Biomedical subjects

Delphine Débarre

Publications and source records attributed to Delphine Débarre.

7 recordsLinked to original sources

PubMed2007

Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy.

The organization of collagen during fibrotic processes is poorly characterized because of the lack of appropriate methodologies. Here we show that multimodal multiphoton microscopy provides novel insights into lung fibrosis. We characterize normal and fibrotic pulmonary tissue in the bleomycin model, and show that second-harmonic generation by fibrillar collagen reveals the micrometer-scale three-dimensional spatial distribution of the fibrosis. We find that combined two-photon excited fluorescence and second-harmonic imaging of unstained lung tissue allows separating the inflammatory and fibrotic steps in this pathology, underlining characteristic features of fibroblastic foci in human Idiopathic Pulmonary Fibrosis samples. Finally, we propose phenomenological scores of lung fibrosis and we show that they unambiguously sort out control and treated mice, with a better sensitivity and reproducibility in the subpleural region. These results should be readily generalized to other organs, as an accurate method to assess extracellular matrix remodeling during fibrosis.

Animals↗

PubMed2006

Quantitative characterization of biological liquids for third-harmonic generation microscopy.

Third-harmonic generation (THG) microscopy provides images of unstained biological samples based on spatial variations in third-order nonlinear susceptibility, refractive index, and dispersion. In this study, we establish quantitative values for the third-order nonlinear susceptibilities of several solvents (water, ethanol, glycerol), physiological aqueous (ions, amino acids, polypeptides, bovine serum albumin, glucose) and lipid (triglycerides, cholesterol) solutions as a function of solute concentration in the 1.05-1.25 microm excitation range. We use these data in conjunction with imaging experiments to show that THG imaging with approximately 1.2 microm excitation lacks specificity and sensitivity to detect physiological ion concentration changes, and that nonaqueous structures such as lipid bodies provide a more robust source of signal. Finally, we illustrate the impact of index-matching liquids in THG images. These data provide a basis for interpreting biological THG images and for developing additional applications.

Animals↗

PubMed2006

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 approximately 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.

Animals↗

PubMed2006

[Second- and third-harmonic generation microscopies for the structural imaging of intact tissues].

One principal advantage of multiphoton excitation microscopy is that it preserves its three-dimensional micrometer resolution when imaging inside light-scattering samples. For that reason two-photon-excited fluorescence microscopy has become an invaluable tool for cellular imaging in intact tissue, with applications in many fields of physiology. This success has driven increasing interest in other forms of nonlinear microscopy that can provide additional information on cells and tissues, such as second- (SHG) and third- (THG) harmonic generation microscopies. In recent years, significant progress has been made in understanding the contrast mechanisms of these recent methodologies, and high-resolution imaging based on intrinsic sources of signal has been demonstrated in cells and tissues. Harmonic generation exhibits structural rather than chemical specificity and can be obtained from a variety of non-fluorescent samples. SHG is observed specifically in dense, non-centrosymmetric arrangements of polarizable molecules, such as collagen fibrils, myofilaments, and polarized microtubule bundles. SHG imaging is therefore emerging as a novel approach for studying processes such as the physiopathological remodelling of the collagen matrix and myofibrillogenesis in intact tissue. THG does not require a non-centrosymmetric system ; however no signal can be obtained from a homogeneous medium. THG imaging therefore provides maps of sub-micrometer heterogeneities (interfaces, inclusions) in unstained samples, and can be used as a general purpose structural imaging tool. Recent studies showed that this technique can be used to image embryo development in small organisms and to characterize the accumulation of large lipid bodies in specialized cells. SHG and THG microscopy both rely on femtosecond laser technology and are easily combined with two-photon microscopy.

Histological Techniques↗

PubMed2005

Structure sensitivity in third-harmonic generation microscopy.

We characterize experimentally the influence of sample structure and beam focusing on signal level in third-harmonic generation (THG) microscopy. In the case of a homogeneous spherical sample, the dependence of the signal on the size of the sphere can be controlled by modifying the Rayleigh length of the excitation beam. More generally, the influence of excitation focusing on the signal depends on sample geometry, allowing one to highlight certain structures within a complex system. We illustrate this point by focusing-based contrast modulation in THG images of Drosophila embryos.

Animals↗

PubMed2005

In vivo modulation of morphogenetic movements in Drosophila embryos with femtosecond laser pulses.

The complex biomechanical events associated with embryo development are investigated in vivo, by using femtosecond laser pulse-induced ablation combined with multimodal nonlinear microscopy. We demonstrate controlled intravital ablations preserving local cytoskeleton dynamics and resulting in the modulation of specific morphogenetic movements in nonmutant Drosophila embryos. A quantitative description of complex movements is obtained both in GFP-expressing systems by using whole-embryo two-photon microscopy and in unlabeled nontransgenic embryos by using third harmonic generation microscopy. This methodology provides insight into the issue of mechano-sensitive gene expression by revealing the correlation of in vivo tissue deformation patterns with Twist protein expression in stomodeal cells at gastrulation.

Animals↗

PubMed2004

Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos.

We demonstrate the association of third-harmonic generation (THG) microscopy and particle image velocimetry (PIV) analysis as a novel functional imaging technique for automated micrometer-scale characterization of morphogenetic movements in developing embryos. Using a combined two-photon-excited fluorescence and THG microscope, we characterize the optical properties of Drosophila embryos and show that sustained THG imaging does not perturb sensitive developmental dynamics. Velocimetric THG imaging provides a quantitative description of the dynamics of internal structures in unstained wild-type and mutant embryos.

Algorithms↗