Working visit to the Department of Pharmacology, Case Western Reserve University, USA.
This working visit took place at Case Western Reserve University in Cleveland. The goals of the visit were twofold: a) to integrate a pulse picker into the existing TPEF set up in order to optimize the signal for limited average excitation power and b) to build the optical setup from scratch for two-photon excitation fluorescence (TPEF) imaging of rodent eye. The TPEF method is used for the investigation of retinal diseases and can help to develop new treatment therapies. The use of infrared light and a two-photon process allows one to avoid substantial light absorption in the 320-400 nm range.
Regarding a) by reducing the repetition rate of the excitation pulses from 80 MHz down to 6 MHz, we achieved a 9-fold increase of the signal with the same average power, which was below the ANSI limit for the human eye. This part of the work was conducted by Patrycjusz Stremplewski.
Regarding b) a long-term goal is to simplify the TPEF setup with respect to the one existing in the lab, which will allow delivering shorter pulses to rodent’s eyes. We expect that TPEF measurement will be possible with lower average laser power, which will further improve the safety of the experiment. To that end, a custom TPEF setup was built by Jakub Bogusławski and Piotr Ciąćka. The setup allows for simultaneous detection of scattered light and fluorescence signals. Another improvement was to include lock-in detection in the fluorescence channel, which will allow detecting much lower light intensities. The first results obtained with the setup, imaging the fluorescent sample in both scattering and TPEF modes, are encouraging.
Further steps will include setting up a dedicated laser source operating at 780 nm and some improvements in the software, which will allow simultaneous detection of scattered and fluorescence light and correction for eye movement. Then, the setup will be ready for measurements with animals.
Jakub Bogusławski, Piotr Ciąćka, Patrycjusz Stremplewski
May 19-30, 2018
The Case Western Reserve University, Cleveland, USA