. The arrows in Figure 2b point out some smaller vessel shadows that were visible in PAOM but invisible in SD-OCT and AF-SLO. Bar: 300 μm.

Figure 3

Figure 3

Comparison of (a) SD-OCT cross-section, (b) PAOM B-scan, and (c) 1D AF-SLO profile acquired from the same position. RNFL/GC: retinal nerve fiber layer and ganglion cell layer; IP: inner plexiform layer; IN: inner nuclear layer; OP: outer plexiform layer; ON: outer nuclear layer; ELM: external limiting membrane; IS/OS: the inner and outer segment of the photoreceptors; RPE/C: the complex of retinal pigment epithelium and choroid. The arrows pointing out the four retinal vessels show good registration among the three imaging modalities. Bar: 100 μm.

Figure 4

Figure 4

Retinal histological of the (a) GFP-expressing eye and the (b) control eye. The anatomic retinal layers were resolved by DAPI staining (blue) in both eyes. The turquoise band locates in ONL represents GFP expression. GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; ONL: outer nuclear layer. The locations pointed out by arrows indicate no GFP expression. Bar: 50 μm.

Figure 5

Figure 5

Comparison between ex vivo flat-mount confocal microscopy results and the in vivo results. (a) Confocal microscopy of the whole-mount retina stained by Brn-3b. The trace of major retinal vessels is labeled by numbers. (b) Confocal microscopy of GFP expression in the whole-mount retina. The photoreceptors with GFP expression appeared as bright turquoise regions. (c) Overlaid in vivo PAOM and AF-SLO images within the region highlighted by the dashed-box in Figure. 2b. The retinal vessels in PAOM are pseudo-colored in red and the AF-SLO image is pseudo-colored in green. Representative photoreceptors in absence of GFP were highlighted by arrows. Bar: 100 μm.

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