Abstract

In vivo two-photon imaging through the pupil of the primate eye has the potential to become a useful tool for functional imaging of the retina. Two-photon excited fluorescence images of the macaque cone mosaic were obtained using a fluorescence adaptive optics scanning laser ophthalmoscope, overcoming the challenges of a low numerical aperture, imperfect optics of the eye, high required light levels, and eye motion. Although the specific fluorophores are as yet unknown, strong in vivo intrinsic fluorescence allowed images of the cone mosaic. Imaging intact ex vivo retina revealed that the strongest two-photon excited fluorescence signal comes from the cone inner segments. The fluorescence response increased following light stimulation, which could provide a functional measure of the effects of light on photoreceptors.

© 2010 OSA

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    [PubMed]

2010

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]

2009

D. Ts’o, J. Schallek, Y. Kwon, R. Kardon, M. Abramoff, and P. Soliz, “Noninvasive functional imaging of the retina reveals outer retinal and hemodynamic intrinsic optical signal origins,” Jpn. J. Ophthalmol. 53(4), 334–344 (2009).
[CrossRef] [PubMed]

K. Tsunoda, G. Hanazono, K. Inomata, Y. Kazato, W. Suzuki, and M. Tanifuji, “Origins of retinal intrinsic signals: a series of experiments on retinas of macaque monkeys,” Jpn. J. Ophthalmol. 53(4), 297–314 (2009).
[CrossRef] [PubMed]

V. J. Srinivasan, Y. Chen, J. S. Duker, and J. G. Fujimoto, “In vivo functional imaging of intrinsic scattering changes in the human retina with high-speed ultrahigh resolution OCT,” Opt. Express 17(5), 3861–3877 (2009).
[CrossRef] [PubMed]

J. S. Wang and V. J. Kefalov, “An alternative pathway mediates the mouse and human cone visual cycle,” Curr. Biol. 19(19), 1665–1669 (2009).
[CrossRef] [PubMed]

2008

O. La Schiazza and J. F. Bille, “High-speed two-photon excited autofluorescence imaging of ex vivo human retinal pigment epithelial cells toward age-related macular degeneration diagnostic,” J. Biomed. Opt. 13(6), 064008 (2008).
[CrossRef] [PubMed]

J. I. Morgan, J. J. Hunter, B. Masella, R. Wolfe, D. C. Gray, W. H. Merigan, F. C. Delori, and D. R. Williams, “Light-induced retinal changes observed with high-resolution autofluorescence imaging of the retinal pigment epithelium,” Invest. Ophthalmol. Vis. Sci. 49(8), 3715–3729 (2008).
[CrossRef] [PubMed]

J. I. 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]

K. Grieve and A. Roorda, “Intrinsic signals from human cone photoreceptors,” Invest. Ophthalmol. Vis. Sci. 49(2), 713–719 (2008).
[CrossRef] [PubMed]

2007

R. S. Jonnal, J. Rha, Y. Zhang, B. Cense, W. Gao, and D. T. Miller, “In vivo functional imaging of human cone photoreceptors,” Opt. Express 15(24), 16141–16160 (2007).
[CrossRef]

F. C. Delori, R. H. Webb, D. H. Sliney, and American National Standards Institute, “Maximum permissible exposures for ocular safety (ANSI 2000), with emphasis on ophthalmic devices,” J. Opt. Soc. Am. A 24(5), 1250–1265 (2007).
[CrossRef] [PubMed]

G. H. Travis, M. Golczak, A. R. Moise, and K. Palczewski, “Diseases caused by defects in the visual cycle: retinoids as potential therapeutic agents,” Annu. Rev. Pharmacol. Toxicol. 47(1), 469–512 (2007).
[CrossRef] [PubMed]

M. Han, G. Giese, S. Schmitz-Valckenberg, A. Bindewald-Wittich, F. G. Holz, J. Yu, J. F. Bille, and M. H. Niemz, “Age-related structural abnormalities in the human retina-choroid complex revealed by two-photon excited autofluorescence imaging,” J. Biomed. Opt. 12(2), 024012 (2007).
[CrossRef] [PubMed]

2006

M. Han, A. Bindewald-Wittich, F. G. Holz, G. Giese, M. H. Niemz, S. Snyder, H. Sun, J. Yu, M. Agopov, O. La Schiazza, and J. F. Bille, “Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells,” J. Biomed. Opt. 11(1), 010501 (2006).
[CrossRef] [PubMed]

A. Bindewald-Wittich, M. Han, S. Schmitz-Valckenberg, S. R. Snyder, G. Giese, J. F. Bille, and F. G. Holz, “Two-photon-excited fluorescence imaging of human RPE cells with a femtosecond Ti:sapphire laser,” Invest. Ophthalmol. Vis. Sci. 47(10), 4553–4557 (2006).
[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. Express 14(16), 7144–7158 (2006).
[CrossRef] [PubMed]

2005

C. Chen, E. Tsina, M. C. Cornwall, R. K. Crouch, S. Vijayaraghavan, and Y. Koutalos, “Reduction of all-trans retinal to all-trans retinol in the outer segments of frog and mouse rod photoreceptors,” Biophys. J. 88(3), 2278–2287 (2005).
[CrossRef] [PubMed]

2004

T. D. Lamb and E. N. Pugh., “Dark adaptation and the retinoid cycle of vision,” Prog. Retin. Eye Res. 23(3), 307–380 (2004).
[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]

2003

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

2002

S. Huang, A. A. Heikal, and W. W. Webb, “Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein,” Biophys. J. 82(5), 2811–2825 (2002).
[CrossRef] [PubMed]

P. Y. Man, D. M. Turnbull, and P. F. Chinnery, “Leber hereditary optic neuropathy,” J. Med. Genet. 39(3), 162–169 (2002).
[CrossRef] [PubMed]

2000

J. Dillon, L. Zheng, J. C. Merriam, and E. R. Gaillard, “Transmission spectra of light to the mammalian retina,” Photochem. Photobiol. 71(2), 225–229 (2000).
[CrossRef] [PubMed]

1995

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]

1990

I. J. Holt, A. E. Harding, R. K. Petty, and J. A. Morgan-Hughes, “A new mitochondrial disease associated with mitochondrial DNA heteroplasmy,” Am. J. Hum. Genet. 46(3), 428–433 (1990).
[PubMed]

1989

R. C. Sears and M. W. Kaplan, “Axial diffusion of retinol in isolated frog rod outer segments following substantial bleaches of visual pigment,” Vision Res. 29(11), 1485–1492 (1989).
[CrossRef] [PubMed]

1979

W. T. Ham, H. A. Mueller, J. J. Ruffolo, and A. M. Clarke, “Sensitivity of the retina to radiation damage as a function of wavelength,” Photochem. Photobiol. 29(4), 735–743 (1979).
[CrossRef] [PubMed]

Abramoff, M.

D. Ts’o, J. Schallek, Y. Kwon, R. Kardon, M. Abramoff, and P. Soliz, “Noninvasive functional imaging of the retina reveals outer retinal and hemodynamic intrinsic optical signal origins,” Jpn. J. Ophthalmol. 53(4), 334–344 (2009).
[CrossRef] [PubMed]

Agopov, M.

M. Han, A. Bindewald-Wittich, F. G. Holz, G. Giese, M. H. Niemz, S. Snyder, H. Sun, J. Yu, M. Agopov, O. La Schiazza, and J. F. Bille, “Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells,” J. Biomed. Opt. 11(1), 010501 (2006).
[CrossRef] [PubMed]

Ahamd, K.

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]

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]

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]

Bille, J. F.

O. La Schiazza and J. F. Bille, “High-speed two-photon excited autofluorescence imaging of ex vivo human retinal pigment epithelial cells toward age-related macular degeneration diagnostic,” J. Biomed. Opt. 13(6), 064008 (2008).
[CrossRef] [PubMed]

M. Han, G. Giese, S. Schmitz-Valckenberg, A. Bindewald-Wittich, F. G. Holz, J. Yu, J. F. Bille, and M. H. Niemz, “Age-related structural abnormalities in the human retina-choroid complex revealed by two-photon excited autofluorescence imaging,” J. Biomed. Opt. 12(2), 024012 (2007).
[CrossRef] [PubMed]

M. Han, A. Bindewald-Wittich, F. G. Holz, G. Giese, M. H. Niemz, S. Snyder, H. Sun, J. Yu, M. Agopov, O. La Schiazza, and J. F. Bille, “Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells,” J. Biomed. Opt. 11(1), 010501 (2006).
[CrossRef] [PubMed]

A. Bindewald-Wittich, M. Han, S. Schmitz-Valckenberg, S. R. Snyder, G. Giese, J. F. Bille, and F. G. Holz, “Two-photon-excited fluorescence imaging of human RPE cells with a femtosecond Ti:sapphire laser,” Invest. Ophthalmol. Vis. Sci. 47(10), 4553–4557 (2006).
[CrossRef] [PubMed]

Bindewald-Wittich, A.

M. Han, G. Giese, S. Schmitz-Valckenberg, A. Bindewald-Wittich, F. G. Holz, J. Yu, J. F. Bille, and M. H. Niemz, “Age-related structural abnormalities in the human retina-choroid complex revealed by two-photon excited autofluorescence imaging,” J. Biomed. Opt. 12(2), 024012 (2007).
[CrossRef] [PubMed]

A. Bindewald-Wittich, M. Han, S. Schmitz-Valckenberg, S. R. Snyder, G. Giese, J. F. Bille, and F. G. Holz, “Two-photon-excited fluorescence imaging of human RPE cells with a femtosecond Ti:sapphire laser,” Invest. Ophthalmol. Vis. Sci. 47(10), 4553–4557 (2006).
[CrossRef] [PubMed]

M. Han, A. Bindewald-Wittich, F. G. Holz, G. Giese, M. H. Niemz, S. Snyder, H. Sun, J. Yu, M. Agopov, O. La Schiazza, and J. F. Bille, “Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells,” J. Biomed. Opt. 11(1), 010501 (2006).
[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]

Cense, B.

Chen, C.

C. Chen, E. Tsina, M. C. Cornwall, R. K. Crouch, S. Vijayaraghavan, and Y. Koutalos, “Reduction of all-trans retinal to all-trans retinol in the outer segments of frog and mouse rod photoreceptors,” Biophys. J. 88(3), 2278–2287 (2005).
[CrossRef] [PubMed]

Chen, Y.

Chinnery, P. F.

P. Y. Man, D. M. Turnbull, and P. F. Chinnery, “Leber hereditary optic neuropathy,” J. Med. Genet. 39(3), 162–169 (2002).
[CrossRef] [PubMed]

Christie, R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

Clarke, A. M.

W. T. Ham, H. A. Mueller, J. J. Ruffolo, and A. M. Clarke, “Sensitivity of the retina to radiation damage as a function of wavelength,” Photochem. Photobiol. 29(4), 735–743 (1979).
[CrossRef] [PubMed]

Cornwall, M. C.

C. Chen, E. Tsina, M. C. Cornwall, R. K. Crouch, S. Vijayaraghavan, and Y. Koutalos, “Reduction of all-trans retinal to all-trans retinol in the outer segments of frog and mouse rod photoreceptors,” Biophys. J. 88(3), 2278–2287 (2005).
[CrossRef] [PubMed]

Crouch, R. K.

C. Chen, E. Tsina, M. C. Cornwall, R. K. Crouch, S. Vijayaraghavan, and Y. Koutalos, “Reduction of all-trans retinal to all-trans retinol in the outer segments of frog and mouse rod photoreceptors,” Biophys. J. 88(3), 2278–2287 (2005).
[CrossRef] [PubMed]

Delori, F. C.

J. I. Morgan, J. J. Hunter, B. Masella, R. Wolfe, D. C. Gray, W. H. Merigan, F. C. Delori, and D. R. Williams, “Light-induced retinal changes observed with high-resolution autofluorescence imaging of the retinal pigment epithelium,” Invest. Ophthalmol. Vis. Sci. 49(8), 3715–3729 (2008).
[CrossRef] [PubMed]

F. C. Delori, R. H. Webb, D. H. Sliney, and American National Standards Institute, “Maximum permissible exposures for ocular safety (ANSI 2000), with emphasis on ophthalmic devices,” J. Opt. Soc. Am. A 24(5), 1250–1265 (2007).
[CrossRef] [PubMed]

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]

Dillon, J.

J. Dillon, L. Zheng, J. C. Merriam, and E. R. Gaillard, “Transmission spectra of light to the mammalian retina,” Photochem. Photobiol. 71(2), 225–229 (2000).
[CrossRef] [PubMed]

Dorey, C. K.

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]

Dubra, A.

J. I. 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. Express 14(16), 7144–7158 (2006).
[CrossRef] [PubMed]

Duker, J. S.

Fujimoto, J. G.

Gaillard, E. R.

J. Dillon, L. Zheng, J. C. Merriam, and E. R. Gaillard, “Transmission spectra of light to the mammalian retina,” Photochem. Photobiol. 71(2), 225–229 (2000).
[CrossRef] [PubMed]

Gao, W.

Gee, B. P.

Giese, G.

M. Han, G. Giese, S. Schmitz-Valckenberg, A. Bindewald-Wittich, F. G. Holz, J. Yu, J. F. Bille, and M. H. Niemz, “Age-related structural abnormalities in the human retina-choroid complex revealed by two-photon excited autofluorescence imaging,” J. Biomed. Opt. 12(2), 024012 (2007).
[CrossRef] [PubMed]

A. Bindewald-Wittich, M. Han, S. Schmitz-Valckenberg, S. R. Snyder, G. Giese, J. F. Bille, and F. G. Holz, “Two-photon-excited fluorescence imaging of human RPE cells with a femtosecond Ti:sapphire laser,” Invest. Ophthalmol. Vis. Sci. 47(10), 4553–4557 (2006).
[CrossRef] [PubMed]

M. Han, A. Bindewald-Wittich, F. G. Holz, G. Giese, M. H. Niemz, S. Snyder, H. Sun, J. Yu, M. Agopov, O. La Schiazza, and J. F. Bille, “Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells,” J. Biomed. Opt. 11(1), 010501 (2006).
[CrossRef] [PubMed]

Goger, D. 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).
[PubMed]

Golczak, M.

G. H. Travis, M. Golczak, A. R. Moise, and K. Palczewski, “Diseases caused by defects in the visual cycle: retinoids as potential therapeutic agents,” Annu. Rev. Pharmacol. Toxicol. 47(1), 469–512 (2007).
[CrossRef] [PubMed]

Gray, D. C.

J. I. Morgan, J. J. Hunter, B. Masella, R. Wolfe, D. C. Gray, W. H. Merigan, F. C. Delori, and D. R. Williams, “Light-induced retinal changes observed with high-resolution autofluorescence imaging of the retinal pigment epithelium,” Invest. Ophthalmol. Vis. Sci. 49(8), 3715–3729 (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. Express 14(16), 7144–7158 (2006).
[CrossRef] [PubMed]

Grieve, K.

K. Grieve and A. Roorda, “Intrinsic signals from human cone photoreceptors,” Invest. Ophthalmol. Vis. Sci. 49(2), 713–719 (2008).
[CrossRef] [PubMed]

Gualda, E. J.

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]

Ham, W. T.

W. T. Ham, H. A. Mueller, J. J. Ruffolo, and A. M. Clarke, “Sensitivity of the retina to radiation damage as a function of wavelength,” Photochem. Photobiol. 29(4), 735–743 (1979).
[CrossRef] [PubMed]

Han, M.

M. Han, G. Giese, S. Schmitz-Valckenberg, A. Bindewald-Wittich, F. G. Holz, J. Yu, J. F. Bille, and M. H. Niemz, “Age-related structural abnormalities in the human retina-choroid complex revealed by two-photon excited autofluorescence imaging,” J. Biomed. Opt. 12(2), 024012 (2007).
[CrossRef] [PubMed]

M. Han, A. Bindewald-Wittich, F. G. Holz, G. Giese, M. H. Niemz, S. Snyder, H. Sun, J. Yu, M. Agopov, O. La Schiazza, and J. F. Bille, “Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells,” J. Biomed. Opt. 11(1), 010501 (2006).
[CrossRef] [PubMed]

A. Bindewald-Wittich, M. Han, S. Schmitz-Valckenberg, S. R. Snyder, G. Giese, J. F. Bille, and F. G. Holz, “Two-photon-excited fluorescence imaging of human RPE cells with a femtosecond Ti:sapphire laser,” Invest. Ophthalmol. Vis. Sci. 47(10), 4553–4557 (2006).
[CrossRef] [PubMed]

Hanazono, G.

K. Tsunoda, G. Hanazono, K. Inomata, Y. Kazato, W. Suzuki, and M. Tanifuji, “Origins of retinal intrinsic signals: a series of experiments on retinas of macaque monkeys,” Jpn. J. Ophthalmol. 53(4), 297–314 (2009).
[CrossRef] [PubMed]

Harding, A. E.

I. J. Holt, A. E. Harding, R. K. Petty, and J. A. Morgan-Hughes, “A new mitochondrial disease associated with mitochondrial DNA heteroplasmy,” Am. J. Hum. Genet. 46(3), 428–433 (1990).
[PubMed]

Heikal, A. A.

S. Huang, A. A. Heikal, and W. W. Webb, “Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein,” Biophys. J. 82(5), 2811–2825 (2002).
[CrossRef] [PubMed]

Holt, I. J.

I. J. Holt, A. E. Harding, R. K. Petty, and J. A. Morgan-Hughes, “A new mitochondrial disease associated with mitochondrial DNA heteroplasmy,” Am. J. Hum. Genet. 46(3), 428–433 (1990).
[PubMed]

Holz, F. G.

M. Han, G. Giese, S. Schmitz-Valckenberg, A. Bindewald-Wittich, F. G. Holz, J. Yu, J. F. Bille, and M. H. Niemz, “Age-related structural abnormalities in the human retina-choroid complex revealed by two-photon excited autofluorescence imaging,” J. Biomed. Opt. 12(2), 024012 (2007).
[CrossRef] [PubMed]

A. Bindewald-Wittich, M. Han, S. Schmitz-Valckenberg, S. R. Snyder, G. Giese, J. F. Bille, and F. G. Holz, “Two-photon-excited fluorescence imaging of human RPE cells with a femtosecond Ti:sapphire laser,” Invest. Ophthalmol. Vis. Sci. 47(10), 4553–4557 (2006).
[CrossRef] [PubMed]

M. Han, A. Bindewald-Wittich, F. G. Holz, G. Giese, M. H. Niemz, S. Snyder, H. Sun, J. Yu, M. Agopov, O. La Schiazza, and J. F. Bille, “Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells,” J. Biomed. Opt. 11(1), 010501 (2006).
[CrossRef] [PubMed]

Huang, S.

S. Huang, A. A. Heikal, and W. W. Webb, “Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein,” Biophys. J. 82(5), 2811–2825 (2002).
[CrossRef] [PubMed]

Hunter, J. J.

J. I. Morgan, J. J. Hunter, B. Masella, R. Wolfe, D. C. Gray, W. H. Merigan, F. C. Delori, and D. R. Williams, “Light-induced retinal changes observed with high-resolution autofluorescence imaging of the retinal pigment epithelium,” Invest. Ophthalmol. Vis. Sci. 49(8), 3715–3729 (2008).
[CrossRef] [PubMed]

Hyman, B. T.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

Imanishi, Y.

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]

Inomata, K.

K. Tsunoda, G. Hanazono, K. Inomata, Y. Kazato, W. Suzuki, and M. Tanifuji, “Origins of retinal intrinsic signals: a series of experiments on retinas of macaque monkeys,” Jpn. J. Ophthalmol. 53(4), 297–314 (2009).
[CrossRef] [PubMed]

Jonnal, R. S.

Kaplan, M. W.

R. C. Sears and M. W. Kaplan, “Axial diffusion of retinol in isolated frog rod outer segments following substantial bleaches of visual pigment,” Vision Res. 29(11), 1485–1492 (1989).
[CrossRef] [PubMed]

Kardon, R.

D. Ts’o, J. Schallek, Y. Kwon, R. Kardon, M. Abramoff, and P. Soliz, “Noninvasive functional imaging of the retina reveals outer retinal and hemodynamic intrinsic optical signal origins,” Jpn. J. Ophthalmol. 53(4), 334–344 (2009).
[CrossRef] [PubMed]

Kazato, Y.

K. Tsunoda, G. Hanazono, K. Inomata, Y. Kazato, W. Suzuki, and M. Tanifuji, “Origins of retinal intrinsic signals: a series of experiments on retinas of macaque monkeys,” Jpn. J. Ophthalmol. 53(4), 297–314 (2009).
[CrossRef] [PubMed]

Kefalov, V. J.

J. S. Wang and V. J. Kefalov, “An alternative pathway mediates the mouse and human cone visual cycle,” Curr. Biol. 19(19), 1665–1669 (2009).
[CrossRef] [PubMed]

Koutalos, Y.

C. Chen, E. Tsina, M. C. Cornwall, R. K. Crouch, S. Vijayaraghavan, and Y. Koutalos, “Reduction of all-trans retinal to all-trans retinol in the outer segments of frog and mouse rod photoreceptors,” Biophys. J. 88(3), 2278–2287 (2005).
[CrossRef] [PubMed]

Kwon, Y.

D. Ts’o, J. Schallek, Y. Kwon, R. Kardon, M. Abramoff, and P. Soliz, “Noninvasive functional imaging of the retina reveals outer retinal and hemodynamic intrinsic optical signal origins,” Jpn. J. Ophthalmol. 53(4), 334–344 (2009).
[CrossRef] [PubMed]

La Schiazza, O.

O. La Schiazza and J. F. Bille, “High-speed two-photon excited autofluorescence imaging of ex vivo human retinal pigment epithelial cells toward age-related macular degeneration diagnostic,” J. Biomed. Opt. 13(6), 064008 (2008).
[CrossRef] [PubMed]

M. Han, A. Bindewald-Wittich, F. G. Holz, G. Giese, M. H. Niemz, S. Snyder, H. Sun, J. Yu, M. Agopov, O. La Schiazza, and J. F. Bille, “Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells,” J. Biomed. Opt. 11(1), 010501 (2006).
[CrossRef] [PubMed]

Lamb, T. D.

T. D. Lamb and E. N. Pugh., “Dark adaptation and the retinoid cycle of vision,” Prog. Retin. Eye Res. 23(3), 307–380 (2004).
[CrossRef] [PubMed]

Man, P. Y.

P. Y. Man, D. M. Turnbull, and P. F. Chinnery, “Leber hereditary optic neuropathy,” J. Med. Genet. 39(3), 162–169 (2002).
[CrossRef] [PubMed]

Masella, B.

J. I. Morgan, J. J. Hunter, B. Masella, R. Wolfe, D. C. Gray, W. H. Merigan, F. C. Delori, and D. R. Williams, “Light-induced retinal changes observed with high-resolution autofluorescence imaging of the retinal pigment epithelium,” Invest. Ophthalmol. Vis. Sci. 49(8), 3715–3729 (2008).
[CrossRef] [PubMed]

Merigan, W.

Merigan, W. H.

J. I. Morgan, J. J. Hunter, B. Masella, R. Wolfe, D. C. Gray, W. H. Merigan, F. C. Delori, and D. R. Williams, “Light-induced retinal changes observed with high-resolution autofluorescence imaging of the retinal pigment epithelium,” Invest. Ophthalmol. Vis. Sci. 49(8), 3715–3729 (2008).
[CrossRef] [PubMed]

J. I. 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]

Merriam, J. C.

J. Dillon, L. Zheng, J. C. Merriam, and E. R. Gaillard, “Transmission spectra of light to the mammalian retina,” Photochem. Photobiol. 71(2), 225–229 (2000).
[CrossRef] [PubMed]

Miller, D. T.

Moise, A. R.

G. H. Travis, M. Golczak, A. R. Moise, and K. Palczewski, “Diseases caused by defects in the visual cycle: retinoids as potential therapeutic agents,” Annu. Rev. Pharmacol. Toxicol. 47(1), 469–512 (2007).
[CrossRef] [PubMed]

Morgan, J. I.

J. I. Morgan, J. J. Hunter, B. Masella, R. Wolfe, D. C. Gray, W. H. Merigan, F. C. Delori, and D. R. Williams, “Light-induced retinal changes observed with high-resolution autofluorescence imaging of the retinal pigment epithelium,” Invest. Ophthalmol. Vis. Sci. 49(8), 3715–3729 (2008).
[CrossRef] [PubMed]

J. I. 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]

Morgan-Hughes, J. A.

I. J. Holt, A. E. Harding, R. K. Petty, and J. A. Morgan-Hughes, “A new mitochondrial disease associated with mitochondrial DNA heteroplasmy,” Am. J. Hum. Genet. 46(3), 428–433 (1990).
[PubMed]

Mueller, H. A.

W. T. Ham, H. A. Mueller, J. J. Ruffolo, and A. M. Clarke, “Sensitivity of the retina to radiation damage as a function of wavelength,” Photochem. Photobiol. 29(4), 735–743 (1979).
[CrossRef] [PubMed]

Niemz, M. H.

M. Han, G. Giese, S. Schmitz-Valckenberg, A. Bindewald-Wittich, F. G. Holz, J. Yu, J. F. Bille, and M. H. Niemz, “Age-related structural abnormalities in the human retina-choroid complex revealed by two-photon excited autofluorescence imaging,” J. Biomed. Opt. 12(2), 024012 (2007).
[CrossRef] [PubMed]

M. Han, A. Bindewald-Wittich, F. G. Holz, G. Giese, M. H. Niemz, S. Snyder, H. Sun, J. Yu, M. Agopov, O. La Schiazza, and J. F. Bille, “Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells,” J. Biomed. Opt. 11(1), 010501 (2006).
[CrossRef] [PubMed]

Nikitin, A. Y.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

Palczewski, K.

G. H. Travis, M. Golczak, A. R. Moise, and K. Palczewski, “Diseases caused by defects in the visual cycle: retinoids as potential therapeutic agents,” Annu. Rev. Pharmacol. Toxicol. 47(1), 469–512 (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]

Petty, R. K.

I. J. Holt, A. E. Harding, R. K. Petty, and J. A. Morgan-Hughes, “A new mitochondrial disease associated with mitochondrial DNA heteroplasmy,” Am. J. Hum. Genet. 46(3), 428–433 (1990).
[PubMed]

Piston, D. 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]

Porter, J.

Pugh, E. N.

T. D. Lamb and E. N. Pugh., “Dark adaptation and the retinoid cycle of vision,” Prog. Retin. Eye Res. 23(3), 307–380 (2004).
[CrossRef] [PubMed]

Reinholz, F.

Rha, J.

Roorda, A.

K. Grieve and A. Roorda, “Intrinsic signals from human cone photoreceptors,” Invest. Ophthalmol. Vis. Sci. 49(2), 713–719 (2008).
[CrossRef] [PubMed]

Ruffolo, J. J.

W. T. Ham, H. A. Mueller, J. J. Ruffolo, and A. M. Clarke, “Sensitivity of the retina to radiation damage as a function of wavelength,” Photochem. Photobiol. 29(4), 735–743 (1979).
[CrossRef] [PubMed]

Schallek, J.

D. Ts’o, J. Schallek, Y. Kwon, R. Kardon, M. Abramoff, and P. Soliz, “Noninvasive functional imaging of the retina reveals outer retinal and hemodynamic intrinsic optical signal origins,” Jpn. J. Ophthalmol. 53(4), 334–344 (2009).
[CrossRef] [PubMed]

Schmitz-Valckenberg, S.

M. Han, G. Giese, S. Schmitz-Valckenberg, A. Bindewald-Wittich, F. G. Holz, J. Yu, J. F. Bille, and M. H. Niemz, “Age-related structural abnormalities in the human retina-choroid complex revealed by two-photon excited autofluorescence imaging,” J. Biomed. Opt. 12(2), 024012 (2007).
[CrossRef] [PubMed]

A. Bindewald-Wittich, M. Han, S. Schmitz-Valckenberg, S. R. Snyder, G. Giese, J. F. Bille, and F. G. Holz, “Two-photon-excited fluorescence imaging of human RPE cells with a femtosecond Ti:sapphire laser,” Invest. Ophthalmol. Vis. Sci. 47(10), 4553–4557 (2006).
[CrossRef] [PubMed]

Sears, R. C.

R. C. Sears and M. W. Kaplan, “Axial diffusion of retinol in isolated frog rod outer segments following substantial bleaches of visual pigment,” Vision Res. 29(11), 1485–1492 (1989).
[CrossRef] [PubMed]

Sliney, D. H.

Snyder, S.

M. Han, A. Bindewald-Wittich, F. G. Holz, G. Giese, M. H. Niemz, S. Snyder, H. Sun, J. Yu, M. Agopov, O. La Schiazza, and J. F. Bille, “Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells,” J. Biomed. Opt. 11(1), 010501 (2006).
[CrossRef] [PubMed]

Snyder, S. R.

A. Bindewald-Wittich, M. Han, S. Schmitz-Valckenberg, S. R. Snyder, G. Giese, J. F. Bille, and F. G. Holz, “Two-photon-excited fluorescence imaging of human RPE cells with a femtosecond Ti:sapphire laser,” Invest. Ophthalmol. Vis. Sci. 47(10), 4553–4557 (2006).
[CrossRef] [PubMed]

Soliz, P.

D. Ts’o, J. Schallek, Y. Kwon, R. Kardon, M. Abramoff, and P. Soliz, “Noninvasive functional imaging of the retina reveals outer retinal and hemodynamic intrinsic optical signal origins,” Jpn. J. Ophthalmol. 53(4), 334–344 (2009).
[CrossRef] [PubMed]

Srinivasan, V. J.

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).
[PubMed]

Sun, H.

M. Han, A. Bindewald-Wittich, F. G. Holz, G. Giese, M. H. Niemz, S. Snyder, H. Sun, J. Yu, M. Agopov, O. La Schiazza, and J. F. Bille, “Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells,” J. Biomed. Opt. 11(1), 010501 (2006).
[CrossRef] [PubMed]

Suzuki, W.

K. Tsunoda, G. Hanazono, K. Inomata, Y. Kazato, W. Suzuki, and M. Tanifuji, “Origins of retinal intrinsic signals: a series of experiments on retinas of macaque monkeys,” Jpn. J. Ophthalmol. 53(4), 297–314 (2009).
[CrossRef] [PubMed]

Tanifuji, M.

K. Tsunoda, G. Hanazono, K. Inomata, Y. Kazato, W. Suzuki, and M. Tanifuji, “Origins of retinal intrinsic signals: a series of experiments on retinas of macaque monkeys,” Jpn. J. Ophthalmol. 53(4), 297–314 (2009).
[CrossRef] [PubMed]

Travis, G. H.

G. H. Travis, M. Golczak, A. R. Moise, and K. Palczewski, “Diseases caused by defects in the visual cycle: retinoids as potential therapeutic agents,” Annu. Rev. Pharmacol. Toxicol. 47(1), 469–512 (2007).
[CrossRef] [PubMed]

Ts’o, D.

D. Ts’o, J. Schallek, Y. Kwon, R. Kardon, M. Abramoff, and P. Soliz, “Noninvasive functional imaging of the retina reveals outer retinal and hemodynamic intrinsic optical signal origins,” Jpn. J. Ophthalmol. 53(4), 334–344 (2009).
[CrossRef] [PubMed]

Tsina, E.

C. Chen, E. Tsina, M. C. Cornwall, R. K. Crouch, S. Vijayaraghavan, and Y. Koutalos, “Reduction of all-trans retinal to all-trans retinol in the outer segments of frog and mouse rod photoreceptors,” Biophys. J. 88(3), 2278–2287 (2005).
[CrossRef] [PubMed]

Tsunoda, K.

K. Tsunoda, G. Hanazono, K. Inomata, Y. Kazato, W. Suzuki, and M. Tanifuji, “Origins of retinal intrinsic signals: a series of experiments on retinas of macaque monkeys,” Jpn. J. Ophthalmol. 53(4), 297–314 (2009).
[CrossRef] [PubMed]

Tumbar, R.

Turnbull, D. M.

P. Y. Man, D. M. Turnbull, and P. F. Chinnery, “Leber hereditary optic neuropathy,” J. Med. Genet. 39(3), 162–169 (2002).
[CrossRef] [PubMed]

Twietmeyer, T. H.

Vijayaraghavan, S.

C. Chen, E. Tsina, M. C. Cornwall, R. K. Crouch, S. Vijayaraghavan, and Y. Koutalos, “Reduction of all-trans retinal to all-trans retinol in the outer segments of frog and mouse rod photoreceptors,” Biophys. J. 88(3), 2278–2287 (2005).
[CrossRef] [PubMed]

Wang, J. S.

J. S. Wang and V. J. Kefalov, “An alternative pathway mediates the mouse and human cone visual cycle,” Curr. Biol. 19(19), 1665–1669 (2009).
[CrossRef] [PubMed]

Webb, R. H.

Webb, W. W.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

S. Huang, A. A. Heikal, and W. W. Webb, “Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein,” Biophys. J. 82(5), 2811–2825 (2002).
[CrossRef] [PubMed]

Weiter, J. J.

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]

Williams, D. R.

J. I. 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]

J. I. Morgan, J. J. Hunter, B. Masella, R. Wolfe, D. C. Gray, W. H. Merigan, F. C. Delori, and D. R. Williams, “Light-induced retinal changes observed with high-resolution autofluorescence imaging of the retinal pigment epithelium,” Invest. Ophthalmol. Vis. Sci. 49(8), 3715–3729 (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. Express 14(16), 7144–7158 (2006).
[CrossRef] [PubMed]

Williams, R. M.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

Wolfe, R.

J. I. Morgan, J. J. Hunter, B. Masella, R. Wolfe, D. C. Gray, W. H. Merigan, F. C. Delori, and D. R. Williams, “Light-induced retinal changes observed with high-resolution autofluorescence imaging of the retinal pigment epithelium,” Invest. Ophthalmol. Vis. Sci. 49(8), 3715–3729 (2008).
[CrossRef] [PubMed]

J. I. 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]

Wolfing, J. I.

Yu, J.

M. Han, G. Giese, S. Schmitz-Valckenberg, A. Bindewald-Wittich, F. G. Holz, J. Yu, J. F. Bille, and M. H. Niemz, “Age-related structural abnormalities in the human retina-choroid complex revealed by two-photon excited autofluorescence imaging,” J. Biomed. Opt. 12(2), 024012 (2007).
[CrossRef] [PubMed]

M. Han, A. Bindewald-Wittich, F. G. Holz, G. Giese, M. H. Niemz, S. Snyder, H. Sun, J. Yu, M. Agopov, O. La Schiazza, and J. F. Bille, “Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells,” J. Biomed. Opt. 11(1), 010501 (2006).
[CrossRef] [PubMed]

Zhang, Y.

Zheng, L.

J. Dillon, L. Zheng, J. C. Merriam, and E. R. Gaillard, “Transmission spectra of light to the mammalian retina,” Photochem. Photobiol. 71(2), 225–229 (2000).
[CrossRef] [PubMed]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

Am. J. Hum. Genet.

I. J. Holt, A. E. Harding, R. K. Petty, and J. A. Morgan-Hughes, “A new mitochondrial disease associated with mitochondrial DNA heteroplasmy,” Am. J. Hum. Genet. 46(3), 428–433 (1990).
[PubMed]

Annu. Rev. Pharmacol. Toxicol.

G. H. Travis, M. Golczak, A. R. Moise, and K. Palczewski, “Diseases caused by defects in the visual cycle: retinoids as potential therapeutic agents,” Annu. Rev. Pharmacol. Toxicol. 47(1), 469–512 (2007).
[CrossRef] [PubMed]

Biophys. J.

C. Chen, E. Tsina, M. C. Cornwall, R. K. Crouch, S. Vijayaraghavan, and Y. Koutalos, “Reduction of all-trans retinal to all-trans retinol in the outer segments of frog and mouse rod photoreceptors,” Biophys. J. 88(3), 2278–2287 (2005).
[CrossRef] [PubMed]

S. Huang, A. A. Heikal, and W. W. Webb, “Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein,” Biophys. J. 82(5), 2811–2825 (2002).
[CrossRef] [PubMed]

Curr. Biol.

J. S. Wang and V. J. Kefalov, “An alternative pathway mediates the mouse and human cone visual cycle,” Curr. Biol. 19(19), 1665–1669 (2009).
[CrossRef] [PubMed]

Invest. Ophthalmol. Vis. Sci.

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]

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[CrossRef] [PubMed]

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Figures (5)

Fig. 1
Fig. 1

Schematic of the FAOSLO system. Changes in the vertical plane are outlined by the dashed lines. Inset: A plot of the transmission spectrum for two-photon emission collection into the detector. DM, deformable mirror; HS, horizontal scanner; LD, laser diode; PMT, photomultiplier tube; SLD, superluminescent diode; VS, vertical scanner; WFS, wavefront sensor.

Fig. 2
Fig. 2

Images of the cone mosaic in the living primate retina. At 2.5° superior, (a) the two-photon image and (b) the reflectance image show good correspondence. The cross correlation coefficient between these images is 0.9. In magnified sections (c) of the larger images, denoted by white rectangles in (a) and (b), the correspondence between individual cones can be observed (white arrows). Black arrows indicate a cone that was not reflecting light but shows a strong fluorescence signal. The images in (c) were low pass filtered to remove frequencies above the diffraction-limit, thereby improving cone visibility. Scale bars, 50 μm. The quadratic nature of the emitted fluorescence as a function of incident excitation power is shown (d). Error bars represent the standard error of the mean gray level among individual frames.

Fig. 3
Fig. 3

Mean cone spacing with eccentricity verifies that two-photon fluorescence originates from the cone mosaic. Shown are two-photon images of the cone mosaic in the living primate at three eccentricities in superior retina, (a) 5.5°, (b) 9.1° and (c) 13°. Scale bars, 50 μm. Cone spacing, as determined in Fourier space, is shown as a function of eccentricity (d) from two-photon (●) and reflectance (Δ) images. Error bars represent the width of the secondary peak in the Fourier spectra.

Fig. 4
Fig. 4

Two-photon fluorescence of photoreceptors in macaque macular region imaged ex vivo. Whole mount view of the photoreceptor inner segment mosaic showing large cones interspersed among the much smaller rods in (a) pre-bleached and (b) post-bleached states. These slices were collapsed across a 2.5 μm depth. (c) Digitally reconstructed transverse view of an ‘average’ cone, computed by averaging the data cropped from 18 identical voxels centered on 18 individual cones, in the post-bleached state showing the bright inner segment (IS) and a much dimmer outer segment (OS). Scale bar, 5 μm.

Fig. 5
Fig. 5

Fluorescence emission response during photoreceptor bleaching. The results for three different retinal locations are shown. Data points are the means of 2 s time intervals and error bars represent the standard error of the means. Lines represent the best fits to the unbinned data.

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