In-vivo optical biopsy at the fundus by measuring the fluorescence intensity and life-time of endogenous fluorophores enables the analysis of physiological processes and pathological changes in vision at the molecular level. In particular, it can be utilized to discover defects in vitamin A metabolism during the regeneration of the visual pig-ments required for the detection of light. In this way, it helps to better understand the development of age-related degenerative diseases and is a possible future method for their early diagnosis before irreversible structural damage has occurred. This will ac-celerate the development of therapies against blinding diseases.
The two-photon excitation of fluorescence enables to use of infrared wavelengths that can penetrate well to the fundus. However, it requires the use of ultra-short light pulses with high peak intensity, which can also cause damage. The irradiation parameters are therefore selected in such a way that maximum fluorescence light yield is associated with a minimum risk of damage. An international team of researchers from the University of California Irvine (USA), the Polish Academy of Sciences, the Nikolaus Kopernikus University in Torun (Poland), and the University of Lübeck in Germany have faced these challenges and now presented the first in-vivo results on mouse eyes. The contribution by Prof. Dr. Alfred Vogel and Dr. Xiaoxuan Liang from Lübeck University concentrates on the analysis of possible photo damage mechanisms and exposure limits, the understanding of which is essential for in-vivo retinal imaging.
Two-photon excitation of fluorophores is easier, the stronger the excitation laser pulses can be focused. The opening angle (numerical aperture) of the mouse eye is NA = 0.4, more than twice as large as that of the human eye, which means that the for the same pulse energy the light irradiance in the focus is more than four times as large. Therefore, it will still take a while before damage-free two-photon optical biopsy can be achieved in humans - but the results that have already been demonstrated on the mouse eye are an important step on this path.
