Quantitative Analysis of Protein-Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy.

Current methods for determining equilibrium constants often operate in three-dimensional environments, which may not accurately reflect interactions with membrane-bound proteins. With our technique, based on single-molecule localization microscopy (SMLM), we directly determine protein-protein association () and dissociation () constants in cellular environments by quantifying associated and isolated molecules and their interaction area. We introduce Kernel Surface Density (ks-density,) a novel method for determining the accessible area for interacting molecules, eliminating the need for user-defined parameters.

Determination of association equilibrium constant from single molecule fluorescence localization microscopy.

Single molecule fluorescence localization microscopy provides molecular localization with a precision in the tens of nanometer range in the plane perpendicular to the light propagation. This opens the possibility to count molecules and correlate their locations, starting from a map of the actual positions in a single molecule super resolution image. Considering molecular pair correlation as an indication of interaction, and a way to discern them from free molecules, we describe a method to calculate thermodynamic equilibrium constants.

Analysis of sparse molecular distributions in fibrous arrangements based on the distance to the first neighbor in single molecule localization microscopy.

Single Molecule Localization Microscopy (SMLM) currently attains a lateral resolution of around 10 nm approaching molecular size. Together with increasingly specific fluorescent labeling, it opens the possibility to quantitatively analyze molecular organization. When the labeling density is high enough, SMLM provides clear images of the molecular organization.

A fluorescence nanoscopy marker for corticotropin-releasing hormone type 1 receptor: computer design, synthesis, signaling effects, super-resolved fluorescence imaging, and in situ affinity constant in cells.

Class B G protein-coupled receptors (GPCRs) are involved in a variety of human pathophysiological states. These groups of membrane receptors are less studied than class A GPCRs due to the lack of structural information, delayed small molecule drug discovery, and scarce fluorescence detection tools available. The class B corticotropin-releasing hormone type 1 receptor (CRHR1) is a key player in the stress response whose dysregulation is critically involved in stress-related disorders: psychiatric conditions (i.

Temperature dependent spectroscopic and excited state dynamics of 3-hydroxychromones with electron donor and acceptor substituents.

We have studied the photophysical and photochemical behavior of three compounds derived from 3-hydroxychromone (3-HC), capable of undergoing excited state proton transfer (ESIPT). The compounds have two substituents, located in positions 2 and 7, one on each ring of the 3-HC heterocycle. The substituent pattern shows different electron donating and acceptor features.

Two-Photon Excitation of a Plasmonic Nanoswitch Monitored by Single-Molecule Fluorescence Microscopy.

Visible-light excitation of the surface plasmon band of silver nanoplates can effectively localize and concentrate the incident electromagnetic field enhancing the photochemical performance of organic molecules. Herein, the first single-molecule study of the plasmon-assisted isomerization of a photochrome-fluorophore dyad, designed to switch between a nonfluorescent and a fluorescent state in response to the photochromic transformation, is reported. The photochemistry of the switchable assembly, consisting of a photochromic benzooxazine chemically conjugated to a coumarin moiety, is examined in real time with total internal reflection fluorescence microscopy in the presence of silver nanoplates excited with a 633 nm laser.

Thermoplasmonic ssDNA Dynamic Release from Gold Nanoparticles Examined with Advanced Fluorescence Microscopy.

Plasmon excitation of spherical gold nanoparticles carrying a fluorescent labeled 30 bp dsDNA cargo, with one chain covalently attached through two S-Au bonds to the surface, results in release of the complementary strand as ssDNA that can be examined in situ using high-resolution fluorescence microscopy. The release is dependent on the total energy delivered, but not the rate of delivery, an important property for plasmonic applications in medicine, sensors, and plasmon-induced PCR.

Chiral power change upon photoisomerization in twisted nematic liquid crystals.

In this work, we use the photoisomerization of azobenzenes, a phenanthrospirooxazine, and a fulgide in a twisted nematic liquid crystalline phase to change the chiral twisting power of the system. The changes are probed by the rotatory power of linearly polarized light. Time resolved and steady state experiments are carried out.

Influence of the glass transition on rotational dynamics of dyes in thin polymer films: single-molecule and ensemble experiments.

We performed polarized fluorescence emission studies of Nile Red (NR) in poly(methyl methacrylate) (PMMA), poly(ethyl methacrylate) (PEMA), and poly(butyl methacrylate) (PBMA) at the single molecule (SM) and at the ensemble level to study the in cage movements of the ground-state molecule in polymer films of nanometric thickness at room temperature. Experiments were performed with wide field irradiation. At the ensemble level, the linearly polarized irradiation was used to induce a photoselection by bleaching, which is compensated by rotational diffusion.

Mapping the fluorescence performance of a photochromic-fluorescent system coupled with gold nanoparticles at the single-molecule-single-particle level.

Single-molecule (SM) fluorescence microscopy was used to investigate the photochromic fluorescent system spiropyran-merocyanine (SP ↔ MC) interacting with gold nanoparticles (AuNPs). We observe a significant increase in the brightness of the emissive MC form, in the duration of its ON time, and in the total number of emitted photons. The spatial distribution of SMs with improved photophysical performance was obtained with 40 nm precision relative to the nearest AuNP.

Fluorescent polymer coatings with tuneable sensitive range for remote temperature sensing.

Polymer films of poly(vinyl alcohol) containing the fluorescent dyes 4-aminophthalimide (AP) or 6-propionyl-2-dimethylamino-naphthalene (Prodan) are used as temperature-sensitive fluorescent coatings for remote temperature sensing. Temperature can be obtained by a two-wavelength ratiometric-based emission intensity measurement. The coatings are sensitive in a 100K temperature range that can be tuned by polymer-solute interactions.

The mechanism of the photochromic transformation of spirorhodamines.

We investigate the equilibrium, kinetics, and mechanism of the photochromic transformation of a series of amido spirorhodamine compounds-differing in the nature of the substituents of the amido group and in the rhodamine chromophore-in ethanol at room temperature in the presence of trifluoroacetic acid. A proton participates in the equilibrium between the spiro form and the open rhodamine form. The relaxation times in the dark or under continuous irradiation show a linear dependence on the proton concentration.

Detection of low quantum yield fluorophores and improved imaging times using metallic nanoparticles.

The behavior of a fluorophore near a gold nanoparticle is rationalized by a theoretical description of the parameters that modify the fluorescence emission: nanoparticle-fluorophore distance, fluorescence quantum yield (φ(0)), and fluorophore absorption and emission spectra, to find optimum conditions for designing fluorophore-nanoparticle probes. The theoretical maximum gain in brightness of the nanoparticle-fluorophore system with respect to the isolated molecule increases almost inversely proportional to φ(0). The brightness enhancement in imaging experiments in vitro was assessed by using Au-SiO(2) core-shell nanoparticles deposited on glass.

Photoisomerization of alfa calcidol by a sensitized quantum chain reaction.

The production of vitamin D3 is a pharmaceutically relevant process, producing high added-value products. Precursors are extracts from vegetal origin but bearing mainly an E geometry in the 5,6 double bond. The synthesis of vitamin D3 (5-E-α-calcidol) with the correct Z stereochemistry in the 5,6 double bond from the E isomer using anthracene and triethylamine (TEA) as the sensitizer system was studied from the kinetic and mechanistic point of view.

Proton transfer from 2-naphthol to aliphatic amines in supercritical CO2.

The proton transfer from 2-naphthol to aliphatic amines was studied in supercritical CO(2) (scCO(2)) and in cyclohexane as reference solvent, by absorption and fluorescence spectroscopy and by time-resolved emission. Irradiation of 2-naphthol in scCO(2) in the presence of ethyldiisopropylamine shows dynamic fluorescence quenching of the acidic form of 2-naphthol and emission from the basic form. Fluorescence excitation spectra show that the emission of the basic form is originated upon excitation of the acidic form.

Critical effects on attractive solutes in binary liquid mixtures close to their consolute point: a new experimental strategy.

The effect of near-criticality upon the properties of dilute solutions of attractive solutes has been previously studied only using pure solvents close to the vapor-liquid critical point. The experimental difficulties that plague this thermodynamic region have somewhat obscured the interpretations of the results. Consequently, the coupling of long-range critical fluctuations with short-range intermolecular interactions is still a matter of debate.

Freezing single molecule dynamics on interfaces and in polymers.

Heterogeneous line broadening and spectral diffusion of the fluorescence emission spectra of perylene diimide molecules have been investigated by means of time dependent single molecule spectroscopy. The influence of temperature and environment has been studied and reveals strong correlation to spectral diffusion processes. We followed the freezing of the molecular mobility of quasi free molecules on the surface upon temperature lowering and by embedding into a poly(methyl methacrylate) (PMMA) polymer.

Photolysis of an asymmetrically substituted diazene in solution and in the crystalline state.

In this work we study the product distribution in the steady state photolysis of a diazene, (1-biphenyl-4-yl-1-methyl-ethyl)-tert-butyl diazene, and a ketone, 2,4-bis(biphenyl-4-yl)-2,4-dimethyl-pentan-3-one, in the solid state and in solution. The two compounds yield 1-biphenyl-4-yl-1-methyl-ethyl (BME ) radicals upon photolysis. The ketone yields two units of this radical, whereas the diazene yields one BME and one tert-butyl radical.

Single photon fluorescent microlithography for live-cell imaging.

Using fluorescent dyes to trigger the polymerization of a commercial polyurethane resin allows a rapid fabrication of micrometer and submicrometer sized fluorescent structures by one-photon absorption. Here, we show that standard He-Ne lasers emitting at 632.8 nm can be used to start the photopolymerization and that very low laser power is required.

Photophysics and photochemistry of an asymmetrically substituted diazene: a suitable cage effect probe.

The photophysics and photochemistry of (1-biphenyl-4-yl-1-methyl-ethyl)-tert-butyl diazene were thoroughly studied by laser flash photolysis from the picosecond to the microsecond time domain. The compound has favorable features as a radical photoinitiator and as a probe for cage effect studies in liquids, supercritical fluids, and compressed gases. The biphenyl moiety acts as an antenna efficiently transferring electronic energy to the dissociative (1)n,pi* state centered on the azo moiety.

Cage effect in supercritical fluids and compressed gases in the photolysis of an asymmetrically substituted diazene.

We studied the photolysis of (1-biphenyl-4-yl-1-methyl-ethyl)-tert-butyl diazene in supercritical CO(2) and Xe, as well as in compressed Kr. The compound has good solubility in the mentioned fluids, allowing the photolysis measurements to be performed in CO(2) at 1.4 K above T(c) and at pressures as low as 70 bar.

10000 times volume reduction for fluorescence correlation spectroscopy using nano-antennas.

We present an experimental and theoretical study of a new scheme for Near-Field Fluorescence Correlation Spectroscopy that, using the field enhancement by optical nanoantennas, allows the reduction of the observation volume 4 orders of magnitude below the diffraction limit. This reduction can be used in two different ways: to increase the sample concentration and to improve the spatial resolution of the dynamics under study. Our experimental results using individual gold nanoparticles and a 150 microM Rose Bengal solution in glycerol confirm the volume reduction.

Luminescent Eu(III) hybrid materials for sensor applications.

In this work we use the sol-gel technique to develop different luminescent Eu(III) porous materials from a bis(trialkoxysilyl) organic precursor synthesized from the amide of the DPA (2,6-pyridinedicarboxylic acid) with APTES (aminopropyltriethoxysilane) in the presence or absence of the non-ionic surfactant F-127. The emission spectrum of the luminescent Eu(III) complex obtained was used to sense and compare the environment of the lanthanide in the amorphous matrices as well as the accessibility of the material by means of the quenching of its luminescence by Cu(II). Solid devices were built to test and compare their performances as potential sensors of Cu(II) in terms of the values of the Stern-Volmer constants in the quenching experiments.

Relaxations in poly(vinyl alcohol) and in poly(vinyl acetate) detected by fluorescence emission of 4-aminophthalimide and prodan.

Steady-state and time-resolved emission spectroscopy (TRES) of the medium-sensitive probes 4-aminophthalimide (4-AP) and 6-propionyl-2-(dimethylamino)naphthalene (Prodan) were performed at 77 and 298 K in vacuum-sealed thin films of poly(vinyl alcohol) (PVA) and poly(vinyl acetate) (PVAc). The two probes show similar red-edge effect in steady state emission and a red shift with time in TRES in PVA. In PVAc the red shifts are much smaller and the spectral shift for 4-AP is slower.