Abstract

While fluorescent contrast is widely used in ophthalmology, three-dimensional (3D) fluorescence retinal imaging over a large field of view (FOV) has been challenging. In this paper, we describe a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging with only one raster scan. The technique utilizes scanned oblique illumination and angled detection to obtain fluorescent cross-sectional images, analogous to optical coherence tomography (OCT) line scans (or B-scans). By breaking the coaxial optical alignment used in conventional retinal imaging modalities, depth resolution is drastically improved. To demonstrate the capability of oSLO, we have performed in vivo volumetric fluorescein angiography (FA) of the rat retina with ~25μm depth resolution and over a 30° FOV. Using depth segmentation, oSLO can obtain high contrast images of the microvasculature down to single capillaries in 3D. The multi-modal nature of oSLO also allows for seamless combination with simultaneous OCT angiography.

© 2017 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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  31. R. S. Shah, B. T. Soetikno, J. Yi, W. Liu, D. Skondra, H. F. Zhang, and A. A. Fawzi, “Visible-Light Optical Coherence Tomography Angiography for Monitoring Laser-Induced Choroidal Neovascularization in Mice,” Invest. Ophthalmol. Vis. Sci. 57(9), OCT86–OCT95 (2016).
    [PubMed]
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    [PubMed]
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    [PubMed]
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    [PubMed]
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2017 (2)

C.-L. Chen and R. K. Wang, “Optical coherence tomography based angiography [Invited],” Biomed. Opt. Express 8(2), 1056–1082 (2017).
[PubMed]

L. Zhang, A. Capilla, W. Song, G. Mostoslavsky, and J. Yi, “Oblique scanning laser microscopy for simultaneously volumetric structural and molecular imaging using only one raster scan,” Sci. Rep. 7(1), 8591 (2017).
[PubMed]

2016 (7)

R. S. Shah, B. T. Soetikno, J. Yi, W. Liu, D. Skondra, H. F. Zhang, and A. A. Fawzi, “Visible-Light Optical Coherence Tomography Angiography for Monitoring Laser-Induced Choroidal Neovascularization in Mice,” Invest. Ophthalmol. Vis. Sci. 57(9), OCT86–OCT95 (2016).
[PubMed]

M. Zhang, T. S. Hwang, J. P. Campbell, S. T. Bailey, D. J. Wilson, D. Huang, and Y. Jia, “Projection-resolved optical coherence tomographic angiography,” Biomed. Opt. Express 7(3), 816–828 (2016).
[PubMed]

M. Cua, D. J. Wahl, Y. Zhao, S. Lee, S. Bonora, R. J. Zawadzki, Y. Jian, and M. V. Sarunic, “Coherence-Gated Sensorless Adaptive Optics Multiphoton Retinal Imaging,” Sci. Rep. 6, 32223 (2016).
[PubMed]

A. S. Bar-Noam, N. Farah, and S. Shoham, “Correction-free remotely scanned two-photon in vivo mouse retinal imaging,” Light: Science &Amp; Applications 5, e16007 (2016).

N. S. Alexander, G. Palczewska, P. Stremplewski, M. Wojtkowski, T. S. Kern, and K. Palczewski, “Image registration and averaging of low laser power two-photon fluorescence images of mouse retina,” Biomed. Opt. Express 7(7), 2671–2691 (2016).
[PubMed]

S. Mo, B. Krawitz, E. Efstathiadis, L. Geyman, R. Weitz, T. Y. P. Chui, J. Carroll, A. Dubra, and R. B. Rosen, “Imaging Foveal Microvasculature: Optical Coherence Tomography Angiography Versus Adaptive Optics Scanning Light Ophthalmoscope Fluorescein Angiography,” Invest. Ophthalmol. Vis. Sci. 57(9), OCT130 (2016).
[PubMed]

M. B. Sikkel, S. Kumar, V. Maioli, C. Rowlands, F. Gordon, S. E. Harding, A. R. Lyon, K. T. MacLeod, and C. Dunsby, “High speed sCMOS-based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes,” J. Biophotonics 9(3), 311–323 (2016).
[PubMed]

2015 (7)

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. C. Hillman, “Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms,” Nat. Photonics 9(2), 113–119 (2015).
[PubMed]

R. J. Zawadzki, P. Zhang, A. Zam, E. B. Miller, M. Goswami, X. Wang, R. S. Jonnal, S.-H. Lee, D. Y. Kim, J. G. Flannery, J. S. Werner, M. E. Burns, and E. N. Pugh., “Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina,” Biomed. Opt. Express 6(6), 2191–2210 (2015).
[PubMed]

P. Zhang, A. Zam, Y. Jian, X. Wang, Y. Li, K. S. Lam, M. E. Burns, M. V. Sarunic, E. N. Pugh, and R. J. Zawadzki, “In vivo wide-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,” J. Biomed. Opt. 20(12), 126005 (2015).
[PubMed]

P. Stremplewski, K. Komar, K. Palczewski, M. Wojtkowski, and G. Palczewska, “Periscope for noninvasive two-photon imaging of murine retina in vivo,” Biomed. Opt. Express 6(9), 3352–3361 (2015).
[PubMed]

T. E. de Carlo, A. Romano, N. K. Waheed, and J. S. Duker, “A review of optical coherence tomography angiography (OCTA),” Int J Retina Vitreous 1(1), 5 (2015).
[PubMed]

A. Ishibazawa, T. Nagaoka, A. Takahashi, T. Omae, T. Tani, K. Sogawa, H. Yokota, and A. Yoshida, “Optical Coherence Tomography Angiography in Diabetic Retinopathy: A Prospective Pilot Study,” Am. J. Ophthalmol. 160(1), 35–44 (2015).
[PubMed]

S. Chen, J. Yi, and H. F. Zhang, “Measuring oxygen saturation in retinal and choroidal circulations in rats using visible light optical coherence tomography angiography,” Biomed. Opt. Express 6(8), 2840–2853 (2015).
[PubMed]

2014 (4)

J. Yi, S. Chen, V. Backman, and H. F. Zhang, “In vivo functional microangiography by visible-light optical coherence tomography,” Biomed. Opt. Express 5(10), 3603–3612 (2014).
[PubMed]

Z. Zhi, J. R. Chao, T. Wietecha, K. L. Hudkins, C. E. Alpers, and R. K. Wang, “Noninvasive Imaging of Retinal Morphology and Microvasculature in Obese Mice Using Optical Coherence Tomography and Optical Microangiography,” Invest. Ophthalmol. Vis. Sci. 55(2), 1024–1030 (2014).
[PubMed]

Y. Jia, S. T. Bailey, D. J. Wilson, O. Tan, M. L. Klein, C. J. Flaxel, B. Potsaid, J. J. Liu, C. D. Lu, M. F. Kraus, J. G. Fujimoto, and D. Huang, “Quantitative Optical Coherence Tomography Angiography of Choroidal Neovascularization in Age-Related Macular Degeneration,” Ophthalmology 121(7), 1435–1444 (2014).
[PubMed]

T. Y. P. Chui, M. Dubow, A. Pinhas, N. Shah, A. Gan, R. Weitz, Y. N. Sulai, A. Dubra, and R. B. Rosen, “Comparison of adaptive optics scanning light ophthalmoscopic fluorescein angiography and offset pinhole imaging,” Biomed. Opt. Express 5(4), 1173–1189 (2014).
[PubMed]

2013 (2)

T. Y. P. Chui, T. J. Gast, and S. A. Burns, “Imaging of Vascular Wall Fine Structure in the Human Retina Using Adaptive Optics Scanning Laser Ophthalmoscopy,” Invest. Ophthalmol. Vis. Sci. 54(10), 7115–7124 (2013).
[PubMed]

J. Schallek, Y. Geng, H. Nguyen, and D. R. Williams, “Morphology and Topography of Retinal Pericytes in the Living Mouse Retina Using In Vivo Adaptive Optics Imaging and Ex Vivo Characterization,” Invest. Ophthalmol. Vis. Sci. 54(13), 8237–8250 (2013).
[PubMed]

2011 (1)

S. Schmitz-Valckenberg, D. Lara, S. Nizari, E. M. Normando, L. Guo, A. R. Wegener, A. Tufail, F. W. Fitzke, F. G. Holz, and M. F. Cordeiro, “Localisation and significance of in vivo near-infrared autofluorescent signal in retinal imaging,” Br. J. Ophthalmol. 95(8), 1134–1139 (2011).
[PubMed]

2010 (2)

J. R. Sparrow, K. D. Yoon, Y. Wu, and K. Yamamoto, “Interpretations of Fundus Autofluorescence from Studies of the Bisretinoids of the Retina,” Invest. Ophthalmol. Vis. Sci. 51(9), 4351–4357 (2010).
[PubMed]

G. Palczewska, T. Maeda, Y. Imanishi, W. Sun, Y. Chen, D. R. Williams, D. W. Piston, A. Maeda, and K. Palczewski, “Noninvasive multiphoton fluorescence microscopy resolves retinol and retinal condensation products in mouse eyes,” Nat. Med. 16(12), 1444–1449 (2010).
[PubMed]

2009 (1)

Y. Geng, K. P. Greenberg, R. Wolfe, D. C. Gray, J. J. Hunter, A. Dubra, J. G. Flannery, D. R. Williams, and J. Porter, “In Vivo Imaging of Microscopic Structures in the Rat Retina,” Invest. Ophthalmol. Vis. Sci. 50(12), 5872–5879 (2009).
[PubMed]

2008 (2)

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy,” Science 322(5904), 1065–1069 (2008).
[PubMed]

C. Dunsby, “Optically sectioned imaging by oblique plane microscopy,” Opt. Express 16(25), 20306–20316 (2008).
[PubMed]

2007 (1)

2006 (2)

C. N. Keilhauer and F. C. Delori, “Near-Infrared Autofluorescence Imaging of the Fundus: Visualization of Ocular Melanin,” Invest. Ophthalmol. Vis. Sci. 47(8), 3556–3564 (2006).
[PubMed]

M. Paques, M. Simonutti, M. J. Roux, S. Picaud, E. Levavasseur, C. Bellman, and J.-A. Sahel, “High resolution fundus imaging by confocal scanning laser ophthalmoscopy in the mouse,” Vision Res. 46(8-9), 1336–1345 (2006).
[PubMed]

2003 (1)

M. Paques, R. Tadayoni, R. Sercombe, P. Laurent, O. Genevois, A. Gaudric, and E. Vicaut, “Structural and Hemodynamic Analysis of the Mouse Retinal Microcirculation,” Invest. Ophthalmol. Vis. Sci. 44(11), 4960–4967 (2003).
[PubMed]

2002 (1)

2000 (1)

F. C. Delori, M. R. Fleckner, D. G. Goger, J. J. Weiter, and C. K. Dorey, “Autofluorescence Distribution Associated with Drusen in Age-Related Macular Degeneration,” Invest. Ophthalmol. Vis. Sci. 41(2), 496–504 (2000).
[PubMed]

1995 (2)

F. C. Delori, C. K. Dorey, G. Staurenghi, O. Arend, D. G. Goger, and J. J. Weiter, “In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics,” Invest. Ophthalmol. Vis. Sci. 36(3), 718–729 (1995).
[PubMed]

A. von Rückmann, F. W. Fitzke, and A. C. Bird, “Distribution of fundus autofluorescence with a scanning laser ophthalmoscope,” Br. J. Ophthalmol. 79(5), 407–412 (1995).
[PubMed]

1987 (1)

1961 (1)

H. R. Novotny and D. L. Alvis, “A method of Photographing Fluorescence in Circulating Blood in the Human Retina,” Circulation 24(1), 82–86 (1961).
[PubMed]

Alexander, N. S.

Alpers, C. E.

Z. Zhi, J. R. Chao, T. Wietecha, K. L. Hudkins, C. E. Alpers, and R. K. Wang, “Noninvasive Imaging of Retinal Morphology and Microvasculature in Obese Mice Using Optical Coherence Tomography and Optical Microangiography,” Invest. Ophthalmol. Vis. Sci. 55(2), 1024–1030 (2014).
[PubMed]

Alvis, D. L.

H. R. Novotny and D. L. Alvis, “A method of Photographing Fluorescence in Circulating Blood in the Human Retina,” Circulation 24(1), 82–86 (1961).
[PubMed]

Arend, O.

F. C. Delori, C. K. Dorey, G. Staurenghi, O. Arend, D. G. Goger, and J. J. Weiter, “In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics,” Invest. Ophthalmol. Vis. Sci. 36(3), 718–729 (1995).
[PubMed]

Backman, V.

Bailey, S. T.

M. Zhang, T. S. Hwang, J. P. Campbell, S. T. Bailey, D. J. Wilson, D. Huang, and Y. Jia, “Projection-resolved optical coherence tomographic angiography,” Biomed. Opt. Express 7(3), 816–828 (2016).
[PubMed]

Y. Jia, S. T. Bailey, D. J. Wilson, O. Tan, M. L. Klein, C. J. Flaxel, B. Potsaid, J. J. Liu, C. D. Lu, M. F. Kraus, J. G. Fujimoto, and D. Huang, “Quantitative Optical Coherence Tomography Angiography of Choroidal Neovascularization in Age-Related Macular Degeneration,” Ophthalmology 121(7), 1435–1444 (2014).
[PubMed]

Bar-Noam, A. S.

A. S. Bar-Noam, N. Farah, and S. Shoham, “Correction-free remotely scanned two-photon in vivo mouse retinal imaging,” Light: Science &Amp; Applications 5, e16007 (2016).

Bellman, C.

M. Paques, M. Simonutti, M. J. Roux, S. Picaud, E. Levavasseur, C. Bellman, and J.-A. Sahel, “High resolution fundus imaging by confocal scanning laser ophthalmoscopy in the mouse,” Vision Res. 46(8-9), 1336–1345 (2006).
[PubMed]

Bird, A. C.

A. von Rückmann, F. W. Fitzke, and A. C. Bird, “Distribution of fundus autofluorescence with a scanning laser ophthalmoscope,” Br. J. Ophthalmol. 79(5), 407–412 (1995).
[PubMed]

Bonora, S.

M. Cua, D. J. Wahl, Y. Zhao, S. Lee, S. Bonora, R. J. Zawadzki, Y. Jian, and M. V. Sarunic, “Coherence-Gated Sensorless Adaptive Optics Multiphoton Retinal Imaging,” Sci. Rep. 6, 32223 (2016).
[PubMed]

Bouchard, M. B.

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. C. Hillman, “Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms,” Nat. Photonics 9(2), 113–119 (2015).
[PubMed]

Bruno, R. M.

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. C. Hillman, “Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms,” Nat. Photonics 9(2), 113–119 (2015).
[PubMed]

Burns, M. E.

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Gast, T. J.

T. Y. P. Chui, T. J. Gast, and S. A. Burns, “Imaging of Vascular Wall Fine Structure in the Human Retina Using Adaptive Optics Scanning Laser Ophthalmoscopy,” Invest. Ophthalmol. Vis. Sci. 54(10), 7115–7124 (2013).
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S. Mo, B. Krawitz, E. Efstathiadis, L. Geyman, R. Weitz, T. Y. P. Chui, J. Carroll, A. Dubra, and R. B. Rosen, “Imaging Foveal Microvasculature: Optical Coherence Tomography Angiography Versus Adaptive Optics Scanning Light Ophthalmoscope Fluorescein Angiography,” Invest. Ophthalmol. Vis. Sci. 57(9), OCT130 (2016).
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M. B. Sikkel, S. Kumar, V. Maioli, C. Rowlands, F. Gordon, S. E. Harding, A. R. Lyon, K. T. MacLeod, and C. Dunsby, “High speed sCMOS-based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes,” J. Biophotonics 9(3), 311–323 (2016).
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Y. Geng, K. P. Greenberg, R. Wolfe, D. C. Gray, J. J. Hunter, A. Dubra, J. G. Flannery, D. R. Williams, and J. Porter, “In Vivo Imaging of Microscopic Structures in the Rat Retina,” Invest. Ophthalmol. Vis. Sci. 50(12), 5872–5879 (2009).
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Harding, S. E.

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Keilhauer, C. N.

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R. S. Shah, B. T. Soetikno, J. Yi, W. Liu, D. Skondra, H. F. Zhang, and A. A. Fawzi, “Visible-Light Optical Coherence Tomography Angiography for Monitoring Laser-Induced Choroidal Neovascularization in Mice,” Invest. Ophthalmol. Vis. Sci. 57(9), OCT86–OCT95 (2016).
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M. B. Sikkel, S. Kumar, V. Maioli, C. Rowlands, F. Gordon, S. E. Harding, A. R. Lyon, K. T. MacLeod, and C. Dunsby, “High speed sCMOS-based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes,” J. Biophotonics 9(3), 311–323 (2016).
[PubMed]

Ma, Z.

MacLeod, K. T.

M. B. Sikkel, S. Kumar, V. Maioli, C. Rowlands, F. Gordon, S. E. Harding, A. R. Lyon, K. T. MacLeod, and C. Dunsby, “High speed sCMOS-based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes,” J. Biophotonics 9(3), 311–323 (2016).
[PubMed]

Maeda, A.

G. Palczewska, T. Maeda, Y. Imanishi, W. Sun, Y. Chen, D. R. Williams, D. W. Piston, A. Maeda, and K. Palczewski, “Noninvasive multiphoton fluorescence microscopy resolves retinol and retinal condensation products in mouse eyes,” Nat. Med. 16(12), 1444–1449 (2010).
[PubMed]

Maeda, T.

G. Palczewska, T. Maeda, Y. Imanishi, W. Sun, Y. Chen, D. R. Williams, D. W. Piston, A. Maeda, and K. Palczewski, “Noninvasive multiphoton fluorescence microscopy resolves retinol and retinal condensation products in mouse eyes,” Nat. Med. 16(12), 1444–1449 (2010).
[PubMed]

Maioli, V.

M. B. Sikkel, S. Kumar, V. Maioli, C. Rowlands, F. Gordon, S. E. Harding, A. R. Lyon, K. T. MacLeod, and C. Dunsby, “High speed sCMOS-based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes,” J. Biophotonics 9(3), 311–323 (2016).
[PubMed]

Mann, R. S.

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. C. Hillman, “Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms,” Nat. Photonics 9(2), 113–119 (2015).
[PubMed]

Mendes, C. S.

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. C. Hillman, “Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms,” Nat. Photonics 9(2), 113–119 (2015).
[PubMed]

Miller, E. B.

Mo, S.

S. Mo, B. Krawitz, E. Efstathiadis, L. Geyman, R. Weitz, T. Y. P. Chui, J. Carroll, A. Dubra, and R. B. Rosen, “Imaging Foveal Microvasculature: Optical Coherence Tomography Angiography Versus Adaptive Optics Scanning Light Ophthalmoscope Fluorescein Angiography,” Invest. Ophthalmol. Vis. Sci. 57(9), OCT130 (2016).
[PubMed]

Mostoslavsky, G.

L. Zhang, A. Capilla, W. Song, G. Mostoslavsky, and J. Yi, “Oblique scanning laser microscopy for simultaneously volumetric structural and molecular imaging using only one raster scan,” Sci. Rep. 7(1), 8591 (2017).
[PubMed]

Nagaoka, T.

A. Ishibazawa, T. Nagaoka, A. Takahashi, T. Omae, T. Tani, K. Sogawa, H. Yokota, and A. Yoshida, “Optical Coherence Tomography Angiography in Diabetic Retinopathy: A Prospective Pilot Study,” Am. J. Ophthalmol. 160(1), 35–44 (2015).
[PubMed]

Nguyen, H.

J. Schallek, Y. Geng, H. Nguyen, and D. R. Williams, “Morphology and Topography of Retinal Pericytes in the Living Mouse Retina Using In Vivo Adaptive Optics Imaging and Ex Vivo Characterization,” Invest. Ophthalmol. Vis. Sci. 54(13), 8237–8250 (2013).
[PubMed]

Nizari, S.

S. Schmitz-Valckenberg, D. Lara, S. Nizari, E. M. Normando, L. Guo, A. R. Wegener, A. Tufail, F. W. Fitzke, F. G. Holz, and M. F. Cordeiro, “Localisation and significance of in vivo near-infrared autofluorescent signal in retinal imaging,” Br. J. Ophthalmol. 95(8), 1134–1139 (2011).
[PubMed]

Normando, E. M.

S. Schmitz-Valckenberg, D. Lara, S. Nizari, E. M. Normando, L. Guo, A. R. Wegener, A. Tufail, F. W. Fitzke, F. G. Holz, and M. F. Cordeiro, “Localisation and significance of in vivo near-infrared autofluorescent signal in retinal imaging,” Br. J. Ophthalmol. 95(8), 1134–1139 (2011).
[PubMed]

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H. R. Novotny and D. L. Alvis, “A method of Photographing Fluorescence in Circulating Blood in the Human Retina,” Circulation 24(1), 82–86 (1961).
[PubMed]

Omae, T.

A. Ishibazawa, T. Nagaoka, A. Takahashi, T. Omae, T. Tani, K. Sogawa, H. Yokota, and A. Yoshida, “Optical Coherence Tomography Angiography in Diabetic Retinopathy: A Prospective Pilot Study,” Am. J. Ophthalmol. 160(1), 35–44 (2015).
[PubMed]

Palczewska, G.

Palczewski, K.

Paques, M.

M. Paques, M. Simonutti, M. J. Roux, S. Picaud, E. Levavasseur, C. Bellman, and J.-A. Sahel, “High resolution fundus imaging by confocal scanning laser ophthalmoscopy in the mouse,” Vision Res. 46(8-9), 1336–1345 (2006).
[PubMed]

M. Paques, R. Tadayoni, R. Sercombe, P. Laurent, O. Genevois, A. Gaudric, and E. Vicaut, “Structural and Hemodynamic Analysis of the Mouse Retinal Microcirculation,” Invest. Ophthalmol. Vis. Sci. 44(11), 4960–4967 (2003).
[PubMed]

Picaud, S.

M. Paques, M. Simonutti, M. J. Roux, S. Picaud, E. Levavasseur, C. Bellman, and J.-A. Sahel, “High resolution fundus imaging by confocal scanning laser ophthalmoscopy in the mouse,” Vision Res. 46(8-9), 1336–1345 (2006).
[PubMed]

Pinhas, A.

Piston, D. W.

G. Palczewska, T. Maeda, Y. Imanishi, W. Sun, Y. Chen, D. R. Williams, D. W. Piston, A. Maeda, and K. Palczewski, “Noninvasive multiphoton fluorescence microscopy resolves retinol and retinal condensation products in mouse eyes,” Nat. Med. 16(12), 1444–1449 (2010).
[PubMed]

Porter, J.

Y. Geng, K. P. Greenberg, R. Wolfe, D. C. Gray, J. J. Hunter, A. Dubra, J. G. Flannery, D. R. Williams, and J. Porter, “In Vivo Imaging of Microscopic Structures in the Rat Retina,” Invest. Ophthalmol. Vis. Sci. 50(12), 5872–5879 (2009).
[PubMed]

Potsaid, B.

Y. Jia, S. T. Bailey, D. J. Wilson, O. Tan, M. L. Klein, C. J. Flaxel, B. Potsaid, J. J. Liu, C. D. Lu, M. F. Kraus, J. G. Fujimoto, and D. Huang, “Quantitative Optical Coherence Tomography Angiography of Choroidal Neovascularization in Age-Related Macular Degeneration,” Ophthalmology 121(7), 1435–1444 (2014).
[PubMed]

Pugh, E. N.

P. Zhang, A. Zam, Y. Jian, X. Wang, Y. Li, K. S. Lam, M. E. Burns, M. V. Sarunic, E. N. Pugh, and R. J. Zawadzki, “In vivo wide-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,” J. Biomed. Opt. 20(12), 126005 (2015).
[PubMed]

R. J. Zawadzki, P. Zhang, A. Zam, E. B. Miller, M. Goswami, X. Wang, R. S. Jonnal, S.-H. Lee, D. Y. Kim, J. G. Flannery, J. S. Werner, M. E. Burns, and E. N. Pugh., “Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina,” Biomed. Opt. Express 6(6), 2191–2210 (2015).
[PubMed]

Queener, H.

Romano, A.

T. E. de Carlo, A. Romano, N. K. Waheed, and J. S. Duker, “A review of optical coherence tomography angiography (OCTA),” Int J Retina Vitreous 1(1), 5 (2015).
[PubMed]

Romero-Borja, F.

Roorda, A.

Rosen, R. B.

S. Mo, B. Krawitz, E. Efstathiadis, L. Geyman, R. Weitz, T. Y. P. Chui, J. Carroll, A. Dubra, and R. B. Rosen, “Imaging Foveal Microvasculature: Optical Coherence Tomography Angiography Versus Adaptive Optics Scanning Light Ophthalmoscope Fluorescein Angiography,” Invest. Ophthalmol. Vis. Sci. 57(9), OCT130 (2016).
[PubMed]

T. Y. P. Chui, M. Dubow, A. Pinhas, N. Shah, A. Gan, R. Weitz, Y. N. Sulai, A. Dubra, and R. B. Rosen, “Comparison of adaptive optics scanning light ophthalmoscopic fluorescein angiography and offset pinhole imaging,” Biomed. Opt. Express 5(4), 1173–1189 (2014).
[PubMed]

Roux, M. J.

M. Paques, M. Simonutti, M. J. Roux, S. Picaud, E. Levavasseur, C. Bellman, and J.-A. Sahel, “High resolution fundus imaging by confocal scanning laser ophthalmoscopy in the mouse,” Vision Res. 46(8-9), 1336–1345 (2006).
[PubMed]

Rowlands, C.

M. B. Sikkel, S. Kumar, V. Maioli, C. Rowlands, F. Gordon, S. E. Harding, A. R. Lyon, K. T. MacLeod, and C. Dunsby, “High speed sCMOS-based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes,” J. Biophotonics 9(3), 311–323 (2016).
[PubMed]

Sahel, J.-A.

M. Paques, M. Simonutti, M. J. Roux, S. Picaud, E. Levavasseur, C. Bellman, and J.-A. Sahel, “High resolution fundus imaging by confocal scanning laser ophthalmoscopy in the mouse,” Vision Res. 46(8-9), 1336–1345 (2006).
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Sarunic, M. V.

M. Cua, D. J. Wahl, Y. Zhao, S. Lee, S. Bonora, R. J. Zawadzki, Y. Jian, and M. V. Sarunic, “Coherence-Gated Sensorless Adaptive Optics Multiphoton Retinal Imaging,” Sci. Rep. 6, 32223 (2016).
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P. Zhang, A. Zam, Y. Jian, X. Wang, Y. Li, K. S. Lam, M. E. Burns, M. V. Sarunic, E. N. Pugh, and R. J. Zawadzki, “In vivo wide-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,” J. Biomed. Opt. 20(12), 126005 (2015).
[PubMed]

Schallek, J.

J. Schallek, Y. Geng, H. Nguyen, and D. R. Williams, “Morphology and Topography of Retinal Pericytes in the Living Mouse Retina Using In Vivo Adaptive Optics Imaging and Ex Vivo Characterization,” Invest. Ophthalmol. Vis. Sci. 54(13), 8237–8250 (2013).
[PubMed]

Schmidt, A. D.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy,” Science 322(5904), 1065–1069 (2008).
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Schmitz-Valckenberg, S.

S. Schmitz-Valckenberg, D. Lara, S. Nizari, E. M. Normando, L. Guo, A. R. Wegener, A. Tufail, F. W. Fitzke, F. G. Holz, and M. F. Cordeiro, “Localisation and significance of in vivo near-infrared autofluorescent signal in retinal imaging,” Br. J. Ophthalmol. 95(8), 1134–1139 (2011).
[PubMed]

Sercombe, R.

M. Paques, R. Tadayoni, R. Sercombe, P. Laurent, O. Genevois, A. Gaudric, and E. Vicaut, “Structural and Hemodynamic Analysis of the Mouse Retinal Microcirculation,” Invest. Ophthalmol. Vis. Sci. 44(11), 4960–4967 (2003).
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Shah, N.

Shah, R. S.

R. S. Shah, B. T. Soetikno, J. Yi, W. Liu, D. Skondra, H. F. Zhang, and A. A. Fawzi, “Visible-Light Optical Coherence Tomography Angiography for Monitoring Laser-Induced Choroidal Neovascularization in Mice,” Invest. Ophthalmol. Vis. Sci. 57(9), OCT86–OCT95 (2016).
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A. S. Bar-Noam, N. Farah, and S. Shoham, “Correction-free remotely scanned two-photon in vivo mouse retinal imaging,” Light: Science &Amp; Applications 5, e16007 (2016).

Sikkel, M. B.

M. B. Sikkel, S. Kumar, V. Maioli, C. Rowlands, F. Gordon, S. E. Harding, A. R. Lyon, K. T. MacLeod, and C. Dunsby, “High speed sCMOS-based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes,” J. Biophotonics 9(3), 311–323 (2016).
[PubMed]

Simonutti, M.

M. Paques, M. Simonutti, M. J. Roux, S. Picaud, E. Levavasseur, C. Bellman, and J.-A. Sahel, “High resolution fundus imaging by confocal scanning laser ophthalmoscopy in the mouse,” Vision Res. 46(8-9), 1336–1345 (2006).
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Skondra, D.

R. S. Shah, B. T. Soetikno, J. Yi, W. Liu, D. Skondra, H. F. Zhang, and A. A. Fawzi, “Visible-Light Optical Coherence Tomography Angiography for Monitoring Laser-Induced Choroidal Neovascularization in Mice,” Invest. Ophthalmol. Vis. Sci. 57(9), OCT86–OCT95 (2016).
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Soetikno, B. T.

R. S. Shah, B. T. Soetikno, J. Yi, W. Liu, D. Skondra, H. F. Zhang, and A. A. Fawzi, “Visible-Light Optical Coherence Tomography Angiography for Monitoring Laser-Induced Choroidal Neovascularization in Mice,” Invest. Ophthalmol. Vis. Sci. 57(9), OCT86–OCT95 (2016).
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A. Ishibazawa, T. Nagaoka, A. Takahashi, T. Omae, T. Tani, K. Sogawa, H. Yokota, and A. Yoshida, “Optical Coherence Tomography Angiography in Diabetic Retinopathy: A Prospective Pilot Study,” Am. J. Ophthalmol. 160(1), 35–44 (2015).
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Song, W.

L. Zhang, A. Capilla, W. Song, G. Mostoslavsky, and J. Yi, “Oblique scanning laser microscopy for simultaneously volumetric structural and molecular imaging using only one raster scan,” Sci. Rep. 7(1), 8591 (2017).
[PubMed]

Sparrow, J. R.

J. R. Sparrow, K. D. Yoon, Y. Wu, and K. Yamamoto, “Interpretations of Fundus Autofluorescence from Studies of the Bisretinoids of the Retina,” Invest. Ophthalmol. Vis. Sci. 51(9), 4351–4357 (2010).
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Staurenghi, G.

F. C. Delori, C. K. Dorey, G. Staurenghi, O. Arend, D. G. Goger, and J. J. Weiter, “In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics,” Invest. Ophthalmol. Vis. Sci. 36(3), 718–729 (1995).
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Stelzer, E. H. K.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy,” Science 322(5904), 1065–1069 (2008).
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Sulai, Y. N.

Sun, W.

G. Palczewska, T. Maeda, Y. Imanishi, W. Sun, Y. Chen, D. R. Williams, D. W. Piston, A. Maeda, and K. Palczewski, “Noninvasive multiphoton fluorescence microscopy resolves retinol and retinal condensation products in mouse eyes,” Nat. Med. 16(12), 1444–1449 (2010).
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Tadayoni, R.

M. Paques, R. Tadayoni, R. Sercombe, P. Laurent, O. Genevois, A. Gaudric, and E. Vicaut, “Structural and Hemodynamic Analysis of the Mouse Retinal Microcirculation,” Invest. Ophthalmol. Vis. Sci. 44(11), 4960–4967 (2003).
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A. Ishibazawa, T. Nagaoka, A. Takahashi, T. Omae, T. Tani, K. Sogawa, H. Yokota, and A. Yoshida, “Optical Coherence Tomography Angiography in Diabetic Retinopathy: A Prospective Pilot Study,” Am. J. Ophthalmol. 160(1), 35–44 (2015).
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Y. Jia, S. T. Bailey, D. J. Wilson, O. Tan, M. L. Klein, C. J. Flaxel, B. Potsaid, J. J. Liu, C. D. Lu, M. F. Kraus, J. G. Fujimoto, and D. Huang, “Quantitative Optical Coherence Tomography Angiography of Choroidal Neovascularization in Age-Related Macular Degeneration,” Ophthalmology 121(7), 1435–1444 (2014).
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A. Ishibazawa, T. Nagaoka, A. Takahashi, T. Omae, T. Tani, K. Sogawa, H. Yokota, and A. Yoshida, “Optical Coherence Tomography Angiography in Diabetic Retinopathy: A Prospective Pilot Study,” Am. J. Ophthalmol. 160(1), 35–44 (2015).
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Tufail, A.

S. Schmitz-Valckenberg, D. Lara, S. Nizari, E. M. Normando, L. Guo, A. R. Wegener, A. Tufail, F. W. Fitzke, F. G. Holz, and M. F. Cordeiro, “Localisation and significance of in vivo near-infrared autofluorescent signal in retinal imaging,” Br. J. Ophthalmol. 95(8), 1134–1139 (2011).
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Vicaut, E.

M. Paques, R. Tadayoni, R. Sercombe, P. Laurent, O. Genevois, A. Gaudric, and E. Vicaut, “Structural and Hemodynamic Analysis of the Mouse Retinal Microcirculation,” Invest. Ophthalmol. Vis. Sci. 44(11), 4960–4967 (2003).
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M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. C. Hillman, “Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms,” Nat. Photonics 9(2), 113–119 (2015).
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A. von Rückmann, F. W. Fitzke, and A. C. Bird, “Distribution of fundus autofluorescence with a scanning laser ophthalmoscope,” Br. J. Ophthalmol. 79(5), 407–412 (1995).
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T. E. de Carlo, A. Romano, N. K. Waheed, and J. S. Duker, “A review of optical coherence tomography angiography (OCTA),” Int J Retina Vitreous 1(1), 5 (2015).
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Wahl, D. J.

M. Cua, D. J. Wahl, Y. Zhao, S. Lee, S. Bonora, R. J. Zawadzki, Y. Jian, and M. V. Sarunic, “Coherence-Gated Sensorless Adaptive Optics Multiphoton Retinal Imaging,” Sci. Rep. 6, 32223 (2016).
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C.-L. Chen and R. K. Wang, “Optical coherence tomography based angiography [Invited],” Biomed. Opt. Express 8(2), 1056–1082 (2017).
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Z. Zhi, J. R. Chao, T. Wietecha, K. L. Hudkins, C. E. Alpers, and R. K. Wang, “Noninvasive Imaging of Retinal Morphology and Microvasculature in Obese Mice Using Optical Coherence Tomography and Optical Microangiography,” Invest. Ophthalmol. Vis. Sci. 55(2), 1024–1030 (2014).
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R. J. Zawadzki, P. Zhang, A. Zam, E. B. Miller, M. Goswami, X. Wang, R. S. Jonnal, S.-H. Lee, D. Y. Kim, J. G. Flannery, J. S. Werner, M. E. Burns, and E. N. Pugh., “Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina,” Biomed. Opt. Express 6(6), 2191–2210 (2015).
[PubMed]

P. Zhang, A. Zam, Y. Jian, X. Wang, Y. Li, K. S. Lam, M. E. Burns, M. V. Sarunic, E. N. Pugh, and R. J. Zawadzki, “In vivo wide-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,” J. Biomed. Opt. 20(12), 126005 (2015).
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Webb, R. H.

Wegener, A. R.

S. Schmitz-Valckenberg, D. Lara, S. Nizari, E. M. Normando, L. Guo, A. R. Wegener, A. Tufail, F. W. Fitzke, F. G. Holz, and M. F. Cordeiro, “Localisation and significance of in vivo near-infrared autofluorescent signal in retinal imaging,” Br. J. Ophthalmol. 95(8), 1134–1139 (2011).
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Weiter, J. J.

F. C. Delori, M. R. Fleckner, D. G. Goger, J. J. Weiter, and C. K. Dorey, “Autofluorescence Distribution Associated with Drusen in Age-Related Macular Degeneration,” Invest. Ophthalmol. Vis. Sci. 41(2), 496–504 (2000).
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Weitz, R.

S. Mo, B. Krawitz, E. Efstathiadis, L. Geyman, R. Weitz, T. Y. P. Chui, J. Carroll, A. Dubra, and R. B. Rosen, “Imaging Foveal Microvasculature: Optical Coherence Tomography Angiography Versus Adaptive Optics Scanning Light Ophthalmoscope Fluorescein Angiography,” Invest. Ophthalmol. Vis. Sci. 57(9), OCT130 (2016).
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T. Y. P. Chui, M. Dubow, A. Pinhas, N. Shah, A. Gan, R. Weitz, Y. N. Sulai, A. Dubra, and R. B. Rosen, “Comparison of adaptive optics scanning light ophthalmoscopic fluorescein angiography and offset pinhole imaging,” Biomed. Opt. Express 5(4), 1173–1189 (2014).
[PubMed]

Werner, J. S.

Wietecha, T.

Z. Zhi, J. R. Chao, T. Wietecha, K. L. Hudkins, C. E. Alpers, and R. K. Wang, “Noninvasive Imaging of Retinal Morphology and Microvasculature in Obese Mice Using Optical Coherence Tomography and Optical Microangiography,” Invest. Ophthalmol. Vis. Sci. 55(2), 1024–1030 (2014).
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Williams, D. R.

J. Schallek, Y. Geng, H. Nguyen, and D. R. Williams, “Morphology and Topography of Retinal Pericytes in the Living Mouse Retina Using In Vivo Adaptive Optics Imaging and Ex Vivo Characterization,” Invest. Ophthalmol. Vis. Sci. 54(13), 8237–8250 (2013).
[PubMed]

G. Palczewska, T. Maeda, Y. Imanishi, W. Sun, Y. Chen, D. R. Williams, D. W. Piston, A. Maeda, and K. Palczewski, “Noninvasive multiphoton fluorescence microscopy resolves retinol and retinal condensation products in mouse eyes,” Nat. Med. 16(12), 1444–1449 (2010).
[PubMed]

Y. Geng, K. P. Greenberg, R. Wolfe, D. C. Gray, J. J. Hunter, A. Dubra, J. G. Flannery, D. R. Williams, and J. Porter, “In Vivo Imaging of Microscopic Structures in the Rat Retina,” Invest. Ophthalmol. Vis. Sci. 50(12), 5872–5879 (2009).
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Wilson, D. J.

M. Zhang, T. S. Hwang, J. P. Campbell, S. T. Bailey, D. J. Wilson, D. Huang, and Y. Jia, “Projection-resolved optical coherence tomographic angiography,” Biomed. Opt. Express 7(3), 816–828 (2016).
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Y. Jia, S. T. Bailey, D. J. Wilson, O. Tan, M. L. Klein, C. J. Flaxel, B. Potsaid, J. J. Liu, C. D. Lu, M. F. Kraus, J. G. Fujimoto, and D. Huang, “Quantitative Optical Coherence Tomography Angiography of Choroidal Neovascularization in Age-Related Macular Degeneration,” Ophthalmology 121(7), 1435–1444 (2014).
[PubMed]

Wittbrodt, J.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy,” Science 322(5904), 1065–1069 (2008).
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Wojtkowski, M.

Wolfe, R.

Y. Geng, K. P. Greenberg, R. Wolfe, D. C. Gray, J. J. Hunter, A. Dubra, J. G. Flannery, D. R. Williams, and J. Porter, “In Vivo Imaging of Microscopic Structures in the Rat Retina,” Invest. Ophthalmol. Vis. Sci. 50(12), 5872–5879 (2009).
[PubMed]

Wu, Y.

J. R. Sparrow, K. D. Yoon, Y. Wu, and K. Yamamoto, “Interpretations of Fundus Autofluorescence from Studies of the Bisretinoids of the Retina,” Invest. Ophthalmol. Vis. Sci. 51(9), 4351–4357 (2010).
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Yamamoto, K.

J. R. Sparrow, K. D. Yoon, Y. Wu, and K. Yamamoto, “Interpretations of Fundus Autofluorescence from Studies of the Bisretinoids of the Retina,” Invest. Ophthalmol. Vis. Sci. 51(9), 4351–4357 (2010).
[PubMed]

Yi, J.

L. Zhang, A. Capilla, W. Song, G. Mostoslavsky, and J. Yi, “Oblique scanning laser microscopy for simultaneously volumetric structural and molecular imaging using only one raster scan,” Sci. Rep. 7(1), 8591 (2017).
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R. S. Shah, B. T. Soetikno, J. Yi, W. Liu, D. Skondra, H. F. Zhang, and A. A. Fawzi, “Visible-Light Optical Coherence Tomography Angiography for Monitoring Laser-Induced Choroidal Neovascularization in Mice,” Invest. Ophthalmol. Vis. Sci. 57(9), OCT86–OCT95 (2016).
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Yokota, H.

A. Ishibazawa, T. Nagaoka, A. Takahashi, T. Omae, T. Tani, K. Sogawa, H. Yokota, and A. Yoshida, “Optical Coherence Tomography Angiography in Diabetic Retinopathy: A Prospective Pilot Study,” Am. J. Ophthalmol. 160(1), 35–44 (2015).
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Zhang, L.

L. Zhang, A. Capilla, W. Song, G. Mostoslavsky, and J. Yi, “Oblique scanning laser microscopy for simultaneously volumetric structural and molecular imaging using only one raster scan,” Sci. Rep. 7(1), 8591 (2017).
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M. Cua, D. J. Wahl, Y. Zhao, S. Lee, S. Bonora, R. J. Zawadzki, Y. Jian, and M. V. Sarunic, “Coherence-Gated Sensorless Adaptive Optics Multiphoton Retinal Imaging,” Sci. Rep. 6, 32223 (2016).
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Supplementary Material (4)

NameDescription
» Visualization 1       Fly through video of oSLO volumetric images of fluorescent microspheres.
» Visualization 2       Cross sectional fly through video of oSLO volumetric image of rat retina in vivo.
» Visualization 3       En face fly through video of simultaneous oSLO and OCT volumetric images of rat retina in vivo.
» Visualization 4       En face fly through video of wide-field oSLO volumetric images of rat retina in vivo.

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Figures (9)

Fig. 1
Fig. 1 Principle of oblique scanning laser ophthalmoscopy (oSLO). (a) Comparison of the optical alignment between the prevalent retinal imaging modalities and oSLO. Blue and green represent the fluorescence excitation and emission detection. oSLO breaks from the coaxial alignment used in other imaging methods, thereby allowing cross-sectional fluorescence imaging. (b-d) The simulated excitation, emission detection, and combined point-spread functions (PSFs) under coaxial alignment. For simplicity, both excitation and detection NA is 0.05. The combined PSF is the multiplication of excitation and detection. (e-g) The same simulation as in (b-d) under oSLO alignment. The excitation has an oblique angle of ~15°. For the purpose of the demonstration, the emission detection and excitation PSFs are symmetrical with respect to the optical axis.

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Fig. 2
Fig. 2 System setup of oSLO combined with OCT. (a) The system schematic. SL: supercontinuum source; DM: dichroic mirror; BT: beam trap; PBS: polarization beam splitter; P: prism; B: block; F: filter; PC: polarization controller; OFC: wide band optical fiber coupler; DC: dispersion control; VNDF: variable neutral density filter; M: mirror; SPEC: spectrometer; L: lens; OL: objective lens; GM: galvanometer mirror. (b, c) 3D model of the oblique laser scanning, and the definition of the volumetric imaging dimensions. (d) The multiplexing spectrum of fluorescein excitation in oSLO and NIR OCT. The blue and pink curves are the transmission spectrum of the dichroic mirror (DM3) and emission filter (F2), respectively.

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Fig. 3
Fig. 3 Feasibility and resolution characterization of oSLO system using fluorescent microspheres under a 6mm ball lens simulating the rat eye. (a) Simultaneously captured OCT and oSLO fluorescence images and their overlay. The fluorescence images were pseudo-colored green. Bar = 100 μm. (b-d) The maximum intensity projection of the volumetric fluorescence microsphere image on the planes of x’-z’, y’-z’, and x’-y’. (e) Magnified images of three representative microspheres in the center of the field of view in (b-d). The locations of three microspheres are labeled in (b-d). (f) The intensity profile across the center of three beads in x’, y’, and z’ directions. Bar = 5μm. A cross sectional fly-through video is included in Visualization 1.

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Fig. 4
Fig. 4 In vivo volumetric oSLO FA and OCTA acquired simultaneously from the rat retina. (a-c) Examples of cross sectional OCT B-scan, and the corresponding OCTA and oSLO FA. (d-f) Depth-resolved oSLO fluorescence images at varying depth ranges. (g-i) The corresponding depth-dependent OCTA images. Red arrows point to OCTA motion artifacts, appearing as vertical stripes. Yellow arrows point to locations where retinal vessels dive down into the deep capillary plexus, seen more clearly on oSLO FA than OCTA. White arrows point to a venule that appears larger in oSLO than OCTA. Blue arrows point to a region where oSLO FA shows better contrast and capillary resolution than OCTA. Bar = 200 μm. An en face fly-through video from both oSLO and OCTA is included in Visualization 3.

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Fig. 5
Fig. 5 Wide-field volumetric fluorescence retinal imaging by oSLO over 30° angle of view. (a-b) Depth-encoded volumetric oSLO FA image and the corresponding conventional SLO image from a rat retina in vivo. (c-d) The magnified images of an ROI between a major retinal arteriole and venule, as outlined in panel (a) and (b). (e-h) The vascular sketch showing the detailed vessel branching and derivation in upper layer in NFL and GCL (e), intermediate layer in OPL (f), deep layer in OPL (g), and their overlay (h). The connections between adjacent layers were labeled by end dots. An enface fly-through video is included in the Visualization 4.

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Fig. 6
Fig. 6 (a) The solid work model for the dove tail mount installed in the optical setup. (b-c) Zoomed in view of the dove tail mount. (d) The photography of the actual setup in oSLO system.

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Fig. 7
Fig. 7 The photography of the phantom experiment using a ball lens mimicking the rat eye. (a) The photograph of the system setup. (b, c) The photography of the in-action oblique scanning laser illumination from two side views.

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Fig. 8
Fig. 8 Illustration of the angle changes of excitation and detection PSFs at various scanning angles over 50° FOV, and the resulting combined PSFs. Bar = 20μm.

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Fig. 9
Fig. 9 In vivo oSLO AF retinal fluorescence imaging from a rat eye. (a) En face maximum value projection of the volumetric oSLO AF data set. (b) The cross sectional image along the dash line in panel (a). Bar = 0.2 mm.

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Tables (1)

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Table 1 The information of the system components.

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Equations (1)

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tan(ϕ)= tan(θ) M ,

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