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Photoswitching kinetics and phase sensitive detection add discriminative dimensions for selective fluorescence imaging

Non-invasive separation-free protocols are attractive to analyze complex mixtures. To increase selectivity, we propose to perform analysis under kinetic control upon exploiting the photochemical reactivity of labeling contrast agents. Our simple protocol is applied in optical fluorescence microscopy, where autofluorescence, light scattering as well as spectral crowding presently bring limitations. We introduce OPIOM (Out-of-Phase Imaging after Optical Modulation), which exploits the rich kinetic signature of a photoswitching fluorescent probe to increase selectively and quantitatively its contrast. Filtering the specific contribution of the probe only requires phase-sensitive detection upon matching the photoswitching dynamics of the probe and the intensity and frequency of a modulated monochromatic light excitation. After in vitro validation, we applied OPIOM for selective imaging in mammalian cells and zebrafish, opening attractive perspectives for multiplexed observations in biological samples.

 

Real-Time Monitoring of Chromophore Isomerization and Deprotonation during the Photoactivation of the Fluorescent Protein Dronpa

 

Dronpa is a GFP-related photochromic fluorescent protein. It is known that the photochromic reaction involves cis/trans isomerization and protonation/deprotonation of the chromophore, but the sequence in time of the two steps and their characteristic timescales are much debated. We followed the entire photoactivation process of Dronpa in real time, by transient absorption spectroscopy from 100 fs to milliseconds.

 

Repair of the (6–4) Photoproduct by DNA Photolyase Requires Two Photons

It takes two (photons) to tango: Single-turnover flash experiments showed that the flavoenzyme (6–4) photolyase uses a successive two-photon mechanism to repair the UV-induced T(6–4)T lesion in DNA (see picture). The intermediate (X) formed by the first photoreaction is likely to be the oxetane-bridged dimer T(ox)T. The enzyme could stabilize the normally short-lived T(ox)T, allowing repair to be completed by the second photoreaction.

Ultraviolet-Induced Fluorescence of Polydopamine-Coated Emulsion Droplets

Polydopamine (PDA), a multifunctional biomaterial with strong adhesion and coating properties, exhibits melanin-like optoelectronic properties but is virtually devoid of intrinsic fluorescence. Herein we disclose the first PDA-based system that can develop fluorescence without chemical manipulation.

Nanosecond motions in proteins revealed by high-resolution relaxometry

A proper description of biological processes at the atomic level require a full characterization of both the structure and the dynamics of biomolecules. Nuclear magnetic resonance (NMR) is a method of choice to access both to the structure and the dynamics of proteins and nucleic acids. One of most powerful NMR probes of biomolecular dynamics is nuclear spin relaxation. Here, we show that nanosecond time scale motions can be revealed with an emerging technique: high-resolution relaxometry.

Mass Transport at Infinite Regular Arrays of Electrodes

Mass transport at infinite regular arrays of microband electrodes was investigated theoretically and experimentally in unstirred solutions. Even in the absence of forced hydrodynamics, natural convection limits the convection-free domain up to which diffusion layers may expand. Hence, several regimes of mass transport may take place according to the electrode size, gap between electrodes, time scale of the experiment, and amplitude of natural convection. They were identified through simulation by establishing zone diagrams that allowed all relative contributions to mass transport to be delineated. Dynamic and steady-state regimes were compared to those achieved at single microband electrodes. These results were validated experimentally by monitoring the chronoamperometric responses of arrays with different ratios of electrode width to gap distance and by mapping steady-state concentration profiles above their surface through scanning electrochemical microscopy.

Biomolecular hydration dynamics: a jump model perspective

The dynamics of water molecules within the hydration shell surrounding a biomolecule can have a crucial influence on its biochemical function. Characterizing their properties and the extent to which they differ from those of bulk water have thus been long-standing questions. Following a tutorial approach, we review the recent advances in this field and the different approaches which have probed the dynamical perturbation experienced by water in the vicinity of proteins or DNA. We discuss the molecular factors causing this perturbation, and describe how they change with temperature. We finally present more biologically relevant cases beyond the dilute aqueous situation. A special focus is on the jump model for water reorientation and hydrogen bond rearrangement.

Membrane tubulation by cell penetrating peptides

Cell penetrating peptides induce membrane invaginations in cellular membranes. They induce negative membrane curvature by a metabolic energy independent pathway. This pathway also called 'Physical endocytosis' could be a new mechanism of cell penetrating peptide uptake

Photochemical properties of Spinach and its use in selective imaging

We propose a dynamic model that accounts for the photochemical behavior of the Spinach system, a recently described fluorescent probe for RNA imaging. We exploit the dynamic fluorogen exchange and the unprecedented photoconversion properties in a non-covalent fluorescence turn-on system to significantly improve signal-to-background ratio during long-term microscopy imaging.