Multi-confocal fluorescence correlation spectroscopy and its application to the study of the cellular response to heat shock

Le noyau d'une cellule est hétérogène par sa structure et son activité et beaucoup de ses composants interagissent de façon dynamique. Lors de l'étude de processus cellulaires comme la réponse au stress thermique, des expériences classiques de spectroscopie de corrélation de fluorescence (FCS), qui sont habituellement limitées à un seul volume d'observation, n'apportent que des résultats partiels à cause des informations spatiales manquantes. Ce mémoire de thèse présente une nouvelle technique de FCS multi-confocale (mFCS) qui permet des mesures FCS simultanées à différents endroits d'une cellule. La technique est basée sur l'emploi d'un modulateur spatial de lumière pour la création de plusieurs volumes d'observations distincts et d'une caméra "electron-multiplying" CCD (EMCCD) pour la détection en parallèle. La résolution spatiale ainsi que la sensibilité du système mFCS sont proches de celles d'un système FCS classique et en utilisant un mode d'acquisition particulier une résolution temporelle de 14µs a pu être atteinte. La technique mFCS est appliquée à l'étude de la réponse cellulaire au stress thermique en observant le facteur de transcription heat shock factor 1 (HSF1), qui est un régulateur clé de la réponse au stress thermique. Des mesures mFCS dans des cellules vivantes révèlent des changements dans la dynamique de HSF1 pendant le choc thermique. Ces changements concernent l'affinité ainsi que l'homogénéité spatiale des interactions avec l'ADN. En outre, nous avons également évalué les performances d'une caméra CMOS-SPAD et testé le dispositif en tant que capteur alternatif pour la mFCS en cellules vivantes.The cell nucleus is heterogeneous in its structure and activity and many of its components are in dynamic interactions with each other. When investigating the cellular response to an external signal, such as heat shock, standard fluorescence correlation spectroscopy (FCS) experiments, which are limited to one observation volume, do only give partial results because of the missing spatial information. This work introduces a novel multi-confocal FCS (mFCS) technique that allows simultaneous FCS measurements in different locations within a cell. It is based on the use of a spatial light modulator (SLM) to create several distinct observation volumes at a time and an electron-multiplying charge coupled device (EMCCD) camera to perform parallel detection. The spatial resolution as well as the sensibility of the mFCS system are close to that of a classical FCS setup and using a special readout mode, a temporal resolution of 14µs is reached. The mFCS technique is applied to study the cellular response to thermal stress by monitoring the transcription factor heat shock factor 1 (HSF1), which is a key regulator of the heat shock response. mFCS experiments in living cells reveal changes in the dynamics of HSF1 upon heat shock. These changes concern the affinity as well as the spatial homogeneity of its interactions with DNA. Additionally, the performance of a CMOS-SPAD camera, consisting of an array of single photon avalanche diodes, is evaluated and the device is tested as an alternative detector for mFCS in living cells

Spectroscopie à corrélation de fluorescence multi-confocale : développement et application à l'étude de la réponse cellulaire au choc thermique

The cell nucleus is heterogeneous in its structure and activity and many of its components are in dynamic interactions with each other. When investigating the cellular response to an external signal, such as heat shock, standard fluorescence correlation spectroscopy (FCS) experiments, which are limited to one observation volume, do only give partial results because of the missing spatial information. This work introduces a novel multi-confocal FCS (mFCS) technique that allows simultaneous FCS measurements in different locations within a cell. It is based on the use of a spatial light modulator (SLM) to create several distinct observation volumes at a time and an electron-multiplying charge coupled device (EMCCD) camera to perform parallel detection. The spatial resolution as well as the sensibility of the mFCS system are close to that of a classical FCS setup and using a special readout mode, a temporal resolution of 14µs is reached. The mFCS technique is applied to study the cellular response to thermal stress by monitoring the transcription factor heat shock factor 1 (HSF1), which is a key regulator of the heat shock response. mFCS experiments in living cells reveal changes in the dynamics of HSF1 upon heat shock. These changes concern the affinity as well as the spatial homogeneity of its interactions with DNA. Additionally, the performance of a CMOS-SPAD camera, consisting of an array of single photon avalanche diodes, is evaluated and the device is tested as an alternative detector for mFCS in living cells.Le noyau d'une cellule est hétérogène par sa structure et son activité et beaucoup de ses composants interagissent de façon dynamique. Lors de l'étude de processus cellulaires comme la réponse au stress thermique, des expériences classiques de spectroscopie de corrélation de fluorescence (FCS), qui sont habituellement limitées à un seul volume d'observation, n'apportent que des résultats partiels à cause des informations spatiales manquantes. Ce mémoire de thèse présente une nouvelle technique de FCS multi-confocale (mFCS) qui permet des mesures FCS simultanées à différents endroits d'une cellule. La technique est basée sur l'emploi d'un modulateur spatial de lumière pour la création de plusieurs volumes d'observations distincts et d'une caméra "electron-multiplying" CCD (EMCCD) pour la détection en parallèle. La résolution spatiale ainsi que la sensibilité du système mFCS sont proches de celles d'un système FCS classique et en utilisant un mode d'acquisition particulier une résolution temporelle de 14µs a pu être atteinte. La technique mFCS est appliquée à l'étude de la réponse cellulaire au stress thermique en observant le facteur de transcription heat shock factor 1 (HSF1), qui est un régulateur clé de la réponse au stress thermique. Des mesures mFCS dans des cellules vivantes révèlent des changements dans la dynamique de HSF1 pendant le choc thermique. Ces changements concernent l'affinité ainsi que l'homogénéité spatiale des interactions avec l'ADN. En outre, nous avons également évalué les performances d'une caméra CMOS-SPAD et testé le dispositif en tant que capteur alternatif pour la mFCS en cellules vivantes

Spectroscopie à corrélation de fluorescence multi-confocale (développement et application à l'étude de la réponse cellulaire au choc thermique)

Le noyau d'une cellule est hétérogène par sa structure et son activité et beaucoup de ses composants interagissent de façon dynamique. Lors de l'étude de processus cellulaires comme la réponse au stress thermique, des expériences classiques de spectroscopie de corrélation de fluorescence (FCS), qui sont habituellement limitées à un seul volume d'observation, n'apportent que des résultats partiels à cause des informations spatiales manquantes. Ce mémoire de thèse présente une nouvelle technique de FCS multi-confocale (mFCS) qui permet des mesures FCS simultanées à différents endroits d'une cellule. La technique est basée sur l'emploi d'un modulateur spatial de lumière pour la création de plusieurs volumes d'observations distincts et d'une caméra electron-multiplying'' CCD (EMCCD) pour la détection en parallèle. La résolution spatiale ainsi que la sensibilité du système mFCS sont proches de celles d'un système FCS classique et en utilisant un mode d'acquisition particulier une résolution temporelle de $14mu s$ a pu être atteinte. La technique mFCS est appliquée à l'étude de la réponse cellulaire au stress thermique en observant le facteur de transcription heat shock factor 1 (HSF1), qui est un régulateur clé de la réponse au stress thermique. Des mesures mFCS dans des cellules vivantes révèlent des changements dans la dynamique de HSF1 pendant le choc thermique. Ces changements concernent l'affinité ainsi que l'homogénéité spatiale des interactions avec l'ADN. En outre, nous avons également évalué les performances d'une caméra CMOS-SPAD et testé le dispositif en tant que capteur alternatif pour la mFCS en cellules vivantes.The cell nucleus is heterogeneous in its structure and activity and many of its components are in dynamic interactions with each other. When investigating the cellular response to an external signal, such as heat shock, standard fluorescence correlation spectroscopy (FCS) experiments, which are limited to one observation volume, do only give partial results because of the missing spatial information. This work introduces a novel multi-confocal FCS (mFCS) technique that allows simultaneous FCS measurements in different locations within a cell. It is based on the use of a spatial light modulator (SLM) to create several distinct observation volumes at a time and an electron-multiplying charge coupled device (EMCCD) camera to perform parallel detection. The spatial resolution as well as the sensibility of the mFCS system are close to that of a classical FCS setup and using a special readout mode, a temporal resolution of $14mu s$ is reached. The mFCS technique is applied to study the cellular response to thermal stress by monitoring the transcription factor heat shock factor 1 (HSF1), which is a key regulator of the heat shock response. mFCS experiments in living cells reveal changes in the dynamics of HSF1 upon heat shock. These changes concern the affinity as well as the spatial homogeneity of its interactions with DNA. Additionally, the performance of a CMOS-SPAD camera, consisting of an array of single photon avalanche diodes, is evaluated and the device is tested as an alternative detector for mFCS in living cells.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Cellular response to heat shock studied by multiconfocal fluorescence correlation spectroscopy.

International audienceHeat shock triggers a transient and ubiquitous response, the function of which is to protect cells against stress-induced damage. The heat-shock response is controlled by a key transcription factor known as heat shock factor 1 (HSF1). We have developed a multiconfocal fluorescence correlation spectroscopy setup to measure the dynamics of HSF1 during the course of the heat-shock response. The system combines a spatial light modulator, to address several points of interest, and an electron-multiplying charge-coupled camera for fast multiconfocal recording of the photon streams. Autocorrelation curves with a temporal resolution of 14 μs were analyzed before and after heat shock on eGFP and HSF1-eGFP-expressing cells. Evaluation of the dynamic parameters of a diffusion-and-binding model showed a slower HSF1 diffusion after heat shock. It is also observed that the dissociation rate decreases after heat shock, whereas the association rate is not affected. In addition, thanks to the multiconfocal fluorescence correlation spectroscopy system, up to five spots could be simultaneously located in each cell nucleus. This made it possible to quantify the intracellular variability of the diffusion constant of HSF1, which is higher than that of inert eGFP molecules and increases after heat shock. This finding is consistent with the fact that heat-shock response is associated with an increase of HSF1 interactions with DNA and cannot be explained even partially by heat-induced modifications of nuclear organization

Dynamics of the Full Length and Mutated Heat Shock Factor 1 in Human Cells

Heat shock factor 1 is the key transcription factor of the heat shock response. Its function is to protect the cell against the deleterious effects of stress. Upon stress, HSF1 binds to and transcribes hsp genes and repeated satellite III (sat III) sequences present at the 9q12 locus. HSF1 binding to pericentric sat III sequences forms structures known as nuclear stress bodies (nSBs). nSBs represent a natural amplification of RNA pol II dependent transcription sites. Dynamics of HSF1 and of deletion mutants were studied in living cells using multi-confocal Fluorescence Correlation Spectroscopy (mFCS) and Fluorescence Recovery After Photobleaching (FRAP). In this paper, we show that HSF1 dynamics modifications upon heat shock result from both formation of high molecular weight complexes and increased HSF1 interactions with chromatin. These interactions involve both DNA binding with Heat Shock Element (HSE) and sat III sequences and a more transient sequence-independent binding likely corresponding to a search for more specific targets. We find that the trimerization domain is required for low affinity interactions with chromatin while the DNA binding domain is required for site-specifi

Fast Raman single bacteria identification: toward a routine in-vitro diagnostic

Conference on Biophotonics - Photonic Solutions for Better Health Care V, Brussels, BELGIUM, APR 04-07, 2016International audienceTimely microbiological results are essential to allow clinicians to optimize the prescribed treatment, ideally at the initial stage of the therapeutic process. Several approaches have been proposed to solve this issue and to provide the microbiological result in a few hours directly from the sample such as molecular biology. However fast and sensitive those methods are not based on single phenotypic information which presents several drawbacks and limitations. Optical methods have the advantage to allow single-cell sensitivity and to probe the phenotype of measured cells. Here we present a process and a prototype that allow automated single bacteria phenotypic analysis. This prototype is based on the use of Digital In-line Holography techniques combined with a specially designed Raman spectrometer using a dedicated device to capture bacteria. The localization of single-cell is finely determined by using holograms and a proper propagation kernel. Holographic images are also used to analyze bacteria in the sample to sort potential pathogens from flora dwelling species or other biological particles. This accurate localization enables the use of a small confocal volume adapted to the measurement of single-cell. Along with the confocal volume adaptation, we also have modified every components of the spectrometer to optimize single-bacteria Raman measurements. This optimization allowed us to acquire informative single-cell spectra using an integration time of 0.5s only. Identification results obtained with this prototype are presented based on a 65144 Raman spectra database acquired automatically on 48 bacteria strains belonging to 8 species

Expression levels of HSF1 and HSF1-eGFP in stable HeLa cell lines.

<p>HSF1 western blotting of full protein extracts before (−) and after one hour heat shock (+), in control cells (Ctl), cell lines expressing the full length HSF1-eGFP (wt), the HSF1-eGFP mutant deleted of the trimerization domain (ΔTRIM), the HSF1-eGFP with the punctual mutation (K80Q) and the HSF1-eGFP mutant deleted for the DNA binding domain (ΔDBD). The shifted bands detected after HS (+) represent the phosphorylated form of HSF1.</p

HSF1-K80Q prevents formation of nSBs and behaves as dominant negative.

<p>Confocal images of WT HSF1- tagRFP-T (A,C) and HSF1-K80Q-eGFP (B,D) after one hour heat shock in single transfected (A,B) and co-transfected (C,D) Hela cells. nSBs are only detected in single transfected WT HSF1-eGFP cells (A).</p

Intracellular localization of HSF1 transcription factor in HeLa cells.

<p>CLSM images of wild type HSF1-eGFP, HSF1-ΔTRIM-eGFP, HSF1-K80Q-eGFP and HSF1-ΔDBD-eGFP before (NHS) and after a one hour heat shock (HS). HSF1 is predominantly nuclear except when the trimerization domain is deleted. After a one-hour heat shock, nuclear stress bodies (nSBs, black arrows) are only formed with HSF1-eGFP full length. Scale bar = 5 µm.</p