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1.
Chemphyschem ; 23(23): e202200295, 2022 12 05.
Article in English | MEDLINE | ID: mdl-35976176

ABSTRACT

We introduce HIGHLIGHT as a simple and general strategy to selectively image a reversibly photoactivatable fluorescent label associated with a given kinetics. The label is submitted to sine-wave illumination of large amplitude, which generates oscillations of its concentration and fluorescence at higher harmonic frequencies. For singularizing a label, HIGHLIGHT uses specific frequencies and mean light intensities associated with resonances of the amplitudes of concentration and fluorescence oscillations at harmonic frequencies. Several non-redundant resonant observables are simultaneously retrieved from a single experiment with phase-sensitive detection. HIGHLIGHT is used for selective imaging of four spectrally similar fluorescent proteins that had not been discriminated so far. Moreover, labels out of targeted locations can be discarded in an inhomogeneous spatial profile of illumination. HIGHLIGHT opens roads for simplified optical setups at reduced cost and easier maintenance.


Subject(s)
Light , Fluorescence , Photochemical Processes
2.
Anal Chim Acta ; 1225: 340180, 2022 Sep 08.
Article in English | MEDLINE | ID: mdl-36038229

ABSTRACT

Titration without separation, e.g. quantification of a target species in living cells, is a challenge of analytical chemistry. We perform the selective detection of a target using the kinetics involved in a photochemical process and develop a correlation method that we illustrate by the titration of a fluorescent photoswitcher and the target of a photoswitching sensor. Correlating an input time series and a well-chosen weighting function associated with a variable characteristic time yields a spectrum of characteristic times. The upper integration limit of the correlation output can be chosen to match the argument of an extremum of the spectrum with a characteristic time of the input time series in order to quantify the target. A similar procedure is followed to optimize the signal-to-noise ratio. Selectivity and signal-to-noise ratio associated with 15 weighting functions are theoretically predicted. The results are applied to the titration of the reversibly photoswitchable fluorescent protein Dronpa-2 and the titration of calcium using a reversibly photoswitchable fluorescent sensor. The performance of the correlation method is favorably compared to the one of other dynamic contrast protocols.


Subject(s)
Microscopy, Fluorescence , Kinetics , Microscopy, Fluorescence/methods , Signal-To-Noise Ratio
3.
Nat Commun ; 13(1): 1482, 2022 03 18.
Article in English | MEDLINE | ID: mdl-35304491

ABSTRACT

Due to its sensitivity and versatility, fluorescence is widely used to detect specifically labeled biomolecules. However, fluorescence is currently limited by label discrimination, which suffers from the broad full width of the absorption/emission bands and the narrow lifetime distribution of the bright fluorophores. We overcome this limitation by introducing extra kinetic dimensions through illuminations of reversibly photoswitchable fluorophores (RSFs) at different light intensities. In this expanded space, each RSF is characterized by a chromatic aberration-free kinetic fingerprint of photochemical reactivity, which can be recovered with limited hardware, excellent photon budget, and minimal data processing. This fingerprint was used to identify and discriminate up to 20 among 22 spectrally similar reversibly photoswitchable fluorescent proteins (RSFPs) in less than 1s. This strategy opens promising perspectives for expanding the multiplexing capabilities of fluorescence imaging.


Subject(s)
Fluorescent Dyes , Optical Imaging , Kinetics , Light , Microscopy, Fluorescence/methods
4.
Math Biosci Eng ; 18(5): 5525-5551, 2021 06 21.
Article in English | MEDLINE | ID: mdl-34517499

ABSTRACT

The competition between cancer cells and immune system cells in inhomogeneous conditions is described at cell scale within the framework of the thermostatted kinetic theory. Cell learning is reproduced by increased cell activity during favorable interactions. The cell activity fluctuations are controlled by a thermostat. The direction of cell velocity is changed according to stochastic rules mimicking a dense fluid. We develop a kinetic Monte Carlo algorithm inspired from the direct simulation Monte Carlo (DSMC) method initially used for dilute gases. The simulations generate stochastic trajectories sampling the kinetic equations for the distributions of the different cell types. The evolution of an initially localized tumor is analyzed. Qualitatively different behaviors are observed as the field regulating activity fluctuations decreases. For high field values, i.e. efficient thermalization, cancer is controlled. For small field values, cancer rapidly and monotonously escapes from immunosurveillance. For the critical field value separating these two domains, the 3E's of immunotherapy are reproduced, with an apparent initial elimination of cancer, a long quasi-equilibrium period followed by large fluctuations, and the final escape of cancer, even for a favored production of immune system cells. For field values slightly smaller than the critical value, more regular oscillations of the number of immune system cells are spontaneously observed in agreement with clinical observations. The antagonistic effects that the stimulation of the immune system may have on oncogenesis are reproduced in the model by activity-weighted rate constants for the autocatalytic productions of immune system cells and cancer cells. Local favorable conditions for the launching of the oscillations are met in the fluctuating inhomogeneous system, able to generate a small cluster of immune system cells with larger activities than those of the surrounding cancer cells.


Subject(s)
Algorithms , Neoplasms , Computer Simulation , Humans , Kinetics , Monte Carlo Method
6.
Methods Mol Biol ; 2350: 191-227, 2021.
Article in English | MEDLINE | ID: mdl-34331287

ABSTRACT

Fluorescence imaging has become a powerful tool for observations in biology. Yet it has also encountered limitations to overcome optical interferences of ambient light, autofluorescence, and spectrally interfering fluorophores. In this account, we first examine the current approaches which address these limitations. Then we more specifically report on Out-of-Phase Imaging after Optical Modulation (OPIOM), which has proved attractive for highly selective multiplexed fluorescence imaging even under adverse optical conditions. After exposing the OPIOM principle, we detail the protocols for successful OPIOM implementation.


Subject(s)
Fluorescent Antibody Technique/methods , Microscopy, Fluorescence, Multiphoton/methods , Optical Imaging/methods , Algorithms , Animals , Fluorescent Dyes , Image Processing, Computer-Assisted , Light , Models, Theoretical , Staining and Labeling
7.
Phys Chem Chem Phys ; 22(36): 20801-20814, 2020 Sep 23.
Article in English | MEDLINE | ID: mdl-32909563

ABSTRACT

A reduced chemical scheme involving a small number of variables is often sufficient to account for the deterministic evolution of the concentration of the main species contributing to a reaction. However, its predictions are questionable in small systems used, for example in fluorescence correlation spectroscopy (FCS) or in explosive systems involving strong nonlinearities such as autocatalytic steps. We make precise dynamical criteria defining the validity domain of the quasi-steady-state approximation and the elimination of a fast concentration in deterministic dynamics. Designing two different three-variable models converging toward the same two-variable model, we show that the variances and covariance of the fluctuations of the slow variables are not correctly predicted using the two-variable model, even in the limit of a large system size. The more striking weaknesses of the reduced scheme are figured out in mesoscaled systems containing a small number of molecules. The results of two stochastic approaches are compared and the shortcomings of the Langevin equations with respect to the master equation are pointed out. We conclude that the description of the fluctuations and their coupling with nonlinearities of deterministic dynamics escape reduced chemical schemes.

8.
Chem Sci ; 11(11): 2882-2887, 2020 Feb 25.
Article in English | MEDLINE | ID: mdl-34122788

ABSTRACT

Interrogating living cells requires sensitive imaging of a large number of components in real time. The state-of-the-art of multiplexed imaging is usually limited to a few components. This review reports on the promise and the challenges of dynamic contrast to overcome this limitation.

9.
Phys Rev E ; 99(2-1): 022205, 2019 Feb.
Article in English | MEDLINE | ID: mdl-30934359

ABSTRACT

The sensitivity to perturbations of the Fisher and Kolmogorov, Petrovskii, Piskunov front is used to find a quantity revealing perturbations of diffusion in a concentrated solution of two chemical species with different diffusivities. The deterministic dynamics includes cross-diffusion terms due to the deviation from the dilution limit. The behaviors of the front speed, the shift between the concentration profiles of the two species, and the width of the reactive zone are investigated, both analytically and numerically. The shift between the two profiles turns out to be a well-adapted criterion presenting noticeable variations with the deviation from the dilution limit in a wide range of parameter values.

10.
Phys Chem Chem Phys ; 20(37): 23998-24010, 2018 Oct 07.
Article in English | MEDLINE | ID: mdl-30215648

ABSTRACT

In order to design a dynamic titration method, we propose a theoretical model harnessing the kinetic properties of the complexation of the titrated species with a titrating photoswitchable reagent. Forced oscillations of illumination are imposed and concentration oscillations of the targeted species are deduced from the equations of chemical kinetics. We determine analytical expressions of the resonance conditions on the control parameters, angular frequency, mean light intensity, and total concentration of the photoswitchable reagent, which optimize the out-of-phase amplitude of concentration oscillations. A user-friendly protocol of dynamic titration is proposed.

11.
J Chem Phys ; 145(15): 154108, 2016 Oct 21.
Article in English | MEDLINE | ID: mdl-27782466

ABSTRACT

In order to mimic the interactions between cancer and the immune system at cell scale, we propose a minimal model of cell interactions that is similar to a chemical mechanism including autocatalytic steps. The cells are supposed to bear a quantity called activity that may increase during the interactions. The fluctuations of cell activity are controlled by a so-called thermostat. We develop a kinetic Monte Carlo algorithm to simulate the cell interactions and thermalization of cell activity. The model is able to reproduce the well-known behavior of tumors treated by immunotherapy: the first apparent elimination of the tumor by the immune system is followed by a long equilibrium period and the final escape of cancer from immunosurveillance.


Subject(s)
Computer Simulation , Immune System , Monte Carlo Method , Neoplasms , Algorithms , Cell Communication , Humans , Kinetics
12.
Phys Rev E ; 93: 042402, 2016 04.
Article in English | MEDLINE | ID: mdl-27176324

ABSTRACT

The adaptation of prevertebra size to embryo size is investigated in the framework of a reaction-diffusion model involving a Turing pattern. The reaction scheme and Fick's first law of diffusion are modified in order to take into account the departure from dilute conditions induced by confinement in smaller embryos. In agreement with the experimental observations of scaling in somitogenesis, our model predicts the formation of smaller prevertebrae or somites in smaller embryos. These results suggest that models based on Turing patterns cannot be automatically disregarded by invoking the question of maintaining proportions in embryonic development. Our approach highlights the nontrivial role that the solvent can play in biology.


Subject(s)
Models, Biological , Somites/embryology , Diffusion
13.
Chemphyschem ; 17(10): 1396-413, 2016 05 18.
Article in English | MEDLINE | ID: mdl-26833808

ABSTRACT

Living cells are chemical mixtures of exceptional interest and significance, whose investigation requires the development of powerful analytical tools fulfilling the demanding constraints resulting from their singular features. In particular, multiplexed observation of a large number of molecular targets with high spatiotemporal resolution appears highly desirable. One attractive road to address this analytical challenge relies on engaging the targets in reactions and exploiting the rich kinetic signature of the resulting reactive module, which originates from its topology and its rate constants. This review explores the various facets of this promising strategy. We first emphasize the singularity of the content of a living cell as a chemical mixture and suggest that its multiplexed observation is significant and timely. Then, we show that exploiting the kinetics of analytical processes is relevant to selectively detect a given analyte: upon perturbing the system, the kinetic window associated to response read-out has to be matched with that of the targeted reactive module. Eventually, we introduce the state-of-the-art of cell imaging exploiting protocols based on reaction kinetics and draw some promising perspectives.


Subject(s)
Molecular Imaging , Kinetics , Pressure , Spectrum Analysis/methods , Temperature
14.
Phys Chem Chem Phys ; 12(40): 13224-31, 2010 Oct 28.
Article in English | MEDLINE | ID: mdl-20820571

ABSTRACT

A master equation approach is used to study the influence of internal fluctuations on the dynamics of three excitable thermochemical systems exhibiting continuous as well as discrete changes of temperature. The systems differ by the types of excitability. The dependences of the relative deviations from mean values of the interspike intervals and escape times from the stable stationary state on the size of the systems calculated from simulations of stochastic trajectories exhibit minima, which testify to the appearance of resonance phenomena. An explanation for the appearance of the stochastic resonances is presented.

15.
Lab Chip ; 8(7): 1205-9, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18584099

ABSTRACT

An integrated approach relying on a microsystem is introduced to easily extract, from a single experiment and with a global robust bi-exponential fit, an extensive set of thermodynamic, kinetic, and diffusion parameters governing associations in solution.


Subject(s)
Fourier Analysis , Lab-On-A-Chip Devices , Oligonucleotides/chemistry , Base Sequence , Diffusion , Equipment Design , Kinetics , Molecular Sequence Data , Oligonucleotides/genetics , Thermodynamics
16.
J Am Chem Soc ; 127(44): 15491-505, 2005 Nov 09.
Article in English | MEDLINE | ID: mdl-16262413

ABSTRACT

The present account is concerned with the measurement of local reactant concentrations by observing specific fluorescent probes in fluorescence correlation spectroscopy (FCS). The Theoretical Analysis section revisits the photophysical, thermodynamic, and kinetic information that is contained in the corresponding FCS correlation curves. In particular, we examine the conditions under which FCS is revealed as a superior tool to measure concentrations of reactive species. Careful molecular engineering of the specific fluorescent probes that simultaneously integrates photophysical, thermodynamic, and kinetic constraints will be required to benefit most from FCS. We illustrate the FCS titration approach with a series of fluorescent probes that we tailored to measure pH at around 4-6 by FCS after two-photon excitation. We show that an optimal design allows one to access pH without any preliminary calibrations such as the determination of the protonation constant or the photophysical properties of the fluorescent probe.


Subject(s)
Fluorescent Dyes , Hydrogen-Ion Concentration , Spectrometry, Fluorescence , Titrimetry
17.
J Phys Chem B ; 109(3): 1318-28, 2005 Jan 27.
Article in English | MEDLINE | ID: mdl-16851097

ABSTRACT

This paper reports on a novel procedure to tune the effective diffusion coefficient of a field-sensitive reactant in the presence of a periodic external field. We investigate the motion of two negatively charged azo dyes interacting with alpha-cyclodextrin (alpha-CD) upon action of a periodic square wave electrical field. We show that the dyes exhibit an effective diffusion coefficient D(eff) that depends on the rate constants for dye complexation within alpha-CD, the period and the amplitude of the field. UV-vis absorption, gradient field (1)H NMR, and fluorescence correlation spectroscopy (FCS) after two photon excitation are used to evidence that D(eff) may be increased far beyond its intrinsic value when specific relations interpreted as a stochastic resonance are fulfilled. The present results may find useful applications in chemical kinetics as well as for molecular sorting.

18.
Proc Natl Acad Sci U S A ; 101(22): 8276-80, 2004 Jun 01.
Article in English | MEDLINE | ID: mdl-15155904

ABSTRACT

To sort a targeted species from a mixture, we introduce a procedure that relies on the enhancement of its effective diffusion coefficient. We use the formation of a host-guest complex between alpha-cyclodextrin and a dye to evidence the dye dispersion when the medium is submitted to an oscillating field. In particular, we demonstrate that the effective diffusion coefficient of the dye may be increased far beyond its intrinsic value by tuning the driving field frequency in the stochastic resonance regime. We use this effect to selectively sort from a mixture a dye that is addressed by its rate constants for association with alpha-cyclodextrin.

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