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Noninvasive two-photon microscopy imaging of mouse retina and retinal pigment epithelium through the pupil of the eye

Nature Medicine volume 20, pages 785–789 (2014)Cite this article

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Abstract

Two-photon excitation microscopy can image retinal molecular processes in vivo. Intrinsically fluorescent retinyl esters in subcellular structures called retinosomes are an integral part of the visual chromophore regeneration pathway. Fluorescent condensation products of all-trans-retinal accumulate in the eye with age and are also associated with age-related macular degeneration (AMD). Here, we report repetitive, dynamic imaging of these compounds in live mice through the pupil of the eye. By leveraging advanced adaptive optics, we developed a data acquisition algorithm that permitted the identification of retinosomes and condensation products in the retinal pigment epithelium by their characteristic localization, spectral properties and absence in genetically modified or drug-treated mice. This imaging approach has the potential to detect early molecular changes in retinoid metabolism that trigger light- and AMD-induced retinal defects and to assess the effectiveness of treatments for these conditions.

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Figure 1: TPM for imaging of mouse retina and RPE.
Figure 2: TPM images of ex vivo albino mouse RPE and retinas obtained through the mouse eye pupil.
Figure 3: Use of TPM imaging for ophthalmic drug screening.
Figure 4: Setup for TPM RPE imaging in living mice.

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Acknowledgements

We thank M. Redmond (National Eye Institute of the US National Institutes of Health (NIH)) for Rpe65−/− mice and J. Wilson (Baylor College of Medicine) for GFP-rhodopsin mice. We also thank the team at Bioptigen for advice on preparation of mice for live animal imaging, D. Piston for stimulating discussion and L.T. Webster Jr. and members of K.P.'s laboratory and Polgenix's team for critical comments on the manuscript. Research reported in this publication was supported by the National Eye Institute of the NIH under award numbers R01EY008061, R01EY009339, R24EY021126 and P30EY11373 and by the National Institute on Aging of the NIH under award number R44AG043645. N.S.A. was supported by NIH institutional training grants 5T32EY007157 and 5T32DK007319.

Author information

Authors and Affiliations

  1. Polgenix, Cleveland, Ohio, USA

    Grazyna Palczewska, Zhiqian Dong & Krzysztof Palczewski

  2. Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, USA

    Marcin Golczak, Nathan S Alexander & Krzysztof Palczewski

  3. Center for Visual Science, University of Rochester, Rochester, New York, USA

    Jennifer J Hunter & David R Williams

  4. Flaum Eye Institute, University of Rochester, Rochester, New York, USA

    Jennifer J Hunter

  5. Department of Biomedical Engineering, University of Rochester, Rochester, New York, USA

    Jennifer J Hunter

  6. The Institute of Optics, University of Rochester, Rochester, New York, USA

    David R Williams

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  1. Grazyna Palczewska
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Contributions

G.P. and K.P. conceived and directed the project. G.P., M.G., Z.D. and N.S.A. designed and conducted experiments, and generated software. G.P., N.S.A. and K.P. prepared the manuscript. J.J.H. and D.R.W. edited the manuscript.

Corresponding author

Correspondence to Krzysztof Palczewski.

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Competing interests

G.P. and Z.D. are employees of Polgenix. K.P. is Chief Scientific Officer at Polgenix. K.P. is an inventor of the U.S. patent no. 7,706,863 and U.S. patent no. 8,346,345, whose values may be affected by this publication. The laboratories of J.J.H and D.R.W. received support from Polgenix.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 (PDF 990 kb)

Imaging retina at different depths.

50 images were obtained ex vivo along the z axis in an eye of a 2-month-old WT mouse. Details of different layers come into focus as the z-axis translation stage moves at even intervals. The first image shows the nerve fiber layer and the 50th image is just behind RPE. The optic disc is in the top right corner. (WMV 3205 kb)

Stochastic parallel gradient descent (SPGD) optimization of imaging Rpe65−/− mice.

Forty steps of optimization were conducted, and the corresponding forty images for the trajectory are shown. The 30th image is the one with the best normalized variance (shown in Fig. 4c). (WMV 6425 kb)

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Palczewska, G., Dong, Z., Golczak, M. et al. Noninvasive two-photon microscopy imaging of mouse retina and retinal pigment epithelium through the pupil of the eye. Nat Med 20, 785–789 (2014). https://doi.org/10.1038/nm.3590

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