Open AccessOptimizing ultrafast illumination for multiphoton-excited fluorescence imaging
Author(s) -
Caleb Stoltzfus,
Aleksander Rebane
Publication year - 2016
Publication title -
biomedical optics express
Language(s) - Uncategorized
Resource type - Journals
SCImago Journal Rank - 1.362
H-Index - 86
ISSN - 2156-7085
DOI - 10.1364/boe.7.001768
Subject(s) - optics , femtosecond , ultrashort pulse , laser , fluorescence lifetime imaging microscopy , excited state , photon counting , materials science , frame rate , fluorescence , photon , throughput , physics , computer science , atomic physics , telecommunications , wireless
We study the optimal conditions for high throughput two-photon excited fluorescence (2PEF) and three-photon excited fluorescence (3PEF) imaging using femtosecond lasers. We derive relations that allow maximization of the rate of imaging depending on the average power, pulse repetition rate, and noise characteristics of the laser, as well as on the size and structure of the sample. We perform our analysis using ~100 MHz, ~1 MHz and 1 kHz pulse rates and using both a tightly-focused illumination beam with diffraction-limited image resolution, as well loosely focused illumination with a relatively low image resolution, where the latter utilizes separate illumination and fluorescence detection beam paths. Our theoretical estimates agree with the experiments, which makes our approach especially useful for optimizing high throughput imaging of large samples with a field-of-view up to 10x10 cm(2).