Nouvelles dimensions pour l’imagerie multiplexée fluorescente

Notre groupe de recherche avait déjà mis au point les protocoles OPIOM pour l’imagerie par fluorescence. En exploitant les sections efficaces de photoswiching de fluorescence, OPIOM permet d’extraire sélectivement la réponse des fluorophores réversiblement photoswitchables (RSFs) en présence de fluorophores interférant spectralement. Cependant, OPIOM nous a permis de ne distinguer que 3 RSFPs spectralement similaires. L’objectif de cette thèse était d’augmenter ce nombre. Pour atteindre cet objectif, un nouvel instrument automatisé appelée photoswichomètre a été mis au point pour cribler la signature photochimique de 22 RSFP en analysant leur réponse de fluorescence aux sauts de lumière dont l’intensité couvre 5 ordres de grandeur. Cette signature a d’abord été exploitée dans un nouveau protocole d’imagerie par fluorescence appelé HIGHLIGHT, qui a capitalisé sur OPIOM et amélioré encore sa sélectivité. Dans HIGHLIGHT, les RSFs sont soumis à une modulation harmonique de la lumière et leur contribution aux signaux d’émission de fluorescence globale est sélectivement récupérée en exploitant leur réponse non linéaire singulière dans des conditions optimisées. HIGHLIGHLIGHT a été implémenté pour l’imagerie des RSFPs dans les cellules sans interférence d’autofluorescence, pour réaliser l’imagerie multiplexée de 3 RSFPs qui ne pouvaient pas être discriminés avec OPIOM, aussi pour améliorer l’effet de sectionnement optique. La signature des RSF a ensuite été utilisée dans un deuxième protocole d’imagerie par fluorescence appelé LIGHTNING. Contrairement à OPIOM et HIGHLIGHT qui exploitent les sections efficaces de la photoswitching en régime permanent de faible intensité lumineuse, LIGHTNING exploite le régime transitoire des RSFs sous de multiples illuminations impliquant diverses gammes d’intensités lumineuses pour la discrimination RSF. Ainsi, LIGHTNING nous a permis d’améliorer le degré de multiplexage du contraste dynamique en imagerie de fluorescence jusqu’à 20 RSFP sur 22 RSFP étudiés.Our research group had previously developed the OPIOM protocols for fluorescence imaging. By exploiting their cross sections of fluorescence photoswiching, OPIOM can selectively extract the response of reversibly photoswitchable fluorophores (RSFs) in the presence of spectrally interfering fluorophores. However, OPIOM allowed us to discriminate only 3 spectrally similar reversibly photoswitchable fluorescent proteins (RSFPs). The goal of this PhD was to augment this number. To reach this goal, a new automated instrumental setup called photoswichometer was first developed to express and screen the rich photochemical signature of 22 RSFPs by analyzing their fluorescence response to light jumps with intensities covering 5 orders of magnitude. This signature has been first exploited in a new fluorescence imaging protocol called HIGHLIGHT, which capitalized on OPIOM and further improved its selectivity. In HIGHLIGHT, the RSFs are submitted to harmonic light modulation and their contribution to the overall fluorescence emission signals is selectively retrieved from exploiting their singular non-linear response under optimized conditions. HIGHLIGHT has been implemented to image RSFPs in cells without interference of autofluorescence, to perform multiplexed imaging of 3 RSFPs which could not be discriminated with OPIOM, and used for its intrinsic optical sectioning. The RSF signature has been then used in a second fluorescence imaging protocol called LIGHTNING. In contrast to OPIOM and HIGHLIGHT which exploit the cross sections of fluorescence photoswitching in a steady-state regime of low light intensity, LIGHTNING exploits the transient time fluorescence response of RSFs under multiple illuminations involving various ranges of light intensities for RSF discrimination. Thus, LIGHTNING allowed us to improve the multiplexing degree of dynamic contrast in fluorescence imaging up to 20 RSFP among 22 studied RSFPs

Dynamic contrast for overcoming spectral interferences in fluorescence imaging

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Dynamic contrast with reversibly photoswitchable fluorescent labels for imaging living cells

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Correction to “Dynamic Contrast for Plant Phenotyping”

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Dynamic Contrast for Plant Phenotyping

International audienceNoninvasiveness, minimal handling, and immediate response are favorable features of fluorescence readout for high-throughput phenotyping of labeled plants.Yet, remote fluorescence imaging may suffer from an autofluorescent background and artificial or natural ambient light. In this work, the latter limitations are overcome by adopting reversibly photoswitchable fluorescent proteins (RSFPs) as labels and Speed OPIOM (out-of-phase imaging after optical modulation), a fluorescence imaging protocol exploiting dynamic contrast. Speed OPIOM can efficiently distinguish the RSFP signal from autofluorescence and other spectrally interfering fluorescent reporters like GFP. It can quantitatively assess gene expressions, even when they are weak. It is as quantitative, sensitive, and robust in dark and bright light conditions. Eventually, it can be used to nondestructively record abiotic stress responses like water or iron limitations in real time at the level of individual plants and even of specific organs. Such Speed OPIOM validation could find numerous applications to identify plant lines in selection programs, design plants as environmental sensors, or ecologically monitor transgenic plants in the environment

Simple imaging protocol for autofluorescence elimination and optical sectioning in fluorescence endomicroscopy

International audienceFiber-optic epifluorescence imaging with one-photon excitation benefits from its ease of use, cheap light sources, and full-frame acquisition, which enables it for favorable temporal resolution of image acquisition. However, it suffers from a lack of robustness against autofluorescence and light scattering. Moreover, it cannot easily eliminate the out-of-focus background, which generally results in low-contrast images. In order to overcome these limitations, we have implemented fast out-of-phase imaging after optical modulation (Speed OPIOM) for dynamic contrast in fluorescence endomicroscopy. Using a simple and cheap optical-fiber bundle-based endomicroscope integrating modulatable light sources, we first showed that Speed OPIOM provides intrinsic optical sectioning, which restricts the observation of fluorescent labels at targeted positions within a sample. We also demonstrated that this imaging protocol efficiently eliminates the interference of autofluorescence arising from both the fiber bundle and the specimen in several biological samples. Finally, we could perform multiplexed observations of two spectrally similar fluorophores differing by their photoswitching dynamics. Such attractive features of Speed OPIOM in fluorescence endomicroscopy should find applications in bioprocessing, clinical diagnostics, plant observation, and surface imaging

Macroscale fluorescence imaging against autofluorescence under ambient light

Bio-imaging: switching signals for clearer fluorescence Fluorescence imaging at the macroscale will become easier and more sensitive thanks to a new optical procedure, which uses photoswitchable fluorescent tags. Until now, observing signals from the fluorescent labels has been hampered by interference due to external light or the natural fluorescence of other chemicals in a biological sample or in the surrounding media. Thomas Le Saux and colleagues at the École Normale Supérieure, in Paris, with co-workers elsewhere in France, overcame the current limitations using fluorescent labels that can be switched between bright “on” and dark “off” states. Improvements in light exposure and processing methods, combined with this unique characteristic of the label molecules, achieved greatly increased sensitivity in identifying the labels and distinguishing their fluorescence from interfering signals such as autofluorescence or ambient light

Extra kinetic dimensions for label discrimination

International audienceAbstract 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

Resonant out-of-phase fluorescence microscopy and remote imaging overcome spectral limitations

We present speed out-of-phase imaging after optical modulation (OPIOM), which exploits reversible photoswitchable fluorophores as fluorescent labels and combines optimized periodic illumination with phase-sensitive detection to specifically retrieve the label signal. Speed OPIOM can extract the fluorescence emission from a targeted label in the presence of spectrally interfering fluorophores and autofluorescence. Up to four fluorescent proteins exhibiting a similar green fluorescence have been distinguished in cells either sequentially or in parallel. Speed OPIOM is compatible with imaging biological processes in real time in live cells. Finally speed OPIOM is not limited to microscopy but is relevant for remote imaging as well, in particular, under ambient light. Thus, speed OPIOM has proved to enable fast and quantitative live microscopic and remote-multiplexed fluorescence imaging of biological samples while filtering out noise, interfering fluorophores, as well as ambient light