Friday, 28-08-2020


Noninvasive two-photon optical biopsy of retinal fluorophores

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. A particular technological challenge is to ensure that the se-lected parameters are also adhered to on the retina after the laser pulses have passed the eye. Another challenge is the establishment of a clear representation of the fluores-cence information, which enables an efficient analysis of the underlying molecular pro-cesses.

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.

In their analysis, they had to consider that a unicellular layer with a high content of mela-nin pigments, the retinal pigment epithelium (RPE), is located directly below the photo-receptors. The strong light absorption in the melanin granules in the RPE heats them up, which can lead to the fragmentation of granules by thermo-elastic stress waves, to chem-ical changes in biomolecules in the vicinity of the granules and to the formation of small vapor bubbles. Moreover, the high peak intensity of the laser pulses generates a certain number of free electrons, which can also cause damage to biomolecules. By carefully ana-lyzing all damage paths using computer simulations, Xiaoxuan Liang and Alfred Vogel came to the conclusion that thermal damage represents the greatest risk with two-photon microscopy on the fundus.

The team's strategy for reducing thermal damage is to reduce the pulse repetition rate of the laser from the commonly used value of 80 MHz to 8 MHz and to increase the energy of the individual pulses. This increases the peak intensity of the pulses and the fluorescence yield with two-photon excitation rises sharply. This means that more fluorescence can be generated at 8 MHz with the same average power in the laser pulse train than at 80 MHz, or, vice versa, that the same fluorescence yield can be achieved with a lower laser power. The reduction in laser power reduces the temperature increase during the laser pulse train and thus also the thermal load. The strategy of reducing the repetition rate reaches its limit when the energy of the individual pulses is so great that the temperature increase per pulse leads to the thermo-elastic fragmentation of many melanin granules. The optimal repetition rate has yet to be determined, but the team was able to demonstrate a damage-free optical biopsy on the mouse eye at 8 MHz.

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.

The paper “Noninvasive two-photon optical biopsy of retinal fluorophores” was pub-lished in the Proceedings of the National Academy of Sciences (PNAS) on August 26, 2020. It can be accessed through

The research in Lübeck was supported by Grant FA9550-18-1-0521 in the Biophysics Basic Science Program of the Air Force Office of Scientific Research (AFOSR).

Example of molecular two-photon imaging in the living mouse eye. The distribution of metabolic products that are important for the visual process was analyzed in mice with different genetic changes. Retinyl esters play an im-portant role in the formation of the visual pigment 11-cis-retinal in cell organelles called retinosomes, while A2E is a harmful degradation product of vitamin A metabolites. The fluorescence life time images evaluated by "phasor analysis" clearly show the different frequency and localization of these metabolic products in the various mouse types. (Fig.: PNAS)