Noninvasive two-photon optical biopsy of retinal fluorophores

Grazyna Palczewska, Jakub Boguslawski, Patrycjusz Stremplewski, Lukasz Kornaszewski, Jianye Zhang, Zhiqian Dong, Xiao Xuan Liang, Enrico Gratton, Alfred Vogel, MacIej Wojtkowski, Krzysztof Palczewski

1 Citation (Scopus)


High-resolution imaging techniques capable of detecting identifiable endogenous fluorophores in the eye along with genetic testing will dramatically improve diagnostic capabilities in the ophthalmology clinic and accelerate the development of new treatments for blinding diseases. Two-photon excitation (TPE)-based imaging overcomes the filtering of ultraviolet light by the lens of the human eye and thus can be utilized to discover defects in vitamin A metabolism during the regeneration of the visual pigments required for the detection of light. Combining TPE with fluorescence lifetime imaging (FLIM) and spectral analyses offers the potential of detecting diseases of the retina at earlier stages before irreversible structural damage has occurred. The main barriers to realizing the benefits of TPE for imaging the human retina arise from concerns about the high light exposure typically needed for informative TPE imaging and the requirement to correlate the ensuing data with different states of health and disease. To overcome these hurdles, we improved TPE efficiency by controlling temporal properties of the excitation light and employed phasor analyses to FLIM and spectral data in mouse models of retinal diseases. Modeling of retinal photodamage revealed that plasma-mediated effects do not play a role and that melanin-related thermal effects are mitigated by reducing pulse repetition frequency. By using noninvasive TPE imaging we identified molecular components of individual granules in the retinal pigment epithelium and present their analytical characteristics.

Original languageEnglish
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number36
Pages (from-to)22532
Number of pages1
Publication statusPublished - 08.09.2020

Research Areas and Centers

  • Academic Focus: Biomedical Engineering


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