Large field-of-view phase and fluorescence mesoscope with microscopic resolution.

Phase and fluorescence are complementary contrasts that are commonly used in biology. However, the coupling of these two modalities is traditionally limited to high magnification and complex imaging systems. For statistical studies of biological populations, a large field-of-view is required. We describe a 30 mm2 field-of-view dual-modality mesoscope with a 4-µm resolution. The potential of the system to address biological questions is illustrated on white blood cell numeration in whole blood and multiwavelength imaging of the human osteosarcoma (U2-OS) cells.

Photothermal Control of Heat-Shock Protein Expression at the Single Cell Level.

Laser heating of individual cells in culture recently led to seminal studies in cell poration, fusion, migration, or nanosurgery, although measuring the local temperature increase in such experiments remains a challenge. Here, the laser-induced dynamical control of the heat-shock response is demonstrated at the single cell level, enabled by the use of light-absorbing gold nanoparticles as nanosources of heat and a temperature mapping technique based on quadriwave lateral shearing interferometry (QLSI) measurements. As it is label-free, this approach does not suffer from artifacts inherent to previously reported fluorescence-based temperature-mapping techniques and enables the use of any standard fluorescent labels to monitor in parallel the cell's response.

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.

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

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.

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.

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.

Negative regulation of estrogen signaling by ERß 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 ERß ectopic expression on 17ß-estradiol (E2)-induced transactivation and cell proliferation in ERα-positive BG1 ovarian cancer cells. As expected, ERß 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 ERß-selective (8ß-vinyl-estradiol) ligands, indicating that ERß activation reinforced the inhibitory effects of ERß. Interestingly, in E2-stimulated BG1 cells, ERß 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 ERß 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 ERß overexpression, and ERß activity was more sensitive to repression by RIP140. Finally, small interfering RNA-mediated knockdown of RIP140 expression abolished the repressive effect exerted by activated ERß on the regulation of ERE-controlled transcription by estrogens. Altogether, these data demonstrate the inhibitory effects of ERß on estrogen signaling in ovarian cancer cells and the key role that RIP140 plays in this phenomenon.

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.

Adriamycin dose and time effects on cell cycle, cell death, and reactive oxygen species generation in leukaemia cells.

We investigate the relative importance of the different mechanisms of Adriamycin, an anthracycline, and their interrelations, in particular the link between cell cycle arrest, cell death, and generation of reactive oxygen species (ROS) that is suspected to be the origin of cardiotoxic side-effects. We introduced a lifetime fluorescence based technology and used videomicrofluorometry, two efficient analytical methods. We show that depending on the doses and time after incubation, ADR will not reach the same compartments (nucleus, mitochondria, cytosol) in the cells, having consequences on the production of ROS, growth arrest pathways and cell death pathways.

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.

Monitoring cell cycle distributions in living cells by videomicrofluorometry and discriminant factorial analysis.

BACKGROUND: The study of the cell cycle of living cells is often based on quantification of nuclear DNA. These studies may be improved by multifactorial analysis evaluating several parameters for each cell. METHODS: Single lymphoblastoid living cells were labeled with three fluorescent markers: Hoechst 33342 for nuclear DNA, Rhodamine 123 for mitochondria, and Nile Red for plasma membrane. Numerical image analysis allowed us to obtain, for each cell, morphological parameters (e.g., cell size, nuclear size, and shape) and functional information (e.g., nuclear DNA content, level of mitochondria energetic state, and the amount and properties of the plasma membrane) by fluorescence intensity. These parameters were used in a typological analysis that separated control cells into four groups. RESULTS: A discriminant factorial analysis (DFA) confirmed the four groups: G0-G1, S, G2+M, and polyploid cells called Gn. These groups were significantly different, with a classification probability of 0.9999; these control cells defined a learning population. Different populations of untreated and adriamycin-treated cells were analyzed as additional individuals within a DFA and were classified into the G0-G1, S, G2+M, and Gn groups by their probability of belonging to each of the groups. CONCLUSIONS: This approach is particularly efficient when it is used to determine variations in cellular properties and to objectively study cellular populations.