Dynamics of mitochondrial membranes under photo-oxidative stress with high spatiotemporal resolution.

In our study, we harnessed an original Enhanced Speed Structured Illumination Microscopy (Fast-SIM) imaging setup to explore the dynamics of mitochondrial and inner membrane ultrastructure under specific photo-oxidation stress induced by Chlorin-e6 and light irradiation. Notably, our Fast-SIM system allowed us to observe and quantify a distinct remodeling and shortening of the mitochondrial structure after 60-80 s of irradiation. These changes were accompanied by fusion events of adjacent inner membrane cristae and global swelling of the organelle. Preceding these alterations, a larger sequence was characterized by heightened dynamics within the mitochondrial network, featuring events such as mitochondrial fission, rapid formation of tubular prolongations, and fluctuations in cristae structure. Our findings provide compelling evidence that, among enhanced-resolution microscopy techniques, Fast-SIM emerges as the most suitable approach for non-invasive dynamic studies of mitochondrial structure in living cells. For the first time, this approach allows quantitative and qualitative characterization of successive steps in the photo-induced oxidation process with sufficient spatial and temporal resolution.

Controlling the optical pathlength in continuous-wave reflectance spectroscopy using polarization.

We investigate potential improvements of continuous-wave diffuse reflectance spectroscopy within highly scattering media by employing polarization gating. Simulations are used to show the extent at which the effective optical pathlength varies in a typical scattering medium as a function of the optical wavelength, the total level of absorption, and the selected polarization channels, including elliptical and circular polarization channels. Experiments then demonstrate that a wavelength dependent polarization gating scheme may reduce the prior knowledge required to solve the problem of chromophore quantification. This is achieved by finding combinations of polarization channels which have similar effective optical pathlengths through the medium at each wavelength.

Enhanced contrast and depth resolution in polarization imaging using elliptically polarized light.

Polarization gating is a popular and widely used technique in biomedical optics to sense superficial tissues (colinear detection), deeper volumes (crosslinear detection), and also selectively probe subsuperficial volumes (using elliptically polarized light). As opposed to the conventional linearly polarized illumination, we propose a new protocol of polarization gating that combines coelliptical and counter-elliptical measurements to selectively enhance the contrast of the images. This new method of eliminating multiple-scattered components from the images shows that it is possible to retrieve a greater signal and a better contrast for subsurface structures. In vivo experiments were performed on skin abnormalities of volunteers to confirm the results of the subtraction method and access subsurface information.

Maintenance of Stemlike Glioma Cells and Microglia in an Organotypic Glioma Slice Model.

BACKGROUND: The therapeutic resistance of gliomas is, at least in part, due to stemlike glioma cells (SLGCs), which self-renew, generate the bulk of tumor cells, and sustain tumor growth. SLGCs from glioblastomas (GB) have been studied in cell cultures or mouse models, whereas little is known about SLGCs from lower grade gliomas. OBJECTIVE: To compare cell and organotypic slice cultures from GBs and lower grade gliomas and study the maintenance of SLGCs. METHODS: Cells and tissue slices from astrocytomas, oligodendrogliomas, oligoastrocytomas, and GBs were cultivated in (1) serum-free medium supplemented with the growth factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), (2) medium containing 10% serum plus EGF and bFGF (F+GF medium), or (3) medium containing 10% fetal calf serum (F medium). Maintenance of cells and cytoarchitecture was addressed, using several candidate SLGC markers (Nestin, Sox2, CD133, CD44, CD49f/integrin α6, and Notch) as well as CD31 (endothelial cells), ionized calcium-binding adapter molecule 1 (microglia), and vimentin. Cell vitality was determined. RESULTS: SLGCs were present in tissue slices from lower and higher grade gliomas. Preservation of the cytoarchitecture in slices was possible for >3 weeks. Maintenance of SLGCs required the presence of EGF/bFGF in cell and slice cultures, in which F+GF appeared superior to N medium. Constraints were observed regarding the preservation of the microglia but not of the endothelial cells. Maintenance of the microglia was improved by addition of the cytokine macrophage colony-stimulating factor. CONCLUSION: Medium supplemented with serum and growth factors EGF, bFGF, and macrophage colony-stimulating factor permits the preservation of SLGCs and non-SLGCs in the original glioma microenvironment.