Abstract

Though in vivo two-photon imaging has been demonstrated in non-human primates, improvements in the signal-to-noise ratio (SNR) would greatly improve its scientific utility. In this study, extrinsic fluorophores, expressed in otherwise transparent retinal ganglion cells, were imaged in the living mouse eye using a two-photon fluorescence adaptive optics scanning laser ophthalmoscope. We recorded two orders of magnitude greater signal levels from extrinsically labeled cells relative to previous work done in two-photon autofluorescence imaging of primates. Features as small as single dendrites in various layers of the retina could be resolved and predictions are made about the feasibility of measuring functional response from cells. In the future, two-photon imaging in the intact eye may allow us to monitor the function of retinal cell classes with infrared light that minimally excites the visual response.

© 2013 OSA

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2013 (1)

L. Yin, Y. Geng, F. Osakada, R. Sharma, A. H. Cetin, E. M. Callaway, D. R. Williams, and W. H. Merigan, “Imaging light responses of retinal ganglion cells in the living mouse eye,” J. Neurophysiol.109(9), 2415–2421 (2013).
[CrossRef] [PubMed]

2012 (1)

2011 (8)

A. Dubra, Y. Sulai, J. L. Norris, R. F. Cooper, A. M. Dubis, D. R. Williams, and J. Carroll, “Noninvasive imaging of the human rod photoreceptor mosaic using a confocal adaptive optics scanning ophthalmoscope,” Biomed. Opt. Express2(7), 1864–1876 (2011).
[CrossRef] [PubMed]

E. A. Rossi, M. Chung, A. Dubra, J. J. Hunter, W. H. Merigan, and D. R. Williams, “Imaging retinal mosaics in the living eye,” Eye (Lond.)25(3), 301–308 (2011).
[CrossRef] [PubMed]

L. Yin, K. Greenberg, J. J. Hunter, D. Dalkara, K. D. Kolstad, B. D. Masella, R. Wolfe, M. Visel, D. Stone, R. T. Libby, D. Diloreto, D. Schaffer, J. Flannery, D. R. Williams, and W. H. Merigan, “Intravitreal injection of AAV2 transduces macaque inner retina,” Invest. Ophthalmol. Vis. Sci.52(5), 2775–2783 (2011).
[CrossRef] [PubMed]

F. Osakada, T. Mori, A. H. Cetin, J. H. Marshel, B. Virgen, and E. M. Callaway, “New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits,” Neuron71(4), 617–631 (2011).
[CrossRef] [PubMed]

M. Pottek, G. C. Knop, R. Weiler, and K. Dedek, “Electrophysiological characterization of GFP-expressing cell populations in the intact retina,” J. Vis. Exp. (57), 13473–13474 (2011).
[CrossRef] [PubMed]

B. G. Borghuis, L. Tian, Y. Xu, S. S. Nikonov, N. Vardi, B. V. Zemelman, and L. L. Looger, “Imaging light responses of targeted neuron populations in the rodent retina,” J. Neurosci.31(8), 2855–2867 (2011).
[CrossRef] [PubMed]

Y. V. Wang, M. Weick, and J. B. Demb, “Spectral and temporal sensitivity of cone-mediated responses in mouse retinal ganglion cells,” J. Neurosci.31(21), 7670–7681 (2011).
[CrossRef] [PubMed]

R.-W. Lu, Y.-C. Li, T. Ye, C. Strang, K. Keyser, C. A. Curcio, and X.-C. Yao, “Two-photon excited autofluorescence imaging of freshly isolated frog retinas,” Biomed. Opt. Express2(6), 1494–1503 (2011).
[CrossRef] [PubMed]

2010 (4)

F. Naarendorp, T. M. Esdaille, S. M. Banden, J. Andrews-Labenski, O. P. Gross, and E. N. Pugh., “Dark light, rod saturation, and the absolute and incremental sensitivity of mouse cone vision,” J. Neurosci.30(37), 12495–12507 (2010).
[CrossRef] [PubMed]

E. J. Gualda, J. M. Bueno, and P. Artal, “Wavefront optimized nonlinear microscopy of ex vivo human retinas,” J. Biomed. Opt.15(2), 026007 (2010).
[CrossRef] [PubMed]

W. Wei, J. Elstrott, and M. B. Feller, “Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina,” Nat. Protoc.5(7), 1347–1352 (2010).
[CrossRef] [PubMed]

J. J. Hunter, B. Masella, A. Dubra, R. Sharma, L. Yin, W. H. Merigan, G. Palczewska, K. Palczewski, and D. R. Williams, “Images of photoreceptors in living primate eyes using adaptive optics two-photon ophthalmoscopy,” Biomed. Opt. Express2(1), 139–148 (2010).
[CrossRef] [PubMed]

2009 (2)

D. Scoles, D. C. Gray, J. J. Hunter, R. Wolfe, B. P. Gee, Y. Geng, B. D. Masella, R. T. Libby, S. Russell, D. R. Williams, and W. H. Merigan, “In vivo imaging of retinal nerve fiber layer vasculature: imaging histology comparison,” BMC Ophthalmol.9(1), 1–9 (2009).
[CrossRef] [PubMed]

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies, and mice with improved GCaMP calcium indicators,” Nat. Methods6(12), 875–881 (2009).
[CrossRef] [PubMed]

2008 (1)

J. I. W. Morgan, A. Dubra, R. Wolfe, W. H. Merigan, and D. R. Williams, “In vivo autofluorescence imaging of the human and macaque retinal pigment epithelial cell mosaic,” Invest. Ophthalmol. Vis. Sci.50(3), 1350–1359 (2008).
[CrossRef] [PubMed]

2007 (1)

Y. Imanishi, K. H. Lodowski, and Y. Koutalos, “Two-photon microscopy: shedding light on the chemistry of vision,” Biochemistry46(34), 9674–9684 (2007).
[CrossRef] [PubMed]

2006 (2)

2005 (1)

K. Ohki, S. Chung, Y. H. Ch’ng, P. Kara, and R. C. Reid, “Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex,” Nature433(7026), 597–603 (2005).
[CrossRef] [PubMed]

2004 (1)

Y. Imanishi, M. L. Batten, D. W. Piston, W. Baehr, and K. Palczewski, “Noninvasive two-photon imaging reveals retinyl ester storage structures in the eye,” J. Cell Biol.164(3), 373–383 (2004).
[CrossRef] [PubMed]

2002 (2)

A. Roorda, F. Romero-Borja, W. Donnelly Iii, H. Queener, T. Hebert, and M. Campbell, “Adaptive optics scanning laser ophthalmoscopy,” Opt. Express10(9), 405–412 (2002).
[CrossRef] [PubMed]

T. Euler, P. B. Detwiler, and W. Denk, “Directionally selective calcium signals in dendrites of starburst amacrine cells,” Nature418(6900), 845–852 (2002).
[CrossRef] [PubMed]

1997 (1)

1995 (1)

T. D. Lamb, “Photoreceptor spectral sensitivities: common shape in the long-wavelength region,” Vision Res.35(22), 3083–3091 (1995).
[CrossRef] [PubMed]

Ahamd, K.

Akerboom, J.

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies, and mice with improved GCaMP calcium indicators,” Nat. Methods6(12), 875–881 (2009).
[CrossRef] [PubMed]

Andrews-Labenski, J.

F. Naarendorp, T. M. Esdaille, S. M. Banden, J. Andrews-Labenski, O. P. Gross, and E. N. Pugh., “Dark light, rod saturation, and the absolute and incremental sensitivity of mouse cone vision,” J. Neurosci.30(37), 12495–12507 (2010).
[CrossRef] [PubMed]

Artal, P.

E. J. Gualda, J. M. Bueno, and P. Artal, “Wavefront optimized nonlinear microscopy of ex vivo human retinas,” J. Biomed. Opt.15(2), 026007 (2010).
[CrossRef] [PubMed]

Baehr, W.

Y. Imanishi, M. L. Batten, D. W. Piston, W. Baehr, and K. Palczewski, “Noninvasive two-photon imaging reveals retinyl ester storage structures in the eye,” J. Cell Biol.164(3), 373–383 (2004).
[CrossRef] [PubMed]

Banden, S. M.

F. Naarendorp, T. M. Esdaille, S. M. Banden, J. Andrews-Labenski, O. P. Gross, and E. N. Pugh., “Dark light, rod saturation, and the absolute and incremental sensitivity of mouse cone vision,” J. Neurosci.30(37), 12495–12507 (2010).
[CrossRef] [PubMed]

Bargmann, C. I.

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies, and mice with improved GCaMP calcium indicators,” Nat. Methods6(12), 875–881 (2009).
[CrossRef] [PubMed]

Batten, M. L.

Y. Imanishi, M. L. Batten, D. W. Piston, W. Baehr, and K. Palczewski, “Noninvasive two-photon imaging reveals retinyl ester storage structures in the eye,” J. Cell Biol.164(3), 373–383 (2004).
[CrossRef] [PubMed]

Borghuis, B. G.

B. G. Borghuis, L. Tian, Y. Xu, S. S. Nikonov, N. Vardi, B. V. Zemelman, and L. L. Looger, “Imaging light responses of targeted neuron populations in the rodent retina,” J. Neurosci.31(8), 2855–2867 (2011).
[CrossRef] [PubMed]

Bueno, J. M.

E. J. Gualda, J. M. Bueno, and P. Artal, “Wavefront optimized nonlinear microscopy of ex vivo human retinas,” J. Biomed. Opt.15(2), 026007 (2010).
[CrossRef] [PubMed]

Callaway, E. M.

L. Yin, Y. Geng, F. Osakada, R. Sharma, A. H. Cetin, E. M. Callaway, D. R. Williams, and W. H. Merigan, “Imaging light responses of retinal ganglion cells in the living mouse eye,” J. Neurophysiol.109(9), 2415–2421 (2013).
[CrossRef] [PubMed]

F. Osakada, T. Mori, A. H. Cetin, J. H. Marshel, B. Virgen, and E. M. Callaway, “New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits,” Neuron71(4), 617–631 (2011).
[CrossRef] [PubMed]

Campbell, M.

Carroll, J.

Cetin, A. H.

L. Yin, Y. Geng, F. Osakada, R. Sharma, A. H. Cetin, E. M. Callaway, D. R. Williams, and W. H. Merigan, “Imaging light responses of retinal ganglion cells in the living mouse eye,” J. Neurophysiol.109(9), 2415–2421 (2013).
[CrossRef] [PubMed]

F. Osakada, T. Mori, A. H. Cetin, J. H. Marshel, B. Virgen, and E. M. Callaway, “New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits,” Neuron71(4), 617–631 (2011).
[CrossRef] [PubMed]

Ch’ng, Y. H.

K. Ohki, S. Chung, Y. H. Ch’ng, P. Kara, and R. C. Reid, “Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex,” Nature433(7026), 597–603 (2005).
[CrossRef] [PubMed]

Chalasani, S. H.

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies, and mice with improved GCaMP calcium indicators,” Nat. Methods6(12), 875–881 (2009).
[CrossRef] [PubMed]

Chalupa, L. M.

J. Coombs, D. van der List, G.-Y. Wang, and L. M. Chalupa, “Morphological properties of mouse retinal ganglion cells,” Neuroscience140(1), 123–136 (2006).
[CrossRef] [PubMed]

Chiappe, M. E.

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies, and mice with improved GCaMP calcium indicators,” Nat. Methods6(12), 875–881 (2009).
[CrossRef] [PubMed]

Chung, M.

E. A. Rossi, M. Chung, A. Dubra, J. J. Hunter, W. H. Merigan, and D. R. Williams, “Imaging retinal mosaics in the living eye,” Eye (Lond.)25(3), 301–308 (2011).
[CrossRef] [PubMed]

Chung, S.

K. Ohki, S. Chung, Y. H. Ch’ng, P. Kara, and R. C. Reid, “Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex,” Nature433(7026), 597–603 (2005).
[CrossRef] [PubMed]

Coombs, J.

J. Coombs, D. van der List, G.-Y. Wang, and L. M. Chalupa, “Morphological properties of mouse retinal ganglion cells,” Neuroscience140(1), 123–136 (2006).
[CrossRef] [PubMed]

Cooper, R. F.

Curcio, C. A.

Dalkara, D.

L. Yin, K. Greenberg, J. J. Hunter, D. Dalkara, K. D. Kolstad, B. D. Masella, R. Wolfe, M. Visel, D. Stone, R. T. Libby, D. Diloreto, D. Schaffer, J. Flannery, D. R. Williams, and W. H. Merigan, “Intravitreal injection of AAV2 transduces macaque inner retina,” Invest. Ophthalmol. Vis. Sci.52(5), 2775–2783 (2011).
[CrossRef] [PubMed]

Dedek, K.

M. Pottek, G. C. Knop, R. Weiler, and K. Dedek, “Electrophysiological characterization of GFP-expressing cell populations in the intact retina,” J. Vis. Exp. (57), 13473–13474 (2011).
[CrossRef] [PubMed]

Demb, J. B.

Y. V. Wang, M. Weick, and J. B. Demb, “Spectral and temporal sensitivity of cone-mediated responses in mouse retinal ganglion cells,” J. Neurosci.31(21), 7670–7681 (2011).
[CrossRef] [PubMed]

Denk, W.

T. Euler, P. B. Detwiler, and W. Denk, “Directionally selective calcium signals in dendrites of starburst amacrine cells,” Nature418(6900), 845–852 (2002).
[CrossRef] [PubMed]

Detwiler, P. B.

T. Euler, P. B. Detwiler, and W. Denk, “Directionally selective calcium signals in dendrites of starburst amacrine cells,” Nature418(6900), 845–852 (2002).
[CrossRef] [PubMed]

Diloreto, D.

L. Yin, K. Greenberg, J. J. Hunter, D. Dalkara, K. D. Kolstad, B. D. Masella, R. Wolfe, M. Visel, D. Stone, R. T. Libby, D. Diloreto, D. Schaffer, J. Flannery, D. R. Williams, and W. H. Merigan, “Intravitreal injection of AAV2 transduces macaque inner retina,” Invest. Ophthalmol. Vis. Sci.52(5), 2775–2783 (2011).
[CrossRef] [PubMed]

Donnelly Iii, W.

Dubis, A. M.

Dubra, A.

Y. Geng, A. Dubra, L. Yin, W. H. Merigan, R. Sharma, R. T. Libby, and D. R. Williams, “Adaptive optics retinal imaging in the living mouse eye,” Biomed. Opt. Express3(4), 715–734 (2012).
[CrossRef] [PubMed]

A. Dubra, Y. Sulai, J. L. Norris, R. F. Cooper, A. M. Dubis, D. R. Williams, and J. Carroll, “Noninvasive imaging of the human rod photoreceptor mosaic using a confocal adaptive optics scanning ophthalmoscope,” Biomed. Opt. Express2(7), 1864–1876 (2011).
[CrossRef] [PubMed]

E. A. Rossi, M. Chung, A. Dubra, J. J. Hunter, W. H. Merigan, and D. R. Williams, “Imaging retinal mosaics in the living eye,” Eye (Lond.)25(3), 301–308 (2011).
[CrossRef] [PubMed]

J. J. Hunter, B. Masella, A. Dubra, R. Sharma, L. Yin, W. H. Merigan, G. Palczewska, K. Palczewski, and D. R. Williams, “Images of photoreceptors in living primate eyes using adaptive optics two-photon ophthalmoscopy,” Biomed. Opt. Express2(1), 139–148 (2010).
[CrossRef] [PubMed]

J. I. W. Morgan, A. Dubra, R. Wolfe, W. H. Merigan, and D. R. Williams, “In vivo autofluorescence imaging of the human and macaque retinal pigment epithelial cell mosaic,” Invest. Ophthalmol. Vis. Sci.50(3), 1350–1359 (2008).
[CrossRef] [PubMed]

D. C. Gray, W. Merigan, J. I. Wolfing, B. P. Gee, J. Porter, A. Dubra, T. H. Twietmeyer, K. Ahamd, R. Tumbar, F. Reinholz, and D. R. Williams, “In vivo fluorescence imaging of primate retinal ganglion cells and retinal pigment epithelial cells,” Opt. Express14(16), 7144–7158 (2006).
[CrossRef] [PubMed]

Elstrott, J.

W. Wei, J. Elstrott, and M. B. Feller, “Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina,” Nat. Protoc.5(7), 1347–1352 (2010).
[CrossRef] [PubMed]

Esdaille, T. M.

F. Naarendorp, T. M. Esdaille, S. M. Banden, J. Andrews-Labenski, O. P. Gross, and E. N. Pugh., “Dark light, rod saturation, and the absolute and incremental sensitivity of mouse cone vision,” J. Neurosci.30(37), 12495–12507 (2010).
[CrossRef] [PubMed]

Euler, T.

T. Euler, P. B. Detwiler, and W. Denk, “Directionally selective calcium signals in dendrites of starburst amacrine cells,” Nature418(6900), 845–852 (2002).
[CrossRef] [PubMed]

Feller, M. B.

W. Wei, J. Elstrott, and M. B. Feller, “Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina,” Nat. Protoc.5(7), 1347–1352 (2010).
[CrossRef] [PubMed]

Flannery, J.

L. Yin, K. Greenberg, J. J. Hunter, D. Dalkara, K. D. Kolstad, B. D. Masella, R. Wolfe, M. Visel, D. Stone, R. T. Libby, D. Diloreto, D. Schaffer, J. Flannery, D. R. Williams, and W. H. Merigan, “Intravitreal injection of AAV2 transduces macaque inner retina,” Invest. Ophthalmol. Vis. Sci.52(5), 2775–2783 (2011).
[CrossRef] [PubMed]

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D. Scoles, D. C. Gray, J. J. Hunter, R. Wolfe, B. P. Gee, Y. Geng, B. D. Masella, R. T. Libby, S. Russell, D. R. Williams, and W. H. Merigan, “In vivo imaging of retinal nerve fiber layer vasculature: imaging histology comparison,” BMC Ophthalmol.9(1), 1–9 (2009).
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D. C. Gray, W. Merigan, J. I. Wolfing, B. P. Gee, J. Porter, A. Dubra, T. H. Twietmeyer, K. Ahamd, R. Tumbar, F. Reinholz, and D. R. Williams, “In vivo fluorescence imaging of primate retinal ganglion cells and retinal pigment epithelial cells,” Opt. Express14(16), 7144–7158 (2006).
[CrossRef] [PubMed]

Geng, Y.

L. Yin, Y. Geng, F. Osakada, R. Sharma, A. H. Cetin, E. M. Callaway, D. R. Williams, and W. H. Merigan, “Imaging light responses of retinal ganglion cells in the living mouse eye,” J. Neurophysiol.109(9), 2415–2421 (2013).
[CrossRef] [PubMed]

Y. Geng, A. Dubra, L. Yin, W. H. Merigan, R. Sharma, R. T. Libby, and D. R. Williams, “Adaptive optics retinal imaging in the living mouse eye,” Biomed. Opt. Express3(4), 715–734 (2012).
[CrossRef] [PubMed]

D. Scoles, D. C. Gray, J. J. Hunter, R. Wolfe, B. P. Gee, Y. Geng, B. D. Masella, R. T. Libby, S. Russell, D. R. Williams, and W. H. Merigan, “In vivo imaging of retinal nerve fiber layer vasculature: imaging histology comparison,” BMC Ophthalmol.9(1), 1–9 (2009).
[CrossRef] [PubMed]

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D. Scoles, D. C. Gray, J. J. Hunter, R. Wolfe, B. P. Gee, Y. Geng, B. D. Masella, R. T. Libby, S. Russell, D. R. Williams, and W. H. Merigan, “In vivo imaging of retinal nerve fiber layer vasculature: imaging histology comparison,” BMC Ophthalmol.9(1), 1–9 (2009).
[CrossRef] [PubMed]

D. C. Gray, W. Merigan, J. I. Wolfing, B. P. Gee, J. Porter, A. Dubra, T. H. Twietmeyer, K. Ahamd, R. Tumbar, F. Reinholz, and D. R. Williams, “In vivo fluorescence imaging of primate retinal ganglion cells and retinal pigment epithelial cells,” Opt. Express14(16), 7144–7158 (2006).
[CrossRef] [PubMed]

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L. Yin, K. Greenberg, J. J. Hunter, D. Dalkara, K. D. Kolstad, B. D. Masella, R. Wolfe, M. Visel, D. Stone, R. T. Libby, D. Diloreto, D. Schaffer, J. Flannery, D. R. Williams, and W. H. Merigan, “Intravitreal injection of AAV2 transduces macaque inner retina,” Invest. Ophthalmol. Vis. Sci.52(5), 2775–2783 (2011).
[CrossRef] [PubMed]

Gross, O. P.

F. Naarendorp, T. M. Esdaille, S. M. Banden, J. Andrews-Labenski, O. P. Gross, and E. N. Pugh., “Dark light, rod saturation, and the absolute and incremental sensitivity of mouse cone vision,” J. Neurosci.30(37), 12495–12507 (2010).
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E. J. Gualda, J. M. Bueno, and P. Artal, “Wavefront optimized nonlinear microscopy of ex vivo human retinas,” J. Biomed. Opt.15(2), 026007 (2010).
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Hires, S. A.

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies, and mice with improved GCaMP calcium indicators,” Nat. Methods6(12), 875–881 (2009).
[CrossRef] [PubMed]

Huber, D.

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies, and mice with improved GCaMP calcium indicators,” Nat. Methods6(12), 875–881 (2009).
[CrossRef] [PubMed]

Hunter, J. J.

E. A. Rossi, M. Chung, A. Dubra, J. J. Hunter, W. H. Merigan, and D. R. Williams, “Imaging retinal mosaics in the living eye,” Eye (Lond.)25(3), 301–308 (2011).
[CrossRef] [PubMed]

L. Yin, K. Greenberg, J. J. Hunter, D. Dalkara, K. D. Kolstad, B. D. Masella, R. Wolfe, M. Visel, D. Stone, R. T. Libby, D. Diloreto, D. Schaffer, J. Flannery, D. R. Williams, and W. H. Merigan, “Intravitreal injection of AAV2 transduces macaque inner retina,” Invest. Ophthalmol. Vis. Sci.52(5), 2775–2783 (2011).
[CrossRef] [PubMed]

J. J. Hunter, B. Masella, A. Dubra, R. Sharma, L. Yin, W. H. Merigan, G. Palczewska, K. Palczewski, and D. R. Williams, “Images of photoreceptors in living primate eyes using adaptive optics two-photon ophthalmoscopy,” Biomed. Opt. Express2(1), 139–148 (2010).
[CrossRef] [PubMed]

D. Scoles, D. C. Gray, J. J. Hunter, R. Wolfe, B. P. Gee, Y. Geng, B. D. Masella, R. T. Libby, S. Russell, D. R. Williams, and W. H. Merigan, “In vivo imaging of retinal nerve fiber layer vasculature: imaging histology comparison,” BMC Ophthalmol.9(1), 1–9 (2009).
[CrossRef] [PubMed]

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Y. Imanishi, K. H. Lodowski, and Y. Koutalos, “Two-photon microscopy: shedding light on the chemistry of vision,” Biochemistry46(34), 9674–9684 (2007).
[CrossRef] [PubMed]

Y. Imanishi, M. L. Batten, D. W. Piston, W. Baehr, and K. Palczewski, “Noninvasive two-photon imaging reveals retinyl ester storage structures in the eye,” J. Cell Biol.164(3), 373–383 (2004).
[CrossRef] [PubMed]

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L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies, and mice with improved GCaMP calcium indicators,” Nat. Methods6(12), 875–881 (2009).
[CrossRef] [PubMed]

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K. Ohki, S. Chung, Y. H. Ch’ng, P. Kara, and R. C. Reid, “Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex,” Nature433(7026), 597–603 (2005).
[CrossRef] [PubMed]

Keyser, K.

Knop, G. C.

M. Pottek, G. C. Knop, R. Weiler, and K. Dedek, “Electrophysiological characterization of GFP-expressing cell populations in the intact retina,” J. Vis. Exp. (57), 13473–13474 (2011).
[CrossRef] [PubMed]

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L. Yin, K. Greenberg, J. J. Hunter, D. Dalkara, K. D. Kolstad, B. D. Masella, R. Wolfe, M. Visel, D. Stone, R. T. Libby, D. Diloreto, D. Schaffer, J. Flannery, D. R. Williams, and W. H. Merigan, “Intravitreal injection of AAV2 transduces macaque inner retina,” Invest. Ophthalmol. Vis. Sci.52(5), 2775–2783 (2011).
[CrossRef] [PubMed]

Koutalos, Y.

Y. Imanishi, K. H. Lodowski, and Y. Koutalos, “Two-photon microscopy: shedding light on the chemistry of vision,” Biochemistry46(34), 9674–9684 (2007).
[CrossRef] [PubMed]

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Liang, J.

Libby, R. T.

Y. Geng, A. Dubra, L. Yin, W. H. Merigan, R. Sharma, R. T. Libby, and D. R. Williams, “Adaptive optics retinal imaging in the living mouse eye,” Biomed. Opt. Express3(4), 715–734 (2012).
[CrossRef] [PubMed]

L. Yin, K. Greenberg, J. J. Hunter, D. Dalkara, K. D. Kolstad, B. D. Masella, R. Wolfe, M. Visel, D. Stone, R. T. Libby, D. Diloreto, D. Schaffer, J. Flannery, D. R. Williams, and W. H. Merigan, “Intravitreal injection of AAV2 transduces macaque inner retina,” Invest. Ophthalmol. Vis. Sci.52(5), 2775–2783 (2011).
[CrossRef] [PubMed]

D. Scoles, D. C. Gray, J. J. Hunter, R. Wolfe, B. P. Gee, Y. Geng, B. D. Masella, R. T. Libby, S. Russell, D. R. Williams, and W. H. Merigan, “In vivo imaging of retinal nerve fiber layer vasculature: imaging histology comparison,” BMC Ophthalmol.9(1), 1–9 (2009).
[CrossRef] [PubMed]

Lodowski, K. H.

Y. Imanishi, K. H. Lodowski, and Y. Koutalos, “Two-photon microscopy: shedding light on the chemistry of vision,” Biochemistry46(34), 9674–9684 (2007).
[CrossRef] [PubMed]

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B. G. Borghuis, L. Tian, Y. Xu, S. S. Nikonov, N. Vardi, B. V. Zemelman, and L. L. Looger, “Imaging light responses of targeted neuron populations in the rodent retina,” J. Neurosci.31(8), 2855–2867 (2011).
[CrossRef] [PubMed]

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies, and mice with improved GCaMP calcium indicators,” Nat. Methods6(12), 875–881 (2009).
[CrossRef] [PubMed]

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Mao, T.

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies, and mice with improved GCaMP calcium indicators,” Nat. Methods6(12), 875–881 (2009).
[CrossRef] [PubMed]

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F. Osakada, T. Mori, A. H. Cetin, J. H. Marshel, B. Virgen, and E. M. Callaway, “New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits,” Neuron71(4), 617–631 (2011).
[CrossRef] [PubMed]

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Masella, B. D.

L. Yin, K. Greenberg, J. J. Hunter, D. Dalkara, K. D. Kolstad, B. D. Masella, R. Wolfe, M. Visel, D. Stone, R. T. Libby, D. Diloreto, D. Schaffer, J. Flannery, D. R. Williams, and W. H. Merigan, “Intravitreal injection of AAV2 transduces macaque inner retina,” Invest. Ophthalmol. Vis. Sci.52(5), 2775–2783 (2011).
[CrossRef] [PubMed]

D. Scoles, D. C. Gray, J. J. Hunter, R. Wolfe, B. P. Gee, Y. Geng, B. D. Masella, R. T. Libby, S. Russell, D. R. Williams, and W. H. Merigan, “In vivo imaging of retinal nerve fiber layer vasculature: imaging histology comparison,” BMC Ophthalmol.9(1), 1–9 (2009).
[CrossRef] [PubMed]

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L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies, and mice with improved GCaMP calcium indicators,” Nat. Methods6(12), 875–881 (2009).
[CrossRef] [PubMed]

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Merigan, W. H.

L. Yin, Y. Geng, F. Osakada, R. Sharma, A. H. Cetin, E. M. Callaway, D. R. Williams, and W. H. Merigan, “Imaging light responses of retinal ganglion cells in the living mouse eye,” J. Neurophysiol.109(9), 2415–2421 (2013).
[CrossRef] [PubMed]

Y. Geng, A. Dubra, L. Yin, W. H. Merigan, R. Sharma, R. T. Libby, and D. R. Williams, “Adaptive optics retinal imaging in the living mouse eye,” Biomed. Opt. Express3(4), 715–734 (2012).
[CrossRef] [PubMed]

E. A. Rossi, M. Chung, A. Dubra, J. J. Hunter, W. H. Merigan, and D. R. Williams, “Imaging retinal mosaics in the living eye,” Eye (Lond.)25(3), 301–308 (2011).
[CrossRef] [PubMed]

L. Yin, K. Greenberg, J. J. Hunter, D. Dalkara, K. D. Kolstad, B. D. Masella, R. Wolfe, M. Visel, D. Stone, R. T. Libby, D. Diloreto, D. Schaffer, J. Flannery, D. R. Williams, and W. H. Merigan, “Intravitreal injection of AAV2 transduces macaque inner retina,” Invest. Ophthalmol. Vis. Sci.52(5), 2775–2783 (2011).
[CrossRef] [PubMed]

J. J. Hunter, B. Masella, A. Dubra, R. Sharma, L. Yin, W. H. Merigan, G. Palczewska, K. Palczewski, and D. R. Williams, “Images of photoreceptors in living primate eyes using adaptive optics two-photon ophthalmoscopy,” Biomed. Opt. Express2(1), 139–148 (2010).
[CrossRef] [PubMed]

D. Scoles, D. C. Gray, J. J. Hunter, R. Wolfe, B. P. Gee, Y. Geng, B. D. Masella, R. T. Libby, S. Russell, D. R. Williams, and W. H. Merigan, “In vivo imaging of retinal nerve fiber layer vasculature: imaging histology comparison,” BMC Ophthalmol.9(1), 1–9 (2009).
[CrossRef] [PubMed]

J. I. W. Morgan, A. Dubra, R. Wolfe, W. H. Merigan, and D. R. Williams, “In vivo autofluorescence imaging of the human and macaque retinal pigment epithelial cell mosaic,” Invest. Ophthalmol. Vis. Sci.50(3), 1350–1359 (2008).
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Miller, D. T.

Morgan, J. I. W.

J. I. W. Morgan, A. Dubra, R. Wolfe, W. H. Merigan, and D. R. Williams, “In vivo autofluorescence imaging of the human and macaque retinal pigment epithelial cell mosaic,” Invest. Ophthalmol. Vis. Sci.50(3), 1350–1359 (2008).
[CrossRef] [PubMed]

Mori, T.

F. Osakada, T. Mori, A. H. Cetin, J. H. Marshel, B. Virgen, and E. M. Callaway, “New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits,” Neuron71(4), 617–631 (2011).
[CrossRef] [PubMed]

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F. Naarendorp, T. M. Esdaille, S. M. Banden, J. Andrews-Labenski, O. P. Gross, and E. N. Pugh., “Dark light, rod saturation, and the absolute and incremental sensitivity of mouse cone vision,” J. Neurosci.30(37), 12495–12507 (2010).
[CrossRef] [PubMed]

Nikonov, S. S.

B. G. Borghuis, L. Tian, Y. Xu, S. S. Nikonov, N. Vardi, B. V. Zemelman, and L. L. Looger, “Imaging light responses of targeted neuron populations in the rodent retina,” J. Neurosci.31(8), 2855–2867 (2011).
[CrossRef] [PubMed]

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Ohki, K.

K. Ohki, S. Chung, Y. H. Ch’ng, P. Kara, and R. C. Reid, “Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex,” Nature433(7026), 597–603 (2005).
[CrossRef] [PubMed]

Osakada, F.

L. Yin, Y. Geng, F. Osakada, R. Sharma, A. H. Cetin, E. M. Callaway, D. R. Williams, and W. H. Merigan, “Imaging light responses of retinal ganglion cells in the living mouse eye,” J. Neurophysiol.109(9), 2415–2421 (2013).
[CrossRef] [PubMed]

F. Osakada, T. Mori, A. H. Cetin, J. H. Marshel, B. Virgen, and E. M. Callaway, “New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits,” Neuron71(4), 617–631 (2011).
[CrossRef] [PubMed]

Palczewska, G.

Palczewski, K.

Petreanu, L.

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies, and mice with improved GCaMP calcium indicators,” Nat. Methods6(12), 875–881 (2009).
[CrossRef] [PubMed]

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Y. Imanishi, M. L. Batten, D. W. Piston, W. Baehr, and K. Palczewski, “Noninvasive two-photon imaging reveals retinyl ester storage structures in the eye,” J. Cell Biol.164(3), 373–383 (2004).
[CrossRef] [PubMed]

Porter, J.

Pottek, M.

M. Pottek, G. C. Knop, R. Weiler, and K. Dedek, “Electrophysiological characterization of GFP-expressing cell populations in the intact retina,” J. Vis. Exp. (57), 13473–13474 (2011).
[CrossRef] [PubMed]

Pugh, E. N.

F. Naarendorp, T. M. Esdaille, S. M. Banden, J. Andrews-Labenski, O. P. Gross, and E. N. Pugh., “Dark light, rod saturation, and the absolute and incremental sensitivity of mouse cone vision,” J. Neurosci.30(37), 12495–12507 (2010).
[CrossRef] [PubMed]

Queener, H.

Reid, R. C.

K. Ohki, S. Chung, Y. H. Ch’ng, P. Kara, and R. C. Reid, “Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex,” Nature433(7026), 597–603 (2005).
[CrossRef] [PubMed]

Reinholz, F.

Romero-Borja, F.

Roorda, A.

Rossi, E. A.

E. A. Rossi, M. Chung, A. Dubra, J. J. Hunter, W. H. Merigan, and D. R. Williams, “Imaging retinal mosaics in the living eye,” Eye (Lond.)25(3), 301–308 (2011).
[CrossRef] [PubMed]

Russell, S.

D. Scoles, D. C. Gray, J. J. Hunter, R. Wolfe, B. P. Gee, Y. Geng, B. D. Masella, R. T. Libby, S. Russell, D. R. Williams, and W. H. Merigan, “In vivo imaging of retinal nerve fiber layer vasculature: imaging histology comparison,” BMC Ophthalmol.9(1), 1–9 (2009).
[CrossRef] [PubMed]

Schaffer, D.

L. Yin, K. Greenberg, J. J. Hunter, D. Dalkara, K. D. Kolstad, B. D. Masella, R. Wolfe, M. Visel, D. Stone, R. T. Libby, D. Diloreto, D. Schaffer, J. Flannery, D. R. Williams, and W. H. Merigan, “Intravitreal injection of AAV2 transduces macaque inner retina,” Invest. Ophthalmol. Vis. Sci.52(5), 2775–2783 (2011).
[CrossRef] [PubMed]

Schreiter, E. R.

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies, and mice with improved GCaMP calcium indicators,” Nat. Methods6(12), 875–881 (2009).
[CrossRef] [PubMed]

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D. Scoles, D. C. Gray, J. J. Hunter, R. Wolfe, B. P. Gee, Y. Geng, B. D. Masella, R. T. Libby, S. Russell, D. R. Williams, and W. H. Merigan, “In vivo imaging of retinal nerve fiber layer vasculature: imaging histology comparison,” BMC Ophthalmol.9(1), 1–9 (2009).
[CrossRef] [PubMed]

Sharma, R.

Stone, D.

L. Yin, K. Greenberg, J. J. Hunter, D. Dalkara, K. D. Kolstad, B. D. Masella, R. Wolfe, M. Visel, D. Stone, R. T. Libby, D. Diloreto, D. Schaffer, J. Flannery, D. R. Williams, and W. H. Merigan, “Intravitreal injection of AAV2 transduces macaque inner retina,” Invest. Ophthalmol. Vis. Sci.52(5), 2775–2783 (2011).
[CrossRef] [PubMed]

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Sulai, Y.

Svoboda, K.

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies, and mice with improved GCaMP calcium indicators,” Nat. Methods6(12), 875–881 (2009).
[CrossRef] [PubMed]

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B. G. Borghuis, L. Tian, Y. Xu, S. S. Nikonov, N. Vardi, B. V. Zemelman, and L. L. Looger, “Imaging light responses of targeted neuron populations in the rodent retina,” J. Neurosci.31(8), 2855–2867 (2011).
[CrossRef] [PubMed]

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies, and mice with improved GCaMP calcium indicators,” Nat. Methods6(12), 875–881 (2009).
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Twietmeyer, T. H.

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J. Coombs, D. van der List, G.-Y. Wang, and L. M. Chalupa, “Morphological properties of mouse retinal ganglion cells,” Neuroscience140(1), 123–136 (2006).
[CrossRef] [PubMed]

Vardi, N.

B. G. Borghuis, L. Tian, Y. Xu, S. S. Nikonov, N. Vardi, B. V. Zemelman, and L. L. Looger, “Imaging light responses of targeted neuron populations in the rodent retina,” J. Neurosci.31(8), 2855–2867 (2011).
[CrossRef] [PubMed]

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F. Osakada, T. Mori, A. H. Cetin, J. H. Marshel, B. Virgen, and E. M. Callaway, “New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits,” Neuron71(4), 617–631 (2011).
[CrossRef] [PubMed]

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L. Yin, K. Greenberg, J. J. Hunter, D. Dalkara, K. D. Kolstad, B. D. Masella, R. Wolfe, M. Visel, D. Stone, R. T. Libby, D. Diloreto, D. Schaffer, J. Flannery, D. R. Williams, and W. H. Merigan, “Intravitreal injection of AAV2 transduces macaque inner retina,” Invest. Ophthalmol. Vis. Sci.52(5), 2775–2783 (2011).
[CrossRef] [PubMed]

Wang, G.-Y.

J. Coombs, D. van der List, G.-Y. Wang, and L. M. Chalupa, “Morphological properties of mouse retinal ganglion cells,” Neuroscience140(1), 123–136 (2006).
[CrossRef] [PubMed]

Wang, Y. V.

Y. V. Wang, M. Weick, and J. B. Demb, “Spectral and temporal sensitivity of cone-mediated responses in mouse retinal ganglion cells,” J. Neurosci.31(21), 7670–7681 (2011).
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W. Wei, J. Elstrott, and M. B. Feller, “Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina,” Nat. Protoc.5(7), 1347–1352 (2010).
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Y. V. Wang, M. Weick, and J. B. Demb, “Spectral and temporal sensitivity of cone-mediated responses in mouse retinal ganglion cells,” J. Neurosci.31(21), 7670–7681 (2011).
[CrossRef] [PubMed]

Weiler, R.

M. Pottek, G. C. Knop, R. Weiler, and K. Dedek, “Electrophysiological characterization of GFP-expressing cell populations in the intact retina,” J. Vis. Exp. (57), 13473–13474 (2011).
[CrossRef] [PubMed]

Williams, D. R.

L. Yin, Y. Geng, F. Osakada, R. Sharma, A. H. Cetin, E. M. Callaway, D. R. Williams, and W. H. Merigan, “Imaging light responses of retinal ganglion cells in the living mouse eye,” J. Neurophysiol.109(9), 2415–2421 (2013).
[CrossRef] [PubMed]

Y. Geng, A. Dubra, L. Yin, W. H. Merigan, R. Sharma, R. T. Libby, and D. R. Williams, “Adaptive optics retinal imaging in the living mouse eye,” Biomed. Opt. Express3(4), 715–734 (2012).
[CrossRef] [PubMed]

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J. J. Hunter, B. Masella, A. Dubra, R. Sharma, L. Yin, W. H. Merigan, G. Palczewska, K. Palczewski, and D. R. Williams, “Images of photoreceptors in living primate eyes using adaptive optics two-photon ophthalmoscopy,” Biomed. Opt. Express2(1), 139–148 (2010).
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B. G. Borghuis, L. Tian, Y. Xu, S. S. Nikonov, N. Vardi, B. V. Zemelman, and L. L. Looger, “Imaging light responses of targeted neuron populations in the rodent retina,” J. Neurosci.31(8), 2855–2867 (2011).
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D. Scoles, D. C. Gray, J. J. Hunter, R. Wolfe, B. P. Gee, Y. Geng, B. D. Masella, R. T. Libby, S. Russell, D. R. Williams, and W. H. Merigan, “In vivo imaging of retinal nerve fiber layer vasculature: imaging histology comparison,” BMC Ophthalmol.9(1), 1–9 (2009).
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Eye (Lond.) (1)

E. A. Rossi, M. Chung, A. Dubra, J. J. Hunter, W. H. Merigan, and D. R. Williams, “Imaging retinal mosaics in the living eye,” Eye (Lond.)25(3), 301–308 (2011).
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Invest. Ophthalmol. Vis. Sci. (2)

L. Yin, K. Greenberg, J. J. Hunter, D. Dalkara, K. D. Kolstad, B. D. Masella, R. Wolfe, M. Visel, D. Stone, R. T. Libby, D. Diloreto, D. Schaffer, J. Flannery, D. R. Williams, and W. H. Merigan, “Intravitreal injection of AAV2 transduces macaque inner retina,” Invest. Ophthalmol. Vis. Sci.52(5), 2775–2783 (2011).
[CrossRef] [PubMed]

J. I. W. Morgan, A. Dubra, R. Wolfe, W. H. Merigan, and D. R. Williams, “In vivo autofluorescence imaging of the human and macaque retinal pigment epithelial cell mosaic,” Invest. Ophthalmol. Vis. Sci.50(3), 1350–1359 (2008).
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L. Yin, Y. Geng, F. Osakada, R. Sharma, A. H. Cetin, E. M. Callaway, D. R. Williams, and W. H. Merigan, “Imaging light responses of retinal ganglion cells in the living mouse eye,” J. Neurophysiol.109(9), 2415–2421 (2013).
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B. G. Borghuis, L. Tian, Y. Xu, S. S. Nikonov, N. Vardi, B. V. Zemelman, and L. L. Looger, “Imaging light responses of targeted neuron populations in the rodent retina,” J. Neurosci.31(8), 2855–2867 (2011).
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Supplementary Material (1)

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Figures (4)

Fig. 1
Fig. 1

Layout of 2PAOSLO for imaging mouse retina. Light from the pulsed ultrafast laser passes through the DeepSee dispersion compensation attachment (not shown) and is then focused through a spatial filter to generate a point source of light. The detection channel for two-photon fluorescence collection as shown in the diagram is close to the eye and fluorescence emitted by cells in the eye is directed into this detector using a dichroic that reflects visible light and transmits infrared wavelengths. For reflectance imaging and wavefront sensing, light scattered from the retina propagates back through the system and is detected near the light sources, as is typical in most AOSLO systems. The rest of the system is identical to the AOSLO described previously by Geng et al [4].

Fig. 2
Fig. 2

Images of GFP labeled cells. Panel (a) is a single frame taken from a video showing the real-time two-photon signal from labeled ganglion cells (Media 1). Panel (b) is a registered image of 5000 frames of the video in panel (a). Two bright cells are visible (labeled 1 and 2 in (a)) as well as many additional fainter cells that are likely at different depths. Panels (c) and (d) show inner retinal cells at the same retinal location but at axial depths separated by 5 μm. Subcellular structures, including dendritic morphology are clearly visible. This shows that the system has sufficient intrinsic axial sectioning capabilities despite the absence of a confocal pinhole. Scale bar is 50 μm. Images are contrast stretched for optimal visualization.

Fig. 3
Fig. 3

(a) Image of ganglion cells labeled with calcium indicator G-CaMP3. Arrow in white points to the cell that was used for analysis of SNR of in vivo two-photon fluorescence imaging of G-CaMP3 labeled cells (outlined detail in the discussion section), (b) Plot of the signal level from the cell marked with the white arrow in the image shown in (a) over time showing stable activation and minimal photobleaching. The raw data for each frame has been plotted in gray. The mean gray level averaged over every 1 second has been plotted in red. Error bars represent the standard deviation of the mean. Scale bar shown in (a) is 50 μm. Image is contrast-stretched for display purposes.

Fig. 4
Fig. 4

(a) Plot of spatio-temporal integration required to capture sufficient signal (SNR ~10 and ~100) from individual ganglion cells labeled with GFP and G-CaMP3 when imaged with single-photon or two-photon imaging modalities. The figure shows a trade-off between spatial and temporal integration for collecting fluorescence signals at equal SNRs. The solid gray dots represent data points from in vivo imaging experiments. Single-photon G-CaMP3 data is from Yin et al [15]. The shaded region between the two dashed horizontal lines indicates the range of cell sizes for ganglion cells from literature [23] (b) Comparison of visual pigment isomerization in the mouse retina between single-photon imaging using 200 μW of 488 nm light and two-photon imaging using 6 mW of 920 nm light for imaging of G-CaMP3 labeled ganglion cells. The black horizontal line corresponds to the commencement of the photopic regime for the mouse visual system (5200 Rh*/photoreceptor/sec) [2426].

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