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Quantitative optical lock‐in detection for quantitative imaging of switchable and non‐switchable components
Author(s) -
Abbandonato Gerardo,
Storti Barbara,
Signore Giovanni,
Beltram Fabio,
Bizzarri Ranieri
Publication year - 2016
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.22724
Subject(s) - förster resonance energy transfer , photochromism , fluorescence , autofluorescence , signal (programming language) , materials science , optics , optical switch , common emitter , contrast (vision) , acceptor , optoelectronics , biological system , computer science , physics , nanotechnology , condensed matter physics , biology , programming language
Abstract Reversible photoswitching has been proposed as a way to identify molecules that are present in small numbers over a large, non‐switching, background. This approach, called optical‐lock‐in‐detection (OLID) requires the deterministic control of the fluorescence of a photochromic emitter through optical modulation between a bright (on) and a dark state (off). OLID yields a high‐contrast map where the switching molecules are pinpointed, but the fractional intensities of the emitters are not returned. The present work presents a modified OLID approach (quantitative OLID or qOLID) that yields quantitative information of the switching (f SW ) and non‐switching (f NS ) components. After the validation of the method with a sample dataset and image sequence, we apply qOLID to measurements in cells that transiently express the photochromic protein EYQ1. We show that qOLID is efficient in separating the modulated from the non‐modulated signal, the latter deriving from background/autofluorescence or fluorophores emitting in the same spectral region. Finally, we apply qOLID to Förster (Fluorescence) Resonance Energy Transfer (FRET) imaging. We here demonstrate that qOLID is able to highlight the distribution of FRET intensity in a sample by using a photochromic donor and a non‐photochromic acceptor.