Bridging biology and optics

Two-photon excitation fluorescence (TPEF) is emerging as a powerful imaging technique with superior penetration power in scattering media, allowing for functional imaging of biological tissues at a subcellular level. TPEF is commonly used in cancer diagnostics, as it enables the direct observation of metabolism within living cells. The technique is now widely used in various medical fields, including ophthalmology. The eye is a complex and delicate organ with multiple layers of different cell types and tissues. Although this structure is ideal for visual perception, it generates aberrations in TPEF eye imaging. However, adaptive optics can now compensate for these aberrations, allowing for improved imaging of the eyes of animal models for human diseases. The eye is naturally built to filter out harmful wavelengths, but these wavelengths can be mimicked and thereby utilized in diagnostics via two-photon (2Ph) excitation. Recent advances in laser-source manufacturing have made it possible to minimize the exposure of in vivo measurements within safety, while achieving sufficient signals to detect functional images, making TPEF a viable option for human application. Recent advances in wavefront-distortion correction in animal models and in the safety of the use of TPEF on human subjects, make TPEF a potentially powerful tool for ophthalmological diagnostics.

Two-photon imaging. (A) Jablonski diagram of fluorophore excitation by single, (B) two-photon, and (C) secondary-harmonic generation (SHG). (D) comparison of one and two-photon (yellow arrow) excitation profiles. (E) focal plane range by single, (F) two-photon, and (G) SHG. (H) FLIM histogram of photon counts versus arrival time after the laser pulse. (I) two-photon excited fluorescence and SHG are isotopically emitted. (J) experimental TPEF-SLO setup, and its (K) imaging processing. (L) experimental SHG setup. (M) relative TPEF intensity as a function of pulse repetition frequency. (N) anatomy of the human eye.

Team:

Dr. Humberto Fernandes

Dr. Vineeta Kaushik

Luca Gessa

Nelam Kumar

Dr. Michał Dąbrowski (former POB member)

Reference:

“Two-photon excitation fluorescence in ophthalmology: safety and improved imaging for functional diagnostics” (2024) V Kaushik, M Dąbrowski, L Gessa, N Kumar, H Fernandes, Frontiers in Medicine 10, 1293640.