Julien Savatier

Thermal Imaging of Nanostructures by Quantitative Optical Phase Analysis

We introduce an optical microscopy technique aimed at characterizing the heat generation arising from nanostructures, in a comprehensive and quantitative manner. Namely, the technique permits (i) mapping the temperature distribution around the source of heat, (ii) mapping the heat power density delivered by the source, and (iii) retrieving the absolute absorption cross section of light-absorbing structures. The technique is based on the measure of the thermal-induced refractive index variation of the medium surrounding the source of heat. The measurement is achieved using an association of a regular CCD camera along with a modified Hartmann diffraction grating. Such a simple association makes this technique straightforward to implement on any conventional microscope with its native broadband illumination conditions. We illustrate this technique on gold nanoparticles illuminated at their plasmonic resonance. The spatial resolution of this technique is diffraction limited, and temperature variations weaker than 1 K can be detected.

Estrogen Receptor Interactions and Dynamics Monitored in Live Cells by Fluorescence Cross-Correlation Spectroscopy

Quantitative characterization of protein interactions in live cells remains one of the most important challenges in modern biology. In the present work we have used two-photon, two-color, fluorescence cross-correlation spectroscopy (FCCS) in transiently transfected COS-7 cells to measure the concentrations and interactions of estrogen receptor (ER) subtypes alpha and beta with one of their transcriptional coactivator proteins, TIF2, as well as heterodimerization between the two ER subtypes. Using this approach in a systematic fashion, we observed a strong ligand-dependent modulation of receptor-coactivator complexation, as well as strong protein concentration dependence for complex formation in the absence of ligand. These quantitative values for protein and complex concentrations provide the first estimates for the ER-TIF2 K(d) for the full-length proteins and in a cellular context (agonist, < approximately 6 nM; antagonist, > approximately 3 microM; unliganded, approximately 200 nM). Coexpression of the two ER subtypes revealed substantial receptor heterodimer formation. They also provide, for the first time, estimated homo- and heterodimerization constants found to be similar and in the low nanomolar range. These results underscore the importance of receptor and coregulator expression levels and stability in the tissue-dependent modulation of receptor function under normal and pathological conditions.

Mapping the Local Organization of Cell Membranes Using Excitation-Polarization-Resolved Confocal Fluorescence Microscopy

Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization.

Optical detection and measurement of living cell morphometric features with single-shot quantitative phase microscopy

We present a quadriwave lateral shearing interferometer used as a wavefront sensor and mounted on a commercial non-modified transmission white-light microscope as a quantitative phase imaging technique. The setup is designed to simultaneously make measurements with both quantitative transmission phase and fluorescence modes: phase enables enhanced contrasted visualization of the cell structure including intracellular organelles, while fluorescence allows a complete and precise identification of each component. After the characterization of the phase measurement reliability and sensitivity on calibrated samples, we use these two imaging modes to measure the characteristic optical path difference between subcellular elements (mitochondria, actin fibers, and vesicles) and cell medium, and demonstrate that phase-only information should be sufficient to identify some organelles without any labeling, like lysosomes. Proof of principle results show that the technique could be used either as a qualitative tool for the control of cells before an experiment, or for quantitative studies on morphology, behavior, and dynamics of cells or cellular components.

Negative Regulation of Estrogen Signaling by ERβ and RIP140 in Ovarian Cancer Cells

In hormone-dependent tissues such as breast and ovary, tumorigenesis is associated with an altered expression ratio between the two estrogen receptor (ER) subtypes. In this study, we investigated the effects of ERβ ectopic expression on 17β-estradiol (E2)-induced transactivation and cell proliferation in ERα-positive BG1 ovarian cancer cells. As expected, ERβ expression strongly decreased the mitogenic effect of E2, significantly reduced E2-dependent transcriptional responses (both on a stably integrated estrogen response element [ERE] reporter gene and on E2-induced mRNAs), and strongly enhanced the formation of ER heterodimers as evidenced by chromatin immunoprecipitation analysis. Inhibition by the ERα-selective ligand propyl pyrazole triol was less marked than with the pan-agonist (E2) or the ERβ-selective (8β-vinyl-estradiol) ligands, indicating that ERβ activation reinforced the inhibitory effects of ERβ. Interestingly, in E2-stimulated BG1 cells, ERβ was more efficient than ERα to regulate the expression of receptor-interacting protein 140 (RIP140), a major ERα transcriptional corepressor. In addition, we found that the RIP140 protein interacted better with ERβ than with ERα (both in vitro and in intact cells by fluorescence cross-correlation spectroscopy). Moreover, RIP140 recruitment on the stably integrated reporter ERE was increased upon ERβ overexpression, and ERβ activity was more sensitive to repression by RIP140. Finally, small interfering RNA-mediated knockdown of RIP140 expression abolished the repressive effect exerted by activated ERβ on the regulation of ERE-controlled transcription by estrogens. Altogether, these data demonstrate the inhibitory effects of ERβ on estrogen signaling in ovarian cancer cells and the key role that RIP140 plays in this phenomenon.

Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy

Significance Central biological processes in cells and tissues are intrinsically governed by the structural order of biomolecular assemblies. It is thus a key factor to decipher how these assemblies organize in complex molecular organizations, from the nanometric to the macroscopic scale. Polarized microscopy can access such information; however, signals are spatially averaged over the optical diffraction limit and are contaminated by the fluorophores’ orientational flexibility of their linker to the biomolecules. By bringing polarized fluorescence down to superresolution microscopy using single-molecule localization, we show that structural imaging can be scaled down to nanometric scales and is able to discriminate fluorophores’ flexibility from biomolecules’ orientational order. We demonstrate nanoscale structural imaging in fundamental biological filament organizations. Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules. Here, we demonstrate that quantitative single-molecule polarized detection in biological filament assemblies allows not only to correct for the rotational flexibility of the label but also to image orientational order of filaments at the nanoscale using superresolution capabilities. The method is based on polarized direct stochastic optical reconstruction microscopy, using dedicated optical scheme and image analysis to determine both molecular localization and orientation with high precision. We apply this method to double-stranded DNA in vitro and microtubules and actin stress fibers in whole cells.

Quantitative retardance imaging of biological samples using quadriwave lateral shearing interferometry

We describe a new technique based on the use of a high-resolution quadri-wave lateral shearing interferometer to perform quantitative linear retardance and birefringence measurements on biological samples. The system combines quantitative phase images with varying polarization excitation to create retardance images. This technique is compatible with living samples and gives information about the local retardance and structure of their anisotropic components. We applied our approach to collagen fibers leading to a birefringence value of (3.4 ± 0.3) · 10(-3) and to living cells, showing that cytoskeleton can be imaged label-free.

Living cell dry mass measurement using quantitative phase imaging with quadriwave lateral shearing interferometry: an accuracy and sensitivity discussion.

Abstract. Single-cell dry mass measurement is used in biology to follow cell cycle, to address effects of drugs, or to investigate cell metabolism. Quantitative phase imaging technique with quadriwave lateral shearing interferometry (QWLSI) allows measuring cell dry mass. The technique is very simple to set up, as it is integrated in a camera-like instrument. It simply plugs onto a standard microscope and uses a white light illumination source. Its working principle is first explained, from image acquisition to automated segmentation algorithm and dry mass quantification. Metrology of the whole process, including its sensitivity, repeatability, reliability, sources of error, over different kinds of samples and under different experimental conditions, is developed. We show that there is no influence of magnification or spatial light coherence on dry mass measurement; effect of defocus is more critical but can be calibrated. As a consequence, QWLSI is a well-suited technique for fast, simple, and reliable cell dry mass study, especially for live cells.

Tomographic incoherent phase imaging, a diffraction tomography alternative for any white-light microscope

In this paper, we discuss the possibility of making tomographic reconstruction of the refractive index of a microscopic sample using a quadriwave lateral shearing interferometer, under incoherent illumination. A Z-stack is performed and the acquired incoherent elecromagnetic fields are deconvoluted before to retrieve in a quantitative manner the refractive index. The results are presented on polystyrene beads and can easily be expanded to biological samples. This technique is suitable to any white-light microscope equipped with nanometric Z-stack module.

Polarized super-resolution structural imaging inside amyloid fibrils using Thioflavine T

Thioflavin T (ThT) is standardly used as a fluorescent marker to detect aggregation of amyloid fibrils by conventional fluorescence microscopy, including polarization resolved imaging that brings information on the orientational order of the fibrils. These techniques are however diffraction limited and cannot provide fine structural details at the fibrils scales of 10–100 nm, which lie beyond the diffraction limit. In this work, we evaluate the capacity of ThT to photoswitch when bound to insulin amyloids by adjusting the redox properties of its environment. We demonstrate that on-off duty cycles, intensity and photostability of the ThT fluorescence emission under adequate buffer conditions permit stochastic super-resolution imaging with a localization precision close to 20 nm. We show moreover that signal to noise conditions allow polarized orientational imaging of single ThT molecules, which reveals ultra-structure signatures related to protofilaments twisting within amyloid fibrils.