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.

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.

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.

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.

Methodology and results of a survey of adverse reactons to a drug in private practice.

A survey of tolerance of a drug, determined in private practice under "naturalistic" conditions by 591 physicians, and involving 22277 patients is presented. The procedure used in private practice to gather systematic information about reactions to the drug involved a system of data sheets with detachable cards for optical reading and computer analysis. The survey was conducted under the responsiblity at regional level of a team of scientific coordinators-hospital pharmacologists and poison control centres. Possible side effects were noticed in 13,82% of patients, a figure similar to known nocebo reactions. Tolerance was significantly related to sex, age, weight, geographical area, duration of treatment, association with other durgs and therapeutic result. When related to individual physicians, the overall number of side effects and the frequency of three of them in particular did not follow a binomial distribution; the rate of adverse reactions was significantly related to the number of years of practice of the physicians.