Open AccessDynamic real-time subtraction of stray-light and background for multiphoton imaging
Author(s) -
A. Fernández,
Andrew Straw,
Martin Distel,
Rainer A. Leitgeb,
Andrius Baltuška,
A. J. Verhoef
Publication year - 2020
Publication title -
biomedical optics express
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.362
H-Index - 86
ISSN - 2156-7085
DOI - 10.1364/boe.403255
Subject(s) - optics , duty cycle , ultrashort pulse , synchronizing , laser , fluorescence , background subtraction , fluorescence lifetime imaging microscopy , subtraction , stray light , computer science , materials science , physics , pixel , telecommunications , power (physics) , arithmetic , mathematics , quantum mechanics , transmission (telecommunications)
We introduce a new approach to reduce uncorrelated background signals from fluorescence imaging data, using real-time subtraction of background light. This approach takes advantage of the short fluorescence lifetime of most popular fluorescent activity reporters, and the low duty-cycle of ultrafast lasers. By synchronizing excitation and recording, laser-induced multiphoton fluorescence can be discriminated from background light levels with each laser pulse. We demonstrate the ability of our method to – in real-time – remove image artifacts that in a conventional imaging setup lead to clipping of the signal. In other words, our method enables imaging under conditions that in a conventional setup would yield corrupted data from which no accurate information can be extracted. This is advantageous in experimental setups requiring additional light sources for applications such as optogenetic stimulation.