Aymeric Leray

Control of pulse-to-pulse fluctuations in visible supercontinuum

Long-pulse supercontinuum sources are initiated by modulation instability and consequently suffer from stochastic shot-to-shot variations of their spectral power density. In this paper, we provide a measurement of pulse-to-pulse fluctuations over the whole supercontinuum spectrum, and we show that their spectral dependence follows the group index curve of the fiber. Then, we demonstrate a significant reduction of supercontinuum pulse-to-pulse fluctuations in the visible by using a photonic crystal fiber with longitudinally tailored guidance properties. We finally show numerically that this new source would allow a significant improvement of the signal-to-noise ratio in fluorescence microscopy.

827Spatio-Temporal Quantification of FRET in Living Cells by Fast Time-Domain FLIM: A Comparative Study of Non-Fitting Methods

Förster Resonance Energy Transfer (FRET) measured with Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique to investigate spatio-temporal regulation of protein-protein interactions in living cells. When using standard fitting methods to analyze time domain FLIM, the correct estimation of the FRET parameters requires a high number of photons and therefore long acquisition times which are incompatible with the observation of dynamic protein-protein interactions. Recently, non-fitting strategies have been developed for the analysis of FLIM images: the polar plot or “phasor” and the minimal fraction of interacting donor mfD. We propose here a novel non-fitting strategy based on the calculation of moments. We then compare the performance of these three methods when shortening the acquisition time: either by reducing the number of counted photons N or the number of temporal channels Nch, which is particularly adapted for the original fast-FLIM prototype presented in this work that employs the time gated approach. Based on theoretical calculations, Monte Carlo simulations and experimental data, we determine the domain of validity of each method. We thus demonstrate that the polar approach remains accurate for a large range of conditions (low N, Nch or small fractions of interacting donor fD). The validity domain of the moments method is more restricted (not applicable when fD<0.25 or when Nch = 4) but it is more precise than the polar approach. We also demonstrate that the mfD is robust in all conditions and it is the most precise strategy; although it does not strictly provide the fraction of interacting donor. We show using the fast-FLIM prototype (with an acquisition rate up to 1 Hz) that these non-fitting strategies are very powerful for on-line analysis on a standard computer and thus for quantifying automatically the spatio-temporal activation of Rac-GTPase in living cells by FRET.

Quantitative comparison of polar approach versus fitting method in time domain FLIM image analysis.

We calculate here analytically the performance of the polar approach (or phasor) in terms of signal‐to‐noise ratio and F values when performing time‐domain Fluorescence Lifetime Imaging Microscopy (FLIM) to determine the minimal number of photons necessary for FLIM measurements (which is directly related to the F value), and compare them to those obtained from a well‐known fitting strategy using the Least Square Method (LSM). The importance of the fluorescence background on the lifetime measurement precision is also investigated. We demonstrate here that the LSM does not provide the best estimator of the lifetime parameter for fluorophores exhibiting mono‐exponential intensity decays as soon as fluorescence background is superior to 5%. The polar approach enables indeed to determine more precisely the lifetime values for a limited range corresponding to usually encountered fluorescence lifetime values. These theoretical results are corroborated with Monte Carlo simulations. We finally demonstrate experimentally that the polar approach allows distinguishing in living cells two fluorophores undetectable with usual time‐domain LSM fitting software.

Three-dimensional polar representation for multispectral fluorescence lifetime imaging microscopy

Multispectral fluorescence lifetime imaging microscopy is a promising and powerful technique for discriminating multiply labeled samples and for detecting molecular interactions inside thick, heterogeneous, and light‐scattering milieu such as tissue. The fast and correct analysis of the spectral and lifetime images constitutes a major challenge, which requires a high level of expertise. We present here a new approach that considerably simplifies this analysis avoiding complex fitting algorithm strategies and permitting a fast and visual graphical representation of the fluorescence lifetimes. By transforming the experimental data from time domain to frequency domain for each spectral channel, we calculate the multispectral polar representation and demonstrate its interest on multiply fluorescent labeled sample. We further apply it on Förster resonance energy transfer (FRET) experiments and demonstrate that FRET measurements with a high level of precision can be performed. With addition of emission wavelength as third dimension in the polar representation, autofluorescence emitted by the sample is thus clearly identified. Analysis artifacts induced by the sample or by fitting algorithm choice become then totally inexistent.

Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements.

Frequency‐domain fluorescence lifetime imaging microscopy (FLIM) has become a commonly used technique to measure lifetimes in biological systems. However, lifetime measurements are strongly dependent on numerous experimental parameters. Here, we describe a complete calibration and characterization of a FLIM system and suggest parameter optimization for minimizing measurement errors during acquisition. We used standard fluorescent molecules and reference biological samples, exhibiting both single and multiple lifetime components, to calibrate and evaluate our frequency domain FLIM system. We identify several sources of lifetime precision degradation that may occur in FLIM measurements. Following a rigorous calibration of the system and a careful optimization of the acquisition parameters, we demonstrate fluorescence lifetime measurements accuracy and reliability. In addition, we show its potential on living cells by visualizing FRET in CHO cells. The proposed calibration and optimization protocol is suitable for the measurement of multiple lifetime components sample and is applicable to any frequency domain FLIM system. Using this method on our FLIM microscope enabled us to obtain the best fluorescence lifetime precision accessible with such a system. Microsc. Res. Tech., 2009.

The Elongation Complex Components BRD4 and MLLT3/AF9 Are Transcriptional Coactivators of Nuclear Retinoid Receptors

Nuclear all-trans retinoic acid receptors (RARs) initiate early transcriptional events which engage pluripotent cells to differentiate into specific lineages. RAR-controlled transactivation depends mostly on agonist-induced structural transitions in RAR C-terminus (AF-2), thus bridging coactivators or corepressors to chromatin, hence controlling preinitiation complex assembly. However, the contribution of other domains of RAR to its overall transcriptional activity remains poorly defined. A proteomic characterization of nuclear proteins interacting with RAR regions distinct from the AF-2 revealed unsuspected functional properties of the RAR N-terminus. Indeed, mass spectrometry fingerprinting identified the Bromodomain-containing protein 4 (BRD4) and ALL1-fused gene from chromosome 9 (AF9/MLLT3), known to associate with and regulates the activity of Positive Transcription Elongation Factor b (P-TEFb), as novel RAR coactivators. In addition to promoter sequences, RAR binds to genomic, transcribed regions of retinoid-regulated genes, in association with RNA polymerase II and as a function of P-TEFb activity. Knockdown of either AF9 or BRD4 expression affected differentially the neural differentiation of stem cell-like P19 cells. Clusters of retinoid-regulated genes were selectively dependent on BRD4 and/or AF9 expression, which correlated with RAR association to transcribed regions. Thus RAR establishes physical and functional links with components of the elongation complex, enabling the rapid retinoid-induced induction of genes required for neuronal differentiation. Our data thereby extends the previously known RAR interactome from classical transcriptional modulators to components of the elongation machinery, and unravel a functional role of RAR in transcriptional elongation.

Generalization of the polar representation in time domain fluorescence lifetime imaging microscopy for biological applications: practical implementation.

The polar representation or phasor, which provides a fast and visual indication on the number of exponentials present in the intensity decay of the fluorescence lifetime images is increasingly used in time domain fluorescence lifetime imaging microscopy experiments. The calculations of the polar coordinates in time domain fluorescence lifetime imaging microscopy experiments involve several experimental parameters (e.g. instrumental response function, background, angular frequency, number of temporal channels) whose role has not been exhaustively investigated. Here, we study theoretically, computationally and experimentally the influence of each parameter on the polar calculations and suggest parameter optimization for minimizing errors. We identify several sources of mistakes that may occur in the calculations of the polar coordinates and propose adapted corrections to compensate for them. For instance, we demonstrate that the numerical integration method employed for integrals calculations may induce errors when the number of temporal channels is low. We report theoretical generalized expressions to compensate for these deviations and conserve the semicircle integrity, facilitating the comparison between fluorescence lifetime imaging microscopy images acquired with distinct channels number. These theoretical generalized expressions were finally corroborated with both Monte Carlo simulations and experiments.

PRC1 components exhibit different binding kinetics in Polycomb bodies

Polycomb group (PcG) proteins keep the memory of cell identity by maintaining the repression of numerous target genes. They accumulate into nuclear foci called Polycomb bodies, which function in Drosophila cells as silencing compartments where PcG target genes convene. PcG proteins also exert their activities elsewhere in the nucleoplasm. In mammalian cells, the dynamic organisation and function of Polycomb bodies remain to be determined.

Label-free microscopy and stress responses reveal the functional organization of Pseudodiaptomus marinus copepod myofibrils.

Pseudodiaptomus marinus copepods are small crustaceans living in estuarine areas endowed with exceptional swimming and adaptative performances. Since the external cuticle acts as an impermeable barrier for most dyes and molecular tools for labeling copepod proteins with fluorescent tags are not available, imaging cellular organelles in these organisms requires label free microscopy. Complementary nonlinear microscopy techniques have been used to investigate the structure and the response of their myofibrils to abrupt changes of temperature or/and salinity. In contrast with previous observations in vertebrates and invertebrates, the flavin autofluorescence which is a signature of mitochondria activity and the Coherent Anti-Stokes Raman Scattering (CARS) pattern assigned to T-tubules overlapped along myofibrils with the second harmonic generation (SHG) striated pattern generated by myosin tails in sarcomeric A bands. Temperature jumps from 18 to 4 °C or salinity jumps from 30 to 15 psu mostly affected flavin autofluorescence. Severe salinity jumps from 30 to 0 psu dismantled myofibril organization with major changes both in the SHG and CARS patterns. After a double stress (from 18 °C/30 psu to 4° C/0 psu) condensed and distended regions appeared within single myofibrils, with flavin autofluorescence bands located between sarcomeric A bands. These results shed light on the interactions between the different functional compartments which provide fast acting excitation-contraction coupling and adequate power supply in copepods muscles.

Measuring the scattering coefficient of turbid media from two-photon microscopy

In this paper, we propose a new and simple method based on two-photon excitation fluorescence (TPEF) microscopy to measure the scattering coefficient µ(s) of thick turbid media. We show, from Monte Carlo simulations, that µ(s) can be derived from the axial profile of the ratio of the TPEF signals epi-collected by the confocal and the non-descanned ports of a scanning microscope, independently of the anisotropy factor g and of the absorption coefficient µ(a) of the medium. The method is validated experimentally on tissue-mimicking optical phantoms, and is shown to have potential for imaging the scattering coefficient of heterogeneous media.