A time-resolved multifocal multiphoton microscope for high speed FRET imaging in vivo

Imaging the spatio-temporal interaction of proteins in vivo is essential to understanding the complexities of biological systems. The highest accuracy monitoring of protein-protein interactions is achieved using FRET measured by fluorescence lifetime imaging with measurements taking minutes to acquire a single frame, limiting their use in dynamic live cell systems. We present a diffraction limited, massively parallel, time-resolved multifocal multiphoton microscope capable of producing fluorescence lifetime images with 55 ps time-resolution giving improvements in acquisition speed of a factor of 64. We present demonstrations with FRET imaging in a model cell system and demonstrate in vivo FLIM using a GTPase biosensor in the zebrafish embryo

New high-speed centre of mass method incorporating background subtraction for accurate determination of fluorescence lifetime

We demonstrate an implementation of a centre-of-mass method (CMM) incorporating background subtraction for use in multifocal fluorescence lifetime imaging microscopy to accurately determine fluorescence lifetime in live cell imaging using the Megaframe camera. The inclusion of background subtraction solves one of the major issues associated with centre-of-mass approaches, namely the sensitivity of the algorithm to background signal. The algorithm, which is predominantly implemented in hardware, provides real-time lifetime output and allows the user to effectively condense large amounts of photon data. Instead of requiring the transfer of thousands of photon arrival times, the lifetime is simply represented by one value which allows the system to collect data up to limit of pulse pile-up without any limitations on data transfer rates. In order to evaluate the performance of this new CMM algorithm with existing techniques (i.e. Rapid lifetime determination and Levenburg-Marquardt), we imaged live MCF-7 human breast carcinoma cells transiently transfected with FRET standards. We show that, it offers significant advantages in terms of lifetime accuracy and insensitivity to variability in dark count rate (DCR) between Megaframe camera pixels. Unlike other algorithms no prior knowledge of the expected lifetime is required to perform lifetime determination. The ability of this technique to provide real-time lifetime readout makes it extremely useful for a number of applications

Microscopie de fluorescence résolue en temps et en polarisation pour le suivi d’interactions protéiques en neurobiologie

In the framework of this thesis, we have used FRET (Forster Resonance Energy Transfer) as a mechanism to follow the interaction of proteins from the plasma membrane to the cytoplasm of cells. To quantify FRET, we have chosen Fluorescence Lifetime Imaging Microscopy (FLIM) since this method is independent of the concentration and intensity of the fluorophores. To have a good axial resolution, a TIRFLIM set-up (Total Internal Reflection Fluorescence Lifetime Imaging Microscopy) was developed and this allowed us to perform wide-field imaging with sub-wavelength axial resolution. This set-up was calibrated and optimized in order to answer biological questions. Different approaches were tested in order to measure the penetration depth of the evanescent field and especially plasmonic surfaces were used to further enhance the axial resolution. Our set-up was dedicated to the study of the effect of cholesterol on the interaction between the Amyloid Precursor Protein (APP), a transmembrane protein involved in Alzheimer Disease, and one of its cleaving enzyme (BACE1). We performed a dynamic tracking of APP and BACE1 proximity under the effect of cholesterol, in HEK-293 cells and primary cultures of embryonic rat hippocampal neurons, thanks to our TIRFLIM set-up.Time-resolved fluorescence anisotropy has been implemented on our set-up. This has enabled us to measure the rotational correlation time of fluorophores and to investigate quantitatively different states of homodimerization of proteins involved in Alzheimer’s disease.Le suivi des interactions entre protéines, localisées à la membrane plasmique ou à l’intérieur de cellules, a été réalisé au cours de cette thèse par imagerie de fluorescence et par l’analyse de processus dits de FRET (Forster Resonance Energy Transfer). Pour quantifier le FRET entre nos protéines d’intérêt, nous avons choisi le contraste de durée de vie de fluorescence car cette méthode est indépendante de la concentration et de l’intensité de fluorescence. Afin d’obtenir une résolution suffisante pour des problématiques neurobiologiques, un microscope TIRFLIM (Total Internal Reflection Fluorescence Lifetime Imaging Microscopy) avait préalablement été développé. Celui-ci nous permet de faire de l’imagerie en plein champ avec une résolution axiale sub-longueur d’onde. Ce dispositif a été calibré et optimisé au cours de cette thèse pour répondre au mieux à des problématiques biologiques. Différentes approches ont ainsi été testées dans le but de calibrer la profondeur de pénétration de l’onde évanescente. Des surfaces plasmoniques ont entre autres été utilisées pour augmenter la sélectivité axiale du montage. Notre microscope a été dédié à l’étude de l’effet du cholestérol sur l’interaction entre la protéine précurseur de l’amyloïde APP, protéine transmembranaire impliquée dans la maladie d’Alzheimer et une de ses enzymes de clivage BACE1. Nous avons ainsi effectué un suivi dynamique de l’effet du cholestérol sur l’interaction entre APP et BACE1 dans des cellules HEK-293 et dans des cultures primaires de neurones d’hippocampe d’embryons de rat, de la membrane plasmique à l’intérieur des cellules grâce à notre dispositif TIRFLIM. La mesure d’anisotropie de fluorescence résolue en temps a également été implémentée sur notre montage. Ces mesures résolues en temps et en polarisation ont permis de mesurer le temps de corrélation rotationnelle de fluorophores et de mettre en évidence de manière qualitative différents niveaux d’homodimérisation de protéines impliquées dans la maladie d’Alzheimer

Time and polarisation resolved microscopy to follow proteins interactions in neurobiology

Le suivi des interactions entre protéines, localisées à la membrane plasmique ou à l’intérieur de cellules, a été réalisé au cours de cette thèse par imagerie de fluorescence et par l’analyse de processus dits de FRET (Forster Resonance Energy Transfer). Pour quantifier le FRET entre nos protéines d’intérêt, nous avons choisi le contraste de durée de vie de fluorescence car cette méthode est indépendante de la concentration et de l’intensité de fluorescence. Afin d’obtenir une résolution suffisante pour des problématiques neurobiologiques, un microscope TIRFLIM (Total Internal Reflection Fluorescence Lifetime Imaging Microscopy) avait préalablement été développé. Celui-ci nous permet de faire de l’imagerie en plein champ avec une résolution axiale sub-longueur d’onde. Ce dispositif a été calibré et optimisé au cours de cette thèse pour répondre au mieux à des problématiques biologiques. Différentes approches ont ainsi été testées dans le but de calibrer la profondeur de pénétration de l’onde évanescente. Des surfaces plasmoniques ont entre autres été utilisées pour augmenter la sélectivité axiale du montage. Notre microscope a été dédié à l’étude de l’effet du cholestérol sur l’interaction entre la protéine précurseur de l’amyloïde APP, protéine transmembranaire impliquée dans la maladie d’Alzheimer et une de ses enzymes de clivage BACE1. Nous avons ainsi effectué un suivi dynamique de l’effet du cholestérol sur l’interaction entre APP et BACE1 dans des cellules HEK-293 et dans des cultures primaires de neurones d’hippocampe d’embryons de rat, de la membrane plasmique à l’intérieur des cellules grâce à notre dispositif TIRFLIM. La mesure d’anisotropie de fluorescence résolue en temps a également été implémentée sur notre montage. Ces mesures résolues en temps et en polarisation ont permis de mesurer le temps de corrélation rotationnelle de fluorophores et de mettre en évidence de manière qualitative différents niveaux d’homodimérisation de protéines impliquées dans la maladie d’Alzheimer.In the framework of this thesis, we have used FRET (Forster Resonance Energy Transfer) as a mechanism to follow the interaction of proteins from the plasma membrane to the cytoplasm of cells. To quantify FRET, we have chosen Fluorescence Lifetime Imaging Microscopy (FLIM) since this method is independent of the concentration and intensity of the fluorophores. To have a good axial resolution, a TIRFLIM set-up (Total Internal Reflection Fluorescence Lifetime Imaging Microscopy) was developed and this allowed us to perform wide-field imaging with sub-wavelength axial resolution. This set-up was calibrated and optimized in order to answer biological questions. Different approaches were tested in order to measure the penetration depth of the evanescent field and especially plasmonic surfaces were used to further enhance the axial resolution. Our set-up was dedicated to the study of the effect of cholesterol on the interaction between the Amyloid Precursor Protein (APP), a transmembrane protein involved in Alzheimer Disease, and one of its cleaving enzyme (BACE1). We performed a dynamic tracking of APP and BACE1 proximity under the effect of cholesterol, in HEK-293 cells and primary cultures of embryonic rat hippocampal neurons, thanks to our TIRFLIM set-up.Time-resolved fluorescence anisotropy has been implemented on our set-up. This has enabled us to measure the rotational correlation time of fluorophores and to investigate quantitatively different states of homodimerization of proteins involved in Alzheimer’s disease

Simulation of breast compression using a new biomechanical model

International audienceMammography is currently the primary imaging modality for breast cancer screening and plays an important role in cancer diagnostics. A standard mammographic image acquisition always includes the compression of the breast prior x-ray exposure. The breast is compressed between two plates (the image receptor and the compression paddle) until a nearly uniform breast thickness is obtained. The breast flattening improves diagnostic image quality 1 and reduces the absorbed dose 2. However, this technique can also be a source of discomfort and might deter some women from attending breast screening by mammography 3,4. Therefore, the characterization of the pain perceived during breast compression is of potential interest to compare different compression approaches. The aim of this work is to develop simulation tools enabling the characterization of existing breast compression techniques in terms of patient comfort, dose delivered to the patient and resulting image quality. A 3D biomechanical model of the breast was developed providing physics-based predictions of tissue motion and internal stress and strain intensity. The internal stress and strain intensity are assumed to be directly correlated with the patient discomfort. The resulting compressed breast model is integrated in an image simulation framework to assess both image quality and average glandular dose. We present the results of compression simulations on two breast geometries, under different compression paddles (flex or rigid)

Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane

We present a novel imaging system combining total internal reflection fluorescence (TIRF) microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET) imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors. This arrangement enabled visualisation of conformational changes of a Raichu-Cdc42 FRET biosensor by measurement of intramolecular FRET between eGFP and mRFP1. Higher activity of the probe was found at the cell plasma membrane compared to intracellularly. Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants. Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor

FRET detection using Total Internal Reflection Fluorescence Lifetime Imaging Microscopy and supercontinuum excitation

International audienceFRET detection using Total Internal Reflection Fluorescence Lifetime Imaging Microscopy and supercontinuum excitatio

Homodimerization of Amyloid Precursor Protein at the Plasma Membrane: A homoFRET Study by Time-Resolved Fluorescence Anisotropy Imaging

Classical FRET (Förster Resonance Energy Transfer) using two fluorescent labels (one for the donor and another one for the acceptor) is not efficient for studying the homodimerization of a protein as only half of the homodimers formed can be identified by this technique. We thus resorted to homoFRET detected by time-resolved Fluorescence Anisotropy IMaging (tr-FAIM). To specifically image the plasma membrane of living cells, an original combination of tr-FAIM and Total Internal Reflection Fluorescence Lifetime Imaging Microscope (TIRFLIM) was implemented. The correcting factor accounting for the depolarization due to the high numerical aperture (NA) objective, mandatory for TIRF microscopy, was quantified on fluorescein solutions and on HEK293 cells expressing enhanced Green Fluorescence Protein (eGFP). Homodimerization of Amyloid Precursor Protein (APP), a key mechanism in the etiology of Alzheimer's disease, was measured on this original set-up. We showed, both in epifluorescence and under TIRF excitation, different energy transfer rates associated with the homodimerization of wild type APP-eGFP or of a mutated APP-eGFP, which forms constitutive dimers. This original set-up thus offers promising prospects for future studies of protein homodimerization in living cells in control and pathological conditions