Francois Lacombe

FLIM FRET Technology for Drug Discovery: Automated Multiwell-Plate High-Content Analysis, Multiplexed Readouts and Application in Situ

A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated.

A fluorescence lifetime imaging scanning confocal endomicroscope

We describe a fluorescence lifetime imaging endomicroscope employing a fibre bundle probe and time correlated single photon counting. Preliminary images of stained pollen grains, eGFP-labelled cells exhibiting Förster resonant energy transfer and tissue autofluorescence are presented.

Hot Very Small dust Grains in NGC 1068 seen in jet induced structures thanks to VLT/NACO adaptive optics

We present K, L and M diffraction-limited images of NGC 1068 obtained with NAOS+CONICA at VLT/YEPUN over a 3.5 field around the central engine. Hot dust (Tcol = 550-650 K) is found in three different regions : (a) in the true nucleus, seen as a slightly NS elongated, core of extremely hot dust, resolved in K and L with respective diameters of ~5 pc and 8.5 pc ; (b) along the NS direction, as a spiral arm and a southern tongue ; (c) as a set of parallel elongatednodules (wave-like) bracketting the jet. Several structures observed on radio maps, mid-IR or HST UV-visible maps are seen, so that a precise registration can be done from UV to 6 cm. These results dosupport the current interpretion that source (a) corresponds to emission from dust near sublimation temperature delimiting the walls of the cavity in the central obscuring torus. Structure (b) is thought tobe a mixture of hot dust and active star forming regions along a micro spiral structure that could trace the tidal mechanism bringing matter to the central engine. Structure c)which was not known, exhibits too high a temperature for classical grains ; it is most probably the signature of transiently heated very small dust grains (VSG) : nano-diamonds, whichare resistant and can form in strong UV field or in shocks, are very attractive candidates. The waves can be condensations triggered by jet induced shocks, as predicted by recent models. First estimates, based on a simple VSG model and on a detailed radiative transfer model, do agree with those interpretations,both qualitatively and quantitatively.

Real Time Autonomous Video Image Registration for Endomicroscopy: Fighting The Compromises

Confocal endomicroscopy provides tools for in vivo imaging of human cell architecture endoscopically. These technologies are a tough challenge since multiple trade-offs have to be overcome: resolution versus field of view, dynamic versus stability, contrast versus low laser power or low contrast agent doses. Many difficult clinical applications, such as lung, bile duct, urethral imaging and NOTES applications, need to optimize miniaturization, resolution, frame rate and contrast agent dose simultaneously. We propose one solution based on real-time video image processing to efficiently address these trade-offs. Dynamic imaging provides a flow of images that we process in real time. Images are aligned using efficientalgorithms specifically adapted to confocal devices. From the displacement that we find across the images, instantaneous velocities are computed and used to compensate for motion distortions. All images are stitched together onto the same reference space and displayed in real-time to reconstruct an image of the entire surface explored during the clinical procedure. This representation brings both stability and an increased field of view. Moreover, because a given area can be imaged by several frames, the contrast can be improved using temporal adaptive averaging. Such processing enhances the visualization of the video sequence, overcoming most classical trade-offs. The stability and increased field of view help the clinician better focus his attention on his practice which improves the patient benefit. Our tools are currently evaluated in a multicenter clinical trial to assess the improvement of the clinical practice.

Coherent femtosecond pulse shaping for the optimization of a non-linear micro-endoscope

A flexible multicore fiber bundle is fed by temporally and spectrally shaped femtosecond pulses allowing for the pre-compensation of both chromatic dispersion and non-linear effects encountered in the bundle. We demonstrate that the pulse duration at the fiber bundle output can be significantly reduced in comparison with linear pre-compensation only. The scheme for femtosecond pulse fiber delivery is applied to the optimization of two-photon fluorescence (TPF) imaging. Experiments and calculations show a five-fold improvement of the TPF signal produced at the end of the fiber bundle in comparison with linear pre-compensation. This is applied to the recording, in real time (12 image/s), of TPF laser-scanning images of human colon cells stained with a fluorescent marker. Further optimizations are discussed.

VLT/NACO infrared adaptive optics images of small scale structures in OMC1 ⋆

Near-infrared observations of line emission from excited H 2 and in the continuum are reported in the direction of the Orion molecular cloud OMC1 , using the European Southern Observatory Very Large Telescope UT4 , equipped with the NAOS adaptive optics system on the CONICA infrared array camera. Spatial resolution has been achieved at close to the diffraction limit of the telescope (0. 08 −0. 12) and images show a wealth of morphological detail. Structure is not fractal but shows two preferred scale sizes of 2. (1100 AU) and 1. 2 (540 AU) , where the larger scale may be associated with star formation. Key words. ISM : individual objects : OMC1 – ISM : circumstellar matter – ISM : kinematics and dynamics – ISM : molecules – infrared : ISM

3D phase diversity: a myopic deconvolution method for short-exposure images: application to retinal imaging

3D deconvolution is an established technique in microscopy that may be useful for low-cost high-resolution imaging of the retina. We report on a myopic 3D deconvolution method developed in a Bayesian framework. This method uses a 3D imaging model, a noise model that accounts for both photon and detector noises, a regularization term that is appropriate for objects that are a mix of sharp edges and smooth areas, a positivity constraint, and a smart parameterization of the point-spread function (PSF) by the pupil phase. It estimates the object and the PSF jointly. The PSF parameterization through the pupil phase constrains the inversion by dramatically reducing the number of unknowns. The joint deconvolution is further constrained by an additional longitudinal support constraint derived from a 3D interpretation of the phase-diversity technique. This method is validated by simulated retinal images.

In vivo fibered confocal reflectance imaging: totally non-invasive morphological cellular imaging brought to the endoscopist

This paper presents a novel fibered confocal reflectance microscopy system (FCRM) specifically designed for the medical observation of biological tissues in vivo and in situ, in real time, at the cellular level: the R-600. Reflectance imaging is based on the refraction index difference between biological components while confocal imaging allow to perform the optical sectioning slice in-depth inside the tissues. The R-600 is based on a proximal scanning system, coupled with a 7 mm diameter probe made of tens of thousands of flexible optical fibers allowing in situ imaging, associated with a dedicated software performing real-time control and image processing. The R-600 provides 12 frames per second at an optical imaging depth of 30 microns, with a high lateral resolution, 1 micron, an axial resolution of 2 microns in a field of view 160 microns in diameter. Thanks to the miniaturization of the optical probe, unprecedented accessibility is made possible in organs such as the cervix or the otolaryngological sphere, in a completely non-invasive fashion. The aim of FCRM is to perform optical biopsy. As a first step towards this goal, we present here results obtained in vivo and in real-time on a human mouth , assessing the ability of the R-600 to perform rapid morphologic examination. Subcellular structures such as nuclei and membranes can be clearly distinguished on the images. Further miniaturization opens perspectives for an integrated endoscope-compatible system with broad medical applications.

To see the unseeable: confocal miniprobes for routine microscopic imaging during endoscopy

Confocal fluorescence high resolution imaging during standard endoscopic procedures has been presented as a very promising tool to enhance patient care and physician practice by providing supplementary diagnostic information in real-time. The purpose of this paper is to show not only potential, but convincing results of endoscopic microscopy using a catheter-based approach. Mauna Kea Technologies core technology, Cellvizio, delivers dynamic imaging at 12 frames/second using confocal miniprobes inserted through the operating channel of regular endoscopes. Cellvizio is composed of 3 parts including (a) a Laser Scanning Unit, (b) Confocal MiniprobeTM with the following characteristics: 5-15 &mgr;m axial resolution, 2-5 &mgr;m lateral resolution, 15-100 &mgr;m depth of penetration, field of view of 600x500 &mgr;m and (c) a software package with onthe- fly processing capabilities. With several tens of patients examined during routine GI endoscopy procedures, the most relevant clinical parameters could be assessed in a doubled-blinded fashion between the endoscopist and a pathologist and results showing very high accuracy in the differentiation of neoplasia from normal and hyperplastic tissue were obtained. In the field of pulmonology, the micro-autofluorescence properties of tissues could be assessed and structures never before accessed in vivo were observed. Cellvizio® may be useful to study bronchial remodeling in asthma and chronic obstructive pulmonary diseases. Using appropriate topical fluorescent dye, the Confocal Miniprobes may also make it possible to perform optical biopsy of precancerous and superficial bronchial cancers. Cellvizio® is as a new tool towards targeted biopsies, leading to earlier, more reliable and cost effective diagnostic procedures. Other applications, specifically in molecular imaging are also made possible by the miniaturization of the probe (combination with biopsy needle for solid organs use or lymph node detection) and by the compatibility of the system with other imaging modalities (auto-fluorescence and narrow-band imaging endoscopy, MRI, PET, etc).

Mauna Kea technologies’ F400 prototype: a new tool for in vivo microscopic imaging during endoscopy

The purpose of this paper is to demonstrate potential for high resolution fluorescence imaging during standard endoscopic procedures using a catheter-based confocal endoscope, compatible with standard video-endoscopes. The instrument, an F400 prototype from Mauna Kea Technologies (Paris, France), may function in various imaging modalities: auto-fluorescence or exogenous fluorescence using topical applications of fluorophores. The system is composed of a Laser Scanning Unit, a range of fibered objectives and a dedicated software, which makes it possible to obtain images at a rate of 12 frames per second. These images have a lateral resolution of 2.5 microns, an axial resolution of 15 microns, a field of view up to 600 microns x 500 microns and can be obtained at depths up to 100 microns. The miniaturized fibered probes offer unique access capabilities, specifically through the operating channel of an endoscope. So far, these studies have demonstrated the safety and efficacy of the F400 in allowing confocal laser imaging of the internal microstructure of tissues in the anatomical tract accessed by the endoscope, thanks to the miniaturization of the system. The device can be considered as a new tool towards optical biopsies and in vivo histology, leading to more physiologically relevant data and cost effective medicine.