Emmanuel Beaurepaire

High-resolution full-field optical coherence tomography with a Linnik microscope

We describe an original microscope for high-resolution optical coherence tomography applications. Our system is based on a Linnik interference microscope with high-numerical-aperture objectives. Lock-in detection of the interference signal is achieved in parallel on a CCD by use of a photoelastic birefringence modulator and full-field stroboscopic illumination with an infrared LED. Transverse cross-section (en-face, or XY) images can be obtained in real time with better than 1-microm axial (Z) resolution and 0.5-microm transverse (XY) resolution. A sensitivity of approximately 80 dB is reached at a 1-image/s acquisition rate, which allows tomography in scattering media such as biological tissues.

Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy

Lipid bodies have an important role in energy storage and lipid regulation. Here we show that lipid bodies are a major source of contrast in third-harmonic generation (THG) microscopy of cells and tissues. In hepatocytes, micrometer-sized lipid bodies produce a THG signal 1–2 orders of magnitude larger than other structures, which allows one to image them with high specificity. THG microscopy with ∼1,200 nm excitation can be used to follow the distribution of lipid bodies in a variety of unstained samples including insect embryos, plant seeds and intact mammalian tissue (liver, lung). We found that epi-THG imaging is possible in weakly absorbing tissues because bulk scattering redirects a substantial fraction of the forward-generated harmonic light toward the objective. Finally, we show that the combination of THG microscopy with two-photon and second-harmonic imaging provides a new tool for exploring the interactions between lipid bodies, extracellular matrix and fluorescent compounds (vitamin A, NADH and others) in tissues.

Two-photon microscopy in brain tissue: parameters influencing the imaging depth

Light scattering by tissue limits the imaging depth of two-photon microscopy and its use for functional brain imaging in vivo. We investigate the influence of scattering on both fluorescence excitation and collection, and identify tissue and instrument parameters that limit the imaging depth in the brain. (i) In brain slices, we measured that the scattering length at lambda=800 nm is a factor 2 higher in juvenile cortical tissue (P14-P18) than in adult tissue (P90). (ii) In a detection geometry typical for in vivo imaging, we show that the collected fraction of fluorescence drops at large depths, and that it is proportional to the square of the effective angular acceptance of the detection optics. Matching the angular acceptance of the microscope to that of the objective lens can result in a gain of approximately 3 in collection efficiency at large depths (>500 microm). A low-magnification (20x), high-numerical aperture objective (0.95) further increases fluorescence collection by a factor of approximately 10 compared with a standard 60x-63x objective without compromising the resolution. This improvement should allow fluorescence measurements related to neuronal or vascular brain activity at >100 microm deeper than with standard objectives.

Full-field optical coherence microscopy

We present a new microscopy system for imaging in turbid media that is based on the spatial coherence gate principle and generates in parallel a complete two-dimensional head-on image without scanning. This system has been implemented in a commercial microscope and preserves the lateral resolution of the optics used. With a spatially incoherent source, speckle-free images with diffraction-limited resolution are recorded at successive depths with shot-noise-limited detection. The setup comprises a photoelastic modulator for path difference modulation and a two-dimensional CCD array and uses a multiplexed lock-in detection scheme.

Cell Lineage Reconstruction of Early Zebrafish Embryos Using Label-Free Nonlinear Microscopy

Zebrafish Development in 3D Vertebrate development has classically been characterized qualitatively, but—by combining expertise in physics, mathematics, and biology—Olivier et al. (p. 967) used label-free conformal nonlinear time-lapse microscopy and image analysis to calculate the spatiotemporal cell lineage of zebrafish embryos throughout their first 10 division cycles. The work reconstructs complete lineage trees, annotated with cell-shape measurements, and allows for visualization with interactive tools. Time-lapse recording characterizes the rhythm and cleavage pattern of the embryo during early stages of development. Quantifying cell behaviors in animal early embryogenesis remains a challenging issue requiring in toto imaging and automated image analysis. We designed a framework for imaging and reconstructing unstained whole zebrafish embryos for their first 10 cell division cycles and report measurements along the cell lineage with micrometer spatial resolution and minute temporal accuracy. Point-scanning multiphoton excitation optimized to preferentially probe the innermost regions of the embryo provided intrinsic signals highlighting all mitotic spindles and cell boundaries. Automated image analysis revealed the phenomenology of cell proliferation. Blastomeres continuously drift out of synchrony. After the 32-cell stage, the cell cycle lengthens according to cell radial position, leading to apparent division waves. Progressive amplification of this process is the rule, contrasting with classical descriptions of abrupt changes in the system dynamics.

Tissue Deformation Modulates Twist Expression to Determine Anterior Midgut Differentiation in Drosophila Embryos

Mechanical deformations associated with embryonic morphogenetic movements have been suggested to actively participate in the signaling cascades regulating developmental gene expression. Here we develop an appropriate experimental approach to ascertain the existence and the physiological relevance of this phenomenon. By combining the use of magnetic tweezers with in vivo laser ablation, we locally control physiologically relevant deformations in wild-type Drosophila embryonic tissues. We demonstrate that the deformations caused by germ band extension upregulate Twist expression in the stomodeal primordium. We find that stomodeal compression triggers Src42A-dependent nuclear translocation of Armadillo/beta-catenin, which is required for Twist mechanical induction in the stomodeum. Finally, stomodeal-specific RNAi-mediated silencing of Twist during compression impairs the differentiation of midgut cells, resulting in larval lethality. These experiments show that mechanically induced Twist upregulation in stomodeal cells is necessary for subsequent midgut differentiation.

Second harmonic imaging and scoring of collagen in fibrotic tissues

We compare second harmonic generation (SHG) to histological and immunohistochemical techniques for the visualization and scoring of collagen in biological tissues. We show that SHG microscopy is highly specific for fibrillar collagens and that combined SHG and two-photon excited fluorescence (2PEF) imaging can provide simultaneous three-dimensional visualization of collagen synthesis and assembly sites in transgenic animal models expressing GFP constructs. Finally, we propose several scores for characterizing collagen accumulation based on SHG images and appropriate for different types of collagen distributions. We illustrate the sensitivity of these scores in a murine model of renal fibrosis using a morphological segmentation of the tissue based on endogenous 2PEF signals.

Use of coherent control for selective two-photon fluorescence microscopy in live organisms

We demonstrate selective fluorescence We demonstrate selective fluorescence excitation of specific molecular species in live organisms by using coherent control of two-photon excitation. We have acquired quasi-simultaneous images in live fluorescently-labeled Drosophila embryos by rapid switching between appropriate pulse shapes. Linear combinations of these images demonstrate that a high degree of fluorophore selectivity is attainable through phase-shaping. Broadband phase-shaped excitation opens up new possibilities for single-laser, multiplex, in-vivo fluorescence microscopy.

Multimodal Nonlinear Imaging of the Human Cornea

PURPOSE To evaluate the potential of third-harmonic generation (THG) microscopy combined with second-harmonic generation (SHG) and two-photon excited fluorescence (2PEF) microscopies for visualizing the microstructure of the human cornea and trabecular meshwork based on their intrinsic nonlinear properties. METHODS Fresh human corneal buttons and corneoscleral discs from an eye bank were observed under a multiphoton microscope incorporating a titanium-sapphire laser and an optical parametric oscillator for the excitation, and equipped with detection channels in the forward and backward directions. RESULTS Original contrast mechanisms of THG signals in cornea with physiological relevance were elucidated. THG microscopy with circular incident polarization detected microscopic anisotropy and revealed the stacking and distribution of stromal collagen lamellae. THG imaging with linear incident polarization also revealed cellular and anchoring structures with micrometer resolution. In edematous tissue, a strong THG signal around cells indicated the local presence of water. Additionally, SHG signals reflected the distribution of fibrillar collagen, and 2PEF imaging revealed the elastic component of the trabecular meshwork and the fluorescence of metabolically active cells. CONCLUSIONS The combined imaging modalities of THG, SHG, and 2PEF provide key information about the physiological state and microstructure of the anterior segment over its entire thickness with remarkable contrast and specificity. This imaging method should prove particularly useful for assessing glaucoma and corneal physiopathologies.

Ultra-deep two-photon fluorescence excitation in turbid media

An important application of two-photon excited fluorescence (TPEF) microscopy is to provide high-resolution images from deep within scattering media. We investigate strategies to further improve TPEF penetration depth by considering the effects of scattering on fluorescence generation and collection separately. In particular, we demonstrate that the redistribution of laser power into higher energy pulses by means of a regenerative amplifier improves the TPEF depth penetration by two to three excitation scattering mean free paths.