title: Quantitative optical lock-in detection for quantitative imaging of switchable and non-switchable components.
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 (fSW) and non-switching (fNS) 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.
authors.affiliation: NEST, Scuola Normale Superiore and Istituto Nanoscienze, piazza San Silvestro 12, I-56127, Italy.
authors.forename: Gerardo
authors.initials: G
authors.lastname: Abbandonato
authors.affiliation: NEST, Scuola Normale Superiore and Istituto Nanoscienze, piazza San Silvestro 12, I-56127, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Storti
authors.affiliation: Center for Nanotechnology Innovation @ NEST, Istituto Italiano di Tecnologia, piazza San Silvestro 12, I-56127, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Signore
authors.affiliation: NEST, Scuola Normale Superiore and Istituto Nanoscienze, piazza San Silvestro 12, I-56127, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Beltram
authors.affiliation: NEST, Scuola Normale Superiore and Istituto Nanoscienze, piazza San Silvestro 12, I-56127, Italy. r.bizzarri@sns.it.
authors.forename: Ranieri
authors.initials: R
authors.lastname: Bizzarri
keywords: TRPV1; optical lock-in detection; photochromic FRET; photochromism; reversibly switchable fluorescent proteins