Abstract

Two-photon fluorescence microscopy (TPM) is now being used routinely to image live cells for extended periods deep within tissues, including the retina and other structures within the eye . However, very low laser power is a requirement to obtain TPM images of the retina safely. Unfortunately, a reduction in laser power also reduces the signal-to-noise ratio of collected images, making it difficult to visualize structural details. Here, image registration and averaging methods applied to TPM images of the eye in living animals (without the need for auxiliary hardware) demonstrate the structural information obtained with laser power down to 1 mW. Image registration provided between 1.4% and 13.0% improvement in image quality compared to averaging images without registrations when using a high-fluorescence template, and between 0.2% and 12.0% when employing the average of collected images as the template. Also, a diminishing return on image quality when more images were used to obtain the averaged image is shown. This work provides a foundation for obtaining informative TPM images with laser powers of 1 mW, compared to previous levels for imaging mice ranging between 6.3 mW [G. Palczewska, Nat Med. 20, 785 (2014) R. Sharma, Biomed. Opt. Express 4, 1285 (2013)].

© 2016 Optical Society of America

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2016 (4)

R. Sharma, C. Schwarz, D. R. Williams, G. Palczewska, K. Palczewski, and J. J. Hunter, “In Vivo Two-Photon Fluorescence Kinetics of Primate Rods and Cones,” Invest. Ophthalmol. Vis. Sci. 57(2), 647–657 (2016).
[Crossref] [PubMed]

R. Sharma, D. R. Williams, G. Palczewska, K. Palczewski, and J. J. Hunter, “Two-Photon Autofluorescence Imaging Reveals Cellular Structures Throughout the Retina of the Living Primate Eye,” Invest. Ophthalmol. Vis. Sci. 57(2), 632–646 (2016).
[Crossref] [PubMed]

A. W. Stitt, T. M. Curtis, M. Chen, R. J. Medina, G. J. McKay, A. Jenkins, T. A. Gardiner, T. J. Lyons, H. P. Hammes, R. Simó, and N. Lois, “The progress in understanding and treatment of diabetic retinopathy,” Prog. Retin. Eye Res. 51, 156–186 (2016).
[Crossref] [PubMed]

D. J. Wahl, Y. Jian, S. Bonora, R. J. Zawadzki, and M. V. Sarunic, “Wavefront sensorless adaptive optics fluorescence biomicroscope for in vivo retinal imaging in mice,” Biomed. Opt. Express 7(1), 1–12 (2016).
[Crossref] [PubMed]

2015 (5)

2014 (5)

G. Kumar and S. T. L. Chung, “Characteristics of fixational eye movements in people with macular disease,” Invest. Ophthalmol. Vis. Sci. 55(8), 5125–5133 (2014).
[Crossref] [PubMed]

B. D. Masella, D. R. Williams, W. S. Fischer, E. A. Rossi, and J. J. Hunter, “Long-Term Reduction in Infrared Autofluorescence Caused by Infrared Light Below the Maximum Permissible Exposure,” Invest. Ophthalmol. Vis. Sci. 55(6), 3929–3938 (2014).
[Crossref] [PubMed]

G. Palczewska, M. Golczak, D. R. Williams, J. J. Hunter, and K. Palczewski, “Endogenous fluorophores enable two-photon imaging of the primate eye,” Invest. Ophthalmol. Vis. Sci. 55(7), 4438–4447 (2014).
[Crossref] [PubMed]

G. Palczewska, Z. Dong, M. Golczak, J. J. Hunter, D. R. Williams, N. S. Alexander, and K. Palczewski, “Noninvasive two-photon microscopy imaging of mouse retina and retinal pigment epithelium through the pupil of the eye,” Nat. Med. 20(7), 785–789 (2014).
[Crossref] [PubMed]

Q. Yang, J. Zhang, K. Nozato, K. Saito, D. R. Williams, A. Roorda, and E. A. Rossi, “Closed-loop optical stabilization and digital image registration in adaptive optics scanning light ophthalmoscopy,” Biomed. Opt. Express 5(9), 3174–3191 (2014).
[Crossref] [PubMed]

2013 (3)

2012 (1)

2011 (2)

2010 (4)

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).
[Crossref] [PubMed]

L. Kessel, J. H. Lundeman, K. Herbst, T. V. Andersen, and M. Larsen, “Age-related changes in the transmission properties of the human lens and their relevance to circadian entrainment,” J. Cataract Refract. Surg. 36(2), 308–312 (2010).
[Crossref] [PubMed]

D. Pestov, Y. Andegeko, V. V. Lozovoy, and M. Dantus, “Photobleaching and photoenhancement of endogenous fluorescence observed in two-photon microscopy with broadband laser sources,” J. Opt. 12, 8 (2010).

R. D. Ferguson, Z. Zhong, D. X. Hammer, M. Mujat, A. H. Patel, C. Deng, W. Zou, and S. A. Burns, “Adaptive optics scanning laser ophthalmoscope with integrated wide-field retinal imaging and tracking,” J. Opt. Soc. Am. A 27, A265–A277 (2010).

2009 (1)

D. S. Greenberg and J. N. D. Kerr, “Automated correction of fast motion artifacts for two-photon imaging of awake animals,” J. Neurosci. Methods 176(1), 1–15 (2009).
[Crossref] [PubMed]

2008 (2)

J. I. W. 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]

L. R. Weisel, P. Xi, Y. Andegeko, V. V. Lovozoy, and M. Dantus, “Greater signal and contrast in two-photon microscopy with ultrashort pulses,” Proc. SPIE 6860, 68601O (2008).

2007 (2)

2006 (2)

2004 (2)

S. Martinez-Conde, S. L. Macknik, and D. H. Hubel, “The role of fixational eye movements in visual perception,” Nat. Rev. Neurosci. 5(3), 229–240 (2004).
[Crossref] [PubMed]

Y. Sun, S. Duthaler, and B. J. Nelson, “Autofocusing in computer microscopy: selecting the optimal focus algorithm,” Microsc. Res. Tech. 65(3), 139–149 (2004).
[Crossref] [PubMed]

2003 (1)

2002 (1)

K. Carlsson and J. Philip, “Theoretical investigation of the signal-to-noise ratio for different fluorescence lifetime imaging techniques,” Proc. SPIE 4622, 70–78 (2002).

2000 (1)

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]

1999 (1)

D. W. Piston, “Imaging living cells and tissues by two-photon excitation microscopy,” Trends Cell Biol. 9(2), 66–69 (1999).
[Crossref] [PubMed]

1998 (1)

V. E. Centonze and J. G. White, “Multiphoton Excitation Provides Optical Sections from Deeper within Scattering Specimens than Confocal Imaging,” Biophys. J. 75(4), 2015–2024 (1998).
[Crossref] [PubMed]

1996 (1)

1995 (1)

M. Hammer, A. Roggan, D. Schweitzer, and G. Müller, “Optical properties of ocular fundus tissues--an in vitro study using the double-integrating-sphere technique and inverse Monte Carlo simulation,” Phys. Med. Biol. 40(6), 963–978 (1995).
[Crossref] [PubMed]

1993 (1)

T. T. E. Yeo, S. H. Ong, Jayasooriah, and R. Sinniah, “Autofocusing for tissue microscopy,” Image Vis. Comput. 11(10), 629–639 (1993).
[Crossref]

1991 (1)

A. Vogel, C. Dlugos, R. Nuffer, and R. Birngruber, “Optical properties of human sclera, and their consequences for transscleral laser applications,” Lasers Surg. Med. 11(4), 331–340 (1991).
[Crossref] [PubMed]

1988 (1)

N. M. Bressler, S. B. Bressler, and S. L. Fine, “Age-related macular degeneration,” Surv. Ophthalmol. 32(6), 375–413 (1988).
[Crossref] [PubMed]

1985 (1)

F. C. A. Groen, I. T. Young, and G. Ligthart, “A comparison of different focus functions for use in autofocus algorithms,” Cytometry 6(2), 81–91 (1985).
[Crossref] [PubMed]

1962 (1)

E. A. Boettner and J. R. Wolter, “Transmission of the Ocular Media,” Invest. Ophthalmol. Vis. Sci. 1, 776–783 (1962).

1950 (1)

F. Ratliff and L. A. Riggs, “Involuntary motions of the eye during monocular fixation,” J. Exp. Psychol. 40(6), 687–701 (1950).
[Crossref] [PubMed]

Ahamd, K.

Alexander, N. S.

G. Palczewska, Z. Dong, M. Golczak, J. J. Hunter, D. R. Williams, N. S. Alexander, and K. Palczewski, “Noninvasive two-photon microscopy imaging of mouse retina and retinal pigment epithelium through the pupil of the eye,” Nat. Med. 20(7), 785–789 (2014).
[Crossref] [PubMed]

Amengual, J.

H. Liu, J. Tang, Y. Du, C. A. Lee, M. Golczak, A. Muthusamy, D. A. Antonetti, A. A. Veenstra, J. Amengual, J. von Lintig, K. Palczewski, and T. S. Kern, “Retinylamine Benefits Early Diabetic Retinopathy in Mice,” J. Biol. Chem. 290(35), 21568–21579 (2015).
[Crossref] [PubMed]

Andegeko, Y.

D. Pestov, Y. Andegeko, V. V. Lozovoy, and M. Dantus, “Photobleaching and photoenhancement of endogenous fluorescence observed in two-photon microscopy with broadband laser sources,” J. Opt. 12, 8 (2010).

L. R. Weisel, P. Xi, Y. Andegeko, V. V. Lovozoy, and M. Dantus, “Greater signal and contrast in two-photon microscopy with ultrashort pulses,” Proc. SPIE 6860, 68601O (2008).

Andersen, T. V.

L. Kessel, J. H. Lundeman, K. Herbst, T. V. Andersen, and M. Larsen, “Age-related changes in the transmission properties of the human lens and their relevance to circadian entrainment,” J. Cataract Refract. Surg. 36(2), 308–312 (2010).
[Crossref] [PubMed]

Antonetti, D. A.

H. Liu, J. Tang, Y. Du, C. A. Lee, M. Golczak, A. Muthusamy, D. A. Antonetti, A. A. Veenstra, J. Amengual, J. von Lintig, K. Palczewski, and T. S. Kern, “Retinylamine Benefits Early Diabetic Retinopathy in Mice,” J. Biol. Chem. 290(35), 21568–21579 (2015).
[Crossref] [PubMed]

Birngruber, R.

A. Vogel, C. Dlugos, R. Nuffer, and R. Birngruber, “Optical properties of human sclera, and their consequences for transscleral laser applications,” Lasers Surg. Med. 11(4), 331–340 (1991).
[Crossref] [PubMed]

Boettner, E. A.

E. A. Boettner and J. R. Wolter, “Transmission of the Ocular Media,” Invest. Ophthalmol. Vis. Sci. 1, 776–783 (1962).

Bonora, S.

Bressler, N. M.

N. M. Bressler, S. B. Bressler, and S. L. Fine, “Age-related macular degeneration,” Surv. Ophthalmol. 32(6), 375–413 (1988).
[Crossref] [PubMed]

Bressler, S. B.

N. M. Bressler, S. B. Bressler, and S. L. Fine, “Age-related macular degeneration,” Surv. Ophthalmol. 32(6), 375–413 (1988).
[Crossref] [PubMed]

Burns, S. A.

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]

Carlsson, K.

K. Carlsson and J. Philip, “Theoretical investigation of the signal-to-noise ratio for different fluorescence lifetime imaging techniques,” Proc. SPIE 4622, 70–78 (2002).

Cattini, S.

Centonze, V. E.

V. E. Centonze and J. G. White, “Multiphoton Excitation Provides Optical Sections from Deeper within Scattering Specimens than Confocal Imaging,” Biophys. J. 75(4), 2015–2024 (1998).
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Cetin, A. H.

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Chung, S. T. L.

G. Kumar and S. T. L. Chung, “Characteristics of fixational eye movements in people with macular disease,” Invest. Ophthalmol. Vis. Sci. 55(8), 5125–5133 (2014).
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L. R. Weisel, P. Xi, Y. Andegeko, V. V. Lovozoy, and M. Dantus, “Greater signal and contrast in two-photon microscopy with ultrashort pulses,” Proc. SPIE 6860, 68601O (2008).

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A. Vogel, C. Dlugos, R. Nuffer, and R. Birngruber, “Optical properties of human sclera, and their consequences for transscleral laser applications,” Lasers Surg. Med. 11(4), 331–340 (1991).
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H. Liu, J. Tang, Y. Du, C. A. Lee, M. Golczak, A. Muthusamy, D. A. Antonetti, A. A. Veenstra, J. Amengual, J. von Lintig, K. Palczewski, and T. S. Kern, “Retinylamine Benefits Early Diabetic Retinopathy in Mice,” J. Biol. Chem. 290(35), 21568–21579 (2015).
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Duthaler, S.

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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).
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A. W. Stitt, T. M. Curtis, M. Chen, R. J. Medina, G. J. McKay, A. Jenkins, T. A. Gardiner, T. J. Lyons, H. P. Hammes, R. Simó, and N. Lois, “The progress in understanding and treatment of diabetic retinopathy,” Prog. Retin. Eye Res. 51, 156–186 (2016).
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Geng, Y.

Golczak, M.

H. Liu, J. Tang, Y. Du, C. A. Lee, M. Golczak, A. Muthusamy, D. A. Antonetti, A. A. Veenstra, J. Amengual, J. von Lintig, K. Palczewski, and T. S. Kern, “Retinylamine Benefits Early Diabetic Retinopathy in Mice,” J. Biol. Chem. 290(35), 21568–21579 (2015).
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G. Palczewska, Z. Dong, M. Golczak, J. J. Hunter, D. R. Williams, N. S. Alexander, and K. Palczewski, “Noninvasive two-photon microscopy imaging of mouse retina and retinal pigment epithelium through the pupil of the eye,” Nat. Med. 20(7), 785–789 (2014).
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J. I. W. 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).
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F. C. A. Groen, I. T. Young, and G. Ligthart, “A comparison of different focus functions for use in autofocus algorithms,” Cytometry 6(2), 81–91 (1985).
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Hammer, M.

M. Hammer, A. Roggan, D. Schweitzer, and G. Müller, “Optical properties of ocular fundus tissues--an in vitro study using the double-integrating-sphere technique and inverse Monte Carlo simulation,” Phys. Med. Biol. 40(6), 963–978 (1995).
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A. W. Stitt, T. M. Curtis, M. Chen, R. J. Medina, G. J. McKay, A. Jenkins, T. A. Gardiner, T. J. Lyons, H. P. Hammes, R. Simó, and N. Lois, “The progress in understanding and treatment of diabetic retinopathy,” Prog. Retin. Eye Res. 51, 156–186 (2016).
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Hasan, M. T.

Herbst, K.

L. Kessel, J. H. Lundeman, K. Herbst, T. V. Andersen, and M. Larsen, “Age-related changes in the transmission properties of the human lens and their relevance to circadian entrainment,” J. Cataract Refract. Surg. 36(2), 308–312 (2010).
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S. Martinez-Conde, S. L. Macknik, and D. H. Hubel, “The role of fixational eye movements in visual perception,” Nat. Rev. Neurosci. 5(3), 229–240 (2004).
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Hunter, J. J.

R. Sharma, C. Schwarz, D. R. Williams, G. Palczewska, K. Palczewski, and J. J. Hunter, “In Vivo Two-Photon Fluorescence Kinetics of Primate Rods and Cones,” Invest. Ophthalmol. Vis. Sci. 57(2), 647–657 (2016).
[Crossref] [PubMed]

R. Sharma, D. R. Williams, G. Palczewska, K. Palczewski, and J. J. Hunter, “Two-Photon Autofluorescence Imaging Reveals Cellular Structures Throughout the Retina of the Living Primate Eye,” Invest. Ophthalmol. Vis. Sci. 57(2), 632–646 (2016).
[Crossref] [PubMed]

G. Palczewska, Z. Dong, M. Golczak, J. J. Hunter, D. R. Williams, N. S. Alexander, and K. Palczewski, “Noninvasive two-photon microscopy imaging of mouse retina and retinal pigment epithelium through the pupil of the eye,” Nat. Med. 20(7), 785–789 (2014).
[Crossref] [PubMed]

G. Palczewska, M. Golczak, D. R. Williams, J. J. Hunter, and K. Palczewski, “Endogenous fluorophores enable two-photon imaging of the primate eye,” Invest. Ophthalmol. Vis. Sci. 55(7), 4438–4447 (2014).
[Crossref] [PubMed]

B. D. Masella, D. R. Williams, W. S. Fischer, E. A. Rossi, and J. J. Hunter, “Long-Term Reduction in Infrared Autofluorescence Caused by Infrared Light Below the Maximum Permissible Exposure,” Invest. Ophthalmol. Vis. Sci. 55(6), 3929–3938 (2014).
[Crossref] [PubMed]

R. Sharma, L. Yin, Y. Geng, W. H. Merigan, G. Palczewska, K. Palczewski, D. R. Williams, and J. J. Hunter, “In vivo two-photon imaging of the mouse retina,” Biomed. Opt. Express 4(8), 1285–1293 (2013).
[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. Express 2(1), 139–148 (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. I. W. 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]

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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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T. T. E. Yeo, S. H. Ong, Jayasooriah, and R. Sinniah, “Autofocusing for tissue microscopy,” Image Vis. Comput. 11(10), 629–639 (1993).
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A. W. Stitt, T. M. Curtis, M. Chen, R. J. Medina, G. J. McKay, A. Jenkins, T. A. Gardiner, T. J. Lyons, H. P. Hammes, R. Simó, and N. Lois, “The progress in understanding and treatment of diabetic retinopathy,” Prog. Retin. Eye Res. 51, 156–186 (2016).
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Kern, T. S.

H. Liu, J. Tang, Y. Du, C. A. Lee, M. Golczak, A. Muthusamy, D. A. Antonetti, A. A. Veenstra, J. Amengual, J. von Lintig, K. Palczewski, and T. S. Kern, “Retinylamine Benefits Early Diabetic Retinopathy in Mice,” J. Biol. Chem. 290(35), 21568–21579 (2015).
[Crossref] [PubMed]

Kerr, J. N. D.

D. S. Greenberg and J. N. D. Kerr, “Automated correction of fast motion artifacts for two-photon imaging of awake animals,” J. Neurosci. Methods 176(1), 1–15 (2009).
[Crossref] [PubMed]

Kessel, L.

L. Kessel, J. H. Lundeman, K. Herbst, T. V. Andersen, and M. Larsen, “Age-related changes in the transmission properties of the human lens and their relevance to circadian entrainment,” J. Cataract Refract. Surg. 36(2), 308–312 (2010).
[Crossref] [PubMed]

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Kumar, G.

G. Kumar and S. T. L. Chung, “Characteristics of fixational eye movements in people with macular disease,” Invest. Ophthalmol. Vis. Sci. 55(8), 5125–5133 (2014).
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L. Kessel, J. H. Lundeman, K. Herbst, T. V. Andersen, and M. Larsen, “Age-related changes in the transmission properties of the human lens and their relevance to circadian entrainment,” J. Cataract Refract. Surg. 36(2), 308–312 (2010).
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Latchney, L. R.

Lee, C. A.

H. Liu, J. Tang, Y. Du, C. A. Lee, M. Golczak, A. Muthusamy, D. A. Antonetti, A. A. Veenstra, J. Amengual, J. von Lintig, K. Palczewski, and T. S. Kern, “Retinylamine Benefits Early Diabetic Retinopathy in Mice,” J. Biol. Chem. 290(35), 21568–21579 (2015).
[Crossref] [PubMed]

Libby, R. T.

Ligthart, G.

F. C. A. Groen, I. T. Young, and G. Ligthart, “A comparison of different focus functions for use in autofocus algorithms,” Cytometry 6(2), 81–91 (1985).
[Crossref] [PubMed]

Liu, H.

H. Liu, J. Tang, Y. Du, C. A. Lee, M. Golczak, A. Muthusamy, D. A. Antonetti, A. A. Veenstra, J. Amengual, J. von Lintig, K. Palczewski, and T. S. Kern, “Retinylamine Benefits Early Diabetic Retinopathy in Mice,” J. Biol. Chem. 290(35), 21568–21579 (2015).
[Crossref] [PubMed]

Lois, N.

A. W. Stitt, T. M. Curtis, M. Chen, R. J. Medina, G. J. McKay, A. Jenkins, T. A. Gardiner, T. J. Lyons, H. P. Hammes, R. Simó, and N. Lois, “The progress in understanding and treatment of diabetic retinopathy,” Prog. Retin. Eye Res. 51, 156–186 (2016).
[Crossref] [PubMed]

Lovozoy, V. V.

L. R. Weisel, P. Xi, Y. Andegeko, V. V. Lovozoy, and M. Dantus, “Greater signal and contrast in two-photon microscopy with ultrashort pulses,” Proc. SPIE 6860, 68601O (2008).

Lozovoy, V. V.

D. Pestov, Y. Andegeko, V. V. Lozovoy, and M. Dantus, “Photobleaching and photoenhancement of endogenous fluorescence observed in two-photon microscopy with broadband laser sources,” J. Opt. 12, 8 (2010).

Lundeman, J. H.

L. Kessel, J. H. Lundeman, K. Herbst, T. V. Andersen, and M. Larsen, “Age-related changes in the transmission properties of the human lens and their relevance to circadian entrainment,” J. Cataract Refract. Surg. 36(2), 308–312 (2010).
[Crossref] [PubMed]

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A. W. Stitt, T. M. Curtis, M. Chen, R. J. Medina, G. J. McKay, A. Jenkins, T. A. Gardiner, T. J. Lyons, H. P. Hammes, R. Simó, and N. Lois, “The progress in understanding and treatment of diabetic retinopathy,” Prog. Retin. Eye Res. 51, 156–186 (2016).
[Crossref] [PubMed]

Macknik, S. L.

S. Martinez-Conde, S. L. Macknik, and D. H. Hubel, “The role of fixational eye movements in visual perception,” Nat. Rev. Neurosci. 5(3), 229–240 (2004).
[Crossref] [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).
[Crossref] [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).
[Crossref] [PubMed]

Martinez-Conde, S.

S. Martinez-Conde, S. L. Macknik, and D. H. Hubel, “The role of fixational eye movements in visual perception,” Nat. Rev. Neurosci. 5(3), 229–240 (2004).
[Crossref] [PubMed]

Masella, B.

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. Express 2(1), 139–148 (2011).
[Crossref] [PubMed]

J. I. W. 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]

Masella, B. D.

B. D. Masella, D. R. Williams, W. S. Fischer, E. A. Rossi, and J. J. Hunter, “Long-Term Reduction in Infrared Autofluorescence Caused by Infrared Light Below the Maximum Permissible Exposure,” Invest. Ophthalmol. Vis. Sci. 55(6), 3929–3938 (2014).
[Crossref] [PubMed]

McKay, G. J.

A. W. Stitt, T. M. Curtis, M. Chen, R. J. Medina, G. J. McKay, A. Jenkins, T. A. Gardiner, T. J. Lyons, H. P. Hammes, R. Simó, and N. Lois, “The progress in understanding and treatment of diabetic retinopathy,” Prog. Retin. Eye Res. 51, 156–186 (2016).
[Crossref] [PubMed]

Medina, R. J.

A. W. Stitt, T. M. Curtis, M. Chen, R. J. Medina, G. J. McKay, A. Jenkins, T. A. Gardiner, T. J. Lyons, H. P. Hammes, R. Simó, and N. Lois, “The progress in understanding and treatment of diabetic retinopathy,” Prog. Retin. Eye Res. 51, 156–186 (2016).
[Crossref] [PubMed]

Merigan, W.

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]

R. Sharma, L. Yin, Y. Geng, W. H. Merigan, G. Palczewska, K. Palczewski, D. R. Williams, and J. J. Hunter, “In vivo two-photon imaging of the mouse retina,” Biomed. Opt. Express 4(8), 1285–1293 (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. Express 3(4), 715–734 (2012).
[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. Express 2(1), 139–148 (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. I. W. 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]

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]

Morgan, J. I. W.

J. I. W. 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]

Mujat, M.

Müller, G.

M. Hammer, A. Roggan, D. Schweitzer, and G. Müller, “Optical properties of ocular fundus tissues--an in vitro study using the double-integrating-sphere technique and inverse Monte Carlo simulation,” Phys. Med. Biol. 40(6), 963–978 (1995).
[Crossref] [PubMed]

Muthusamy, A.

H. Liu, J. Tang, Y. Du, C. A. Lee, M. Golczak, A. Muthusamy, D. A. Antonetti, A. A. Veenstra, J. Amengual, J. von Lintig, K. Palczewski, and T. S. Kern, “Retinylamine Benefits Early Diabetic Retinopathy in Mice,” J. Biol. Chem. 290(35), 21568–21579 (2015).
[Crossref] [PubMed]

Nelson, B. J.

Y. Sun, S. Duthaler, and B. J. Nelson, “Autofocusing in computer microscopy: selecting the optimal focus algorithm,” Microsc. Res. Tech. 65(3), 139–149 (2004).
[Crossref] [PubMed]

Nozato, K.

Nuffer, R.

A. Vogel, C. Dlugos, R. Nuffer, and R. Birngruber, “Optical properties of human sclera, and their consequences for transscleral laser applications,” Lasers Surg. Med. 11(4), 331–340 (1991).
[Crossref] [PubMed]

Ong, S. H.

T. T. E. Yeo, S. H. Ong, Jayasooriah, and R. Sinniah, “Autofocusing for tissue microscopy,” Image Vis. Comput. 11(10), 629–639 (1993).
[Crossref]

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]

Palczewska, G.

R. Sharma, D. R. Williams, G. Palczewska, K. Palczewski, and J. J. Hunter, “Two-Photon Autofluorescence Imaging Reveals Cellular Structures Throughout the Retina of the Living Primate Eye,” Invest. Ophthalmol. Vis. Sci. 57(2), 632–646 (2016).
[Crossref] [PubMed]

R. Sharma, C. Schwarz, D. R. Williams, G. Palczewska, K. Palczewski, and J. J. Hunter, “In Vivo Two-Photon Fluorescence Kinetics of Primate Rods and Cones,” Invest. Ophthalmol. Vis. Sci. 57(2), 647–657 (2016).
[Crossref] [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).
[Crossref] [PubMed]

G. Palczewska, M. Golczak, D. R. Williams, J. J. Hunter, and K. Palczewski, “Endogenous fluorophores enable two-photon imaging of the primate eye,” Invest. Ophthalmol. Vis. Sci. 55(7), 4438–4447 (2014).
[Crossref] [PubMed]

G. Palczewska, Z. Dong, M. Golczak, J. J. Hunter, D. R. Williams, N. S. Alexander, and K. Palczewski, “Noninvasive two-photon microscopy imaging of mouse retina and retinal pigment epithelium through the pupil of the eye,” Nat. Med. 20(7), 785–789 (2014).
[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. Express 2(1), 139–148 (2011).
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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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R. Sharma, C. Schwarz, D. R. Williams, G. Palczewska, K. Palczewski, and J. J. Hunter, “In Vivo Two-Photon Fluorescence Kinetics of Primate Rods and Cones,” Invest. Ophthalmol. Vis. Sci. 57(2), 647–657 (2016).
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R. Sharma, D. R. Williams, G. Palczewska, K. Palczewski, and J. J. Hunter, “Two-Photon Autofluorescence Imaging Reveals Cellular Structures Throughout the Retina of the Living Primate Eye,” Invest. Ophthalmol. Vis. Sci. 57(2), 632–646 (2016).
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[Crossref] [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).
[Crossref] [PubMed]

G. Palczewska, M. Golczak, D. R. Williams, J. J. Hunter, and K. Palczewski, “Endogenous fluorophores enable two-photon imaging of the primate eye,” Invest. Ophthalmol. Vis. Sci. 55(7), 4438–4447 (2014).
[Crossref] [PubMed]

G. Palczewska, Z. Dong, M. Golczak, J. J. Hunter, D. R. Williams, N. S. Alexander, and K. Palczewski, “Noninvasive two-photon microscopy imaging of mouse retina and retinal pigment epithelium through the pupil of the eye,” Nat. Med. 20(7), 785–789 (2014).
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R. Sharma, L. Yin, Y. Geng, W. H. Merigan, G. Palczewska, K. Palczewski, D. R. Williams, and J. J. Hunter, “In vivo two-photon imaging of the mouse retina,” Biomed. Opt. Express 4(8), 1285–1293 (2013).
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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. Express 2(1), 139–148 (2011).
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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. Express 2(1), 139–148 (2011).
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R. Sharma, D. R. Williams, G. Palczewska, K. Palczewski, and J. J. Hunter, “Two-Photon Autofluorescence Imaging Reveals Cellular Structures Throughout the Retina of the Living Primate Eye,” Invest. Ophthalmol. Vis. Sci. 57(2), 632–646 (2016).
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Q. Yang, J. Zhang, K. Nozato, K. Saito, D. R. Williams, A. Roorda, and E. A. Rossi, “Closed-loop optical stabilization and digital image registration in adaptive optics scanning light ophthalmoscopy,” Biomed. Opt. Express 5(9), 3174–3191 (2014).
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B. D. Masella, D. R. Williams, W. S. Fischer, E. A. Rossi, and J. J. Hunter, “Long-Term Reduction in Infrared Autofluorescence Caused by Infrared Light Below the Maximum Permissible Exposure,” Invest. Ophthalmol. Vis. Sci. 55(6), 3929–3938 (2014).
[Crossref] [PubMed]

G. Palczewska, M. Golczak, D. R. Williams, J. J. Hunter, and K. Palczewski, “Endogenous fluorophores enable two-photon imaging of the primate eye,” Invest. Ophthalmol. Vis. Sci. 55(7), 4438–4447 (2014).
[Crossref] [PubMed]

G. Palczewska, Z. Dong, M. Golczak, J. J. Hunter, D. R. Williams, N. S. Alexander, and K. Palczewski, “Noninvasive two-photon microscopy imaging of mouse retina and retinal pigment epithelium through the pupil of the eye,” Nat. Med. 20(7), 785–789 (2014).
[Crossref] [PubMed]

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]

R. Sharma, L. Yin, Y. Geng, W. H. Merigan, G. Palczewska, K. Palczewski, D. R. Williams, and J. J. Hunter, “In vivo two-photon imaging of the mouse retina,” Biomed. Opt. Express 4(8), 1285–1293 (2013).
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E. A. Rossi, P. Rangel-Fonseca, K. Parkins, W. Fischer, L. R. Latchney, M. A. Folwell, D. R. Williams, A. Dubra, and M. M. Chung, “In vivo imaging of retinal pigment epithelium cells in age related macular degeneration,” Biomed. Opt. Express 4(11), 2527–2539 (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. Express 3(4), 715–734 (2012).
[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. Express 2(1), 139–148 (2011).
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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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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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J. I. W. 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).
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Biomed. Opt. Express (9)

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. Express 2(1), 139–148 (2011).
[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. Express 3(4), 715–734 (2012).
[Crossref] [PubMed]

R. Sharma, L. Yin, Y. Geng, W. H. Merigan, G. Palczewska, K. Palczewski, D. R. Williams, and J. J. Hunter, “In vivo two-photon imaging of the mouse retina,” Biomed. Opt. Express 4(8), 1285–1293 (2013).
[Crossref] [PubMed]

E. A. Rossi, P. Rangel-Fonseca, K. Parkins, W. Fischer, L. R. Latchney, M. A. Folwell, D. R. Williams, A. Dubra, and M. M. Chung, “In vivo imaging of retinal pigment epithelium cells in age related macular degeneration,” Biomed. Opt. Express 4(11), 2527–2539 (2013).
[Crossref] [PubMed]

Q. Yang, J. Zhang, K. Nozato, K. Saito, D. R. Williams, A. Roorda, and E. A. Rossi, “Closed-loop optical stabilization and digital image registration in adaptive optics scanning light ophthalmoscopy,” Biomed. Opt. Express 5(9), 3174–3191 (2014).
[Crossref] [PubMed]

K. S. K. Wong, Y. Jian, M. Cua, S. Bonora, R. J. Zawadzki, and M. V. Sarunic, “In vivo imaging of human photoreceptor mosaic with wavefront sensorless adaptive optics optical coherence tomography,” Biomed. Opt. Express 6(2), 580–590 (2015).
[Crossref] [PubMed]

J. Zhang, Q. Yang, K. Saito, K. Nozato, D. R. Williams, and E. A. Rossi, “An adaptive optics imaging system designed for clinical use,” Biomed. Opt. Express 6(6), 2120–2137 (2015).
[Crossref] [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).
[Crossref] [PubMed]

D. J. Wahl, Y. Jian, S. Bonora, R. J. Zawadzki, and M. V. Sarunic, “Wavefront sensorless adaptive optics fluorescence biomicroscope for in vivo retinal imaging in mice,” Biomed. Opt. Express 7(1), 1–12 (2016).
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Biophys. J. (1)

V. E. Centonze and J. G. White, “Multiphoton Excitation Provides Optical Sections from Deeper within Scattering Specimens than Confocal Imaging,” Biophys. J. 75(4), 2015–2024 (1998).
[Crossref] [PubMed]

Cytometry (1)

F. C. A. Groen, I. T. Young, and G. Ligthart, “A comparison of different focus functions for use in autofocus algorithms,” Cytometry 6(2), 81–91 (1985).
[Crossref] [PubMed]

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).
[Crossref] [PubMed]

Image Vis. Comput. (1)

T. T. E. Yeo, S. H. Ong, Jayasooriah, and R. Sinniah, “Autofocusing for tissue microscopy,” Image Vis. Comput. 11(10), 629–639 (1993).
[Crossref]

Invest. Ophthalmol. Vis. Sci. (7)

R. Sharma, D. R. Williams, G. Palczewska, K. Palczewski, and J. J. Hunter, “Two-Photon Autofluorescence Imaging Reveals Cellular Structures Throughout the Retina of the Living Primate Eye,” Invest. Ophthalmol. Vis. Sci. 57(2), 632–646 (2016).
[Crossref] [PubMed]

R. Sharma, C. Schwarz, D. R. Williams, G. Palczewska, K. Palczewski, and J. J. Hunter, “In Vivo Two-Photon Fluorescence Kinetics of Primate Rods and Cones,” Invest. Ophthalmol. Vis. Sci. 57(2), 647–657 (2016).
[Crossref] [PubMed]

B. D. Masella, D. R. Williams, W. S. Fischer, E. A. Rossi, and J. J. Hunter, “Long-Term Reduction in Infrared Autofluorescence Caused by Infrared Light Below the Maximum Permissible Exposure,” Invest. Ophthalmol. Vis. Sci. 55(6), 3929–3938 (2014).
[Crossref] [PubMed]

J. I. W. 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]

E. A. Boettner and J. R. Wolter, “Transmission of the Ocular Media,” Invest. Ophthalmol. Vis. Sci. 1, 776–783 (1962).

G. Palczewska, M. Golczak, D. R. Williams, J. J. Hunter, and K. Palczewski, “Endogenous fluorophores enable two-photon imaging of the primate eye,” Invest. Ophthalmol. Vis. Sci. 55(7), 4438–4447 (2014).
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G. Kumar and S. T. L. Chung, “Characteristics of fixational eye movements in people with macular disease,” Invest. Ophthalmol. Vis. Sci. 55(8), 5125–5133 (2014).
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J. Biol. Chem. (1)

H. Liu, J. Tang, Y. Du, C. A. Lee, M. Golczak, A. Muthusamy, D. A. Antonetti, A. A. Veenstra, J. Amengual, J. von Lintig, K. Palczewski, and T. S. Kern, “Retinylamine Benefits Early Diabetic Retinopathy in Mice,” J. Biol. Chem. 290(35), 21568–21579 (2015).
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J. Cataract Refract. Surg. (1)

L. Kessel, J. H. Lundeman, K. Herbst, T. V. Andersen, and M. Larsen, “Age-related changes in the transmission properties of the human lens and their relevance to circadian entrainment,” J. Cataract Refract. Surg. 36(2), 308–312 (2010).
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J. Exp. Psychol. (1)

F. Ratliff and L. A. Riggs, “Involuntary motions of the eye during monocular fixation,” J. Exp. Psychol. 40(6), 687–701 (1950).
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J. Neurophysiol. (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).
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J. Neurosci. Methods (1)

D. S. Greenberg and J. N. D. Kerr, “Automated correction of fast motion artifacts for two-photon imaging of awake animals,” J. Neurosci. Methods 176(1), 1–15 (2009).
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Figures (9)

Fig. 1
Fig. 1

Flow chart of alignment procedure. A) A template image is obtained either from averaging all images in a series together without alignment, or a highly fluorescent image. B) The image of interest is aligned to the template image in a three step procedure. 1) Both the image and the template are blurred by averaging a two pixel radius around each pixel. 2) The orientation of the image maximizing its cross correlation with the template is determined using Fourier transform- based alignment. 3) The non-rigid registration algorithm based on Lukas and Kanade is used to register the image to the template. D) Steps B and C are repeated for every image in an imaging series producing a set of aligned images. These images are then averaged together to produce the average image.

Fig. 2
Fig. 2

Single images collected with laser powers of A) 6.2 mW, C) 3.2 mW, E) 2.1 mW. On the right are images resulting from averaging 150 images collected with laser powers of B) 6.2 mW C) 3.2 mW, F) 2.1 mW. To reveal image content better, images were brightened as described in Methods.

Fig. 3
Fig. 3

Single images collected with laser powers of A) 1.5 mW, C) 1.0 mW. On the right are images resulting from averaging 150 images collected with laser powers of B) 1.5 mW D) 1.0 mW. To reveal image content better, images were brightened as described in Methods.

Fig. 4
Fig. 4

Images collected of retina vasculature. A), C), E), G) Single images. Average of B) 9, D) 5, F) 20, H) 40 images without any registration. All images were collected at 5 mW of laser power.

Fig. 5
Fig. 5

Ideal versus average template comparison for images of RPE. Comparison of registration to an unregistered average image (A, C, E) versus image registration to an ideal template image (B, D, F). Laser powers used to collect 150 images for averaging were 6.2 mW (A), 3.2 mW (C), 2.1 mW (E). To reveal image content better, images were brightened as described in Methods. Ideal template images used for registration in (B, D, F) were collected with laser power of 31mW.

Fig. 6
Fig. 6

Ideal versus average template comparison for images of RPE. Comparison of registration to an unregistered average image (A, C) versus image registration to an ideal template image (B, D). Ideal template images used for registration in (B, D) were collected with laser power of 31mW. Laser powers used to collect 150 images for averaging were 1.5 mW (A, B), and 1.0 mW (C, D). To reveal image content better, brightness of images was increased as described in Methods.

Fig. 7
Fig. 7

Average images of capillary fluorescence after injection of fluorescent dye. A, C, E, G) Average images created after images were registered using the unaligned average image as the template. B, D, F, H) Average of collected images using a single manually selected image as the template. A and B included 10 images. C and E included 5 images. E and F included 20 images. G and H included 40 images.

Fig. 8
Fig. 8

Image quality as between 1 and 150 images were included in the image average data series collected with 1.5 and 1.0 mW. Image quality is calculated as SNR / NV . Images were registered to the unregistered average image. Vertical bars indicate standard deviations of five random accumulation trials for a given number of images.

Fig. 9
Fig. 9

High laser-power images collected at 31 mW. A, B, C, D, E) correspond with the RPE images collected at 6.2, 3.2, 2.1, 1.5, 1.0 mW, respectively.

Tables (4)

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Table 1 Normalized variance of RPE image averagesa under differing registration conditions

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Table 2 Normalized variance of retina vessel image averages under differing registration conditions

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Table 3 Template to target Pearson correlation and pixel displacement of RPE imagesa

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Table 4 Template to target Pearson correlation and pixel displacement of RPE imagesa

Equations (2)

Equations on this page are rendered with MathJax. Learn more.

NV= 1 ( HW μ 2 ) H W ( ( i( w,h )μ ) 2 ) ,
SNR= N N ,

Metrics