Basic investigations for 2-dimensional time-resolved fluorescence measurements at the fundus

Area of Science:

• Biophotonics
• Ophthalmology
• Fluorescence Spectroscopy

Background:

• Natural fluorophores possess characteristic decay times, independent of fluorescence intensity.
• This property allows for the detection of weak, short-lived fluorescence even when obscured by intense, long-lived fluorescence.
• Decay time is influenced by the embedding matrix, suggesting potential for cellular stage information.

Purpose of the Study:

• To develop and validate a method for discriminating fluorophores in the eye based on fluorescence decay time.
• To investigate the potential of fluorescence decay time analysis for cellular stage determination.
• To establish optimal techniques for detecting short-lived fluorophores, such as A2E, a marker in age-related macular degeneration.

Main Methods:

• Utilizing laser scanning microscopy combined with time-correlated single photon counting (TCSPC).
• Developing an experimental setup for in vivo measurements on structured fluorescent tests under conditions simulating the living eye.
• Implementing tau mapping to visualize fields of equal decay times.

Main Results:

• Demonstrated the feasibility of discriminating fluorophores based on decay time.
• Basic experiments on fluorescent tests under simulated in vivo conditions yielded promising results.
• Identified the need for a light source with approximately 50 ps pulses for detecting the short decay time (120 ps) of A2E.

Conclusions:

• Fluorescence decay time analysis is a promising method for fluorophore discrimination in the eye.
• The developed laser scanning and TCSPC technique offers optimal discrimination based on decay time.
• Further development, including a suitable pulsed light source, is required for detecting specific short-lived fluorophores like A2E.