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Noninvasive multiphoton fluorescence microscopy resolves retinol and retinal condensation products in mouse eyes

Nature Medicine volume 16, pages 1444–1449 (2010)Cite this article

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Abstract

Multiphoton excitation fluorescence microscopy (MPM) can image certain molecular processes in vivo. In the eye, fluorescent retinyl esters in subcellular structures called retinosomes mediate regeneration of the visual chromophore, 11-cis-retinal, by the visual cycle. But harmful fluorescent condensation products of retinoids also occur in the retina. We report that in wild-type mice, excitation with a wavelength of ∼730 nm identified retinosomes in the retinal pigment epithelium, and excitation with a wavelength of ∼910 nm revealed at least one additional retinal fluorophore. The latter fluorescence was absent in eyes of genetically modified mice lacking a functional visual cycle, but accentuated in eyes of older wild-type mice and mice with defective clearance of all-trans-retinal, an intermediate in the visual cycle. MPM, a noninvasive imaging modality that facilitates concurrent monitoring of retinosomes along with potentially harmful products in aging eyes, has the potential to detect early molecular changes due to age-related macular degeneration and other defects in retinoid metabolism.

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Figure 1: Multiphoton excitation of a 6-month-old WT mouse eye at 730 and 910 nm produced emission spectra indicating more than one fluorophore.
Figure 2: Multiphoton excitation of the RPE in an intact 6-month-old WT mouse eye at different wavelengths of excitation light.
Figure 3: Visualization of retinosomes by three-photon excitation spectroscopy in the intact 7-week-old Rpe65−/− mouse eye.
Figure 4: Two-photon excitation of 3-month-old Abca4−/−;Rdh8−/− (DKO) intact mouse eye.
Figure 5: Age-dependent changes in fluorophore accumulation in mouse eyes.

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Acknowledgements

We would like to thank M. Golczak for help during the course of this study and Z. Dong for expert handling of mice. We also thank L.T. Webster and members of Palczewski laboratory for critical comments on the manuscript. Rpe65−/– mice were a kind gift from M. Redmond (US National Eye Institute). This research was supported in part by grants EY008061, EY009339, EY019880, EY019031, EY020715 and P30 EY11373 from the US National Institutes of Health, TECH 09-004 from the State of Ohio Department of Development and Third Frontier Commission, the European Life Scientist Organization and the Klaus Tschira Foundation.

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Authors and Affiliations

  1. Polgenix, Cleveland, Ohio, USA

    Grazyna Palczewska, Wenyu Sun & Krzysztof Palczewski

  2. Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio, USA

    Tadao Maeda & Akiko Maeda

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

    Tadao Maeda, Yoshikazu Imanishi, Yu Chen, Akiko Maeda & Krzysztof Palczewski

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

    David R Williams

  5. Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA

    David W Piston

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  1. Grazyna Palczewska
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Contributions

G.P. and K.P. conceived of and directed the project. G.P., T.M., Y.I., W.S., Y.C. and A.M. designed and conducted experiments. G.P. and K.P. prepared the manuscript. Y.I., D.W.P. analyzed the data and edited the manuscript. D.R.W. edited the manuscript.

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Correspondence to Grazyna Palczewska or Krzysztof Palczewski.

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Competing interests

G.P. and W.S. are employees of Polgenix. K.P. is chief scientific officer at Polgenix. K.P. and Y.M. are inventors of the US Patent No. 7,706,863, “Methods for assessing a physiological state of a mammalian retina,” whose value may be affected by this publication. The laboratory of D.R.W. received support from Polgenix.

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Palczewska, G., Maeda, T., Imanishi, Y. et al. Noninvasive multiphoton fluorescence microscopy resolves retinol and retinal condensation products in mouse eyes. Nat Med 16, 1444–1449 (2010). https://doi.org/10.1038/nm.2260

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