A Novel Microfluidic-Based Fluorescence Detection Method Reveals Heavy Atom Effects on Photophysics of Fluorophores With High Triplet Quantum Yield: A Numerical Simulation Study.

The present study introduces the idea of a novel fluorescence-based imaging technique combined with a microfluidic platform that enables a precise control of dark transient state populations of fluorescent probes flowing over a uniform, top flat supergaussian excitation field with a constant flow rate. To demonstrate the imaging capability of the proposed detection method, numerical simulations have been performed by considering laser, microscope and flow parameters of experimental setup together with photophysical model and electronic transition rates of fluorescent dyes. As an output data to be assessed, fluorescence image data is simulated numerically for bromine-free carboxyfluorescein and its brominated derivatives having different numbers of bromine atoms.

Fluorescence Bar-Coding and Flowmetry Based on Dark State Transitions in Fluorescence Emitters.

Reversible dark state transitions in fluorophores represent a limiting factor in fluorescence-based ultrasensitive spectroscopy, are a necessary basis for fluorescence-based super-resolution imaging, but may also offer additional, largely orthogonal fluorescence-based readout parameters. In this work, we analyzed the blinking kinetics of Cyanine5 (Cy5) as a bar-coding feature distinguishing Cy5 from rhodamine fluorophores having largely overlapping emission spectra. First, fluorescence correlation spectroscopy (FCS) solution measurements on mixtures of free fluorophores and fluorophore-labeled small unilamellar vesicles (SUVs) showed that Cy5 could be readily distinguished from the rhodamines by its reversible, largely excitation-driven isomerization.

Photo-physical characterization of high triplet yield brominated fluoresceins by transient state (TRAST) spectroscopy.

Photo-induced dark transient states of fluorophores can pose a problem in fluorescence spectroscopy. However, their typically long lifetimes also make them highly environment sensitive, suggesting fluorophores with prominent dark-state formation yields to be used as microenvironmental sensors in bio-molecular spectroscopy and imaging. In this work, we analyzed the singlet-triplet transitions of fluorescein and three synthesized carboxy-fluorescein derivatives, with one, two or four bromines linked to the anthracence backbone.

Trans-cis isomerization kinetics of cyanine dyes reports on the folding states of exogeneous RNA G-quadruplexes in live cells.

Guanine (G)-rich nucleic acids are prone to assemble into four-stranded structures, so-called G-quadruplexes. Abnormal GGGGCC repeat elongations, and in particular their folding states, are associated with amyotrophic lateral sclerosis and frontotemporal dementia. Due to methodological constraints however, most studies of G quadruplex structures are restricted to in vitro conditions.

Imaging Fluorescence Blinking of a Mitochondrial Localization Probe: Cellular Localization Probes Turned into Multifunctional Sensors.

Mitochondrial membranes and their microenvironments directly influence and reflect cellular metabolic states but are difficult to probe on site in live cells. Here, we demonstrate a strategy, showing how the widely used mitochondrial membrane localization fluorophore 10-nonyl acridine orange (NAO) can be transformed into a multifunctional probe of membrane microenvironments by monitoring its blinking kinetics. By transient state (TRAST) studies of NAO in small unilamellar vesicles (SUVs), together with computational simulations, we found that NAO exhibits prominent reversible singlet-triplet state transitions and can act as a light-induced Lewis acid forming a red-emissive doublet radical.