Advanced nonlinear optical (NLO) microscopy enables quantitative in situ mapping of parchment degradation at the micrometer scale. We show that measurements of the ratio of fluorescence over second harmonic generation (SHG) signals probes severe collagen degradation and could help to identify the parchments most at risk, close to the irreversible collagen denaturation into gelatin. Conversely, the anisotropy parameter obtained from polarization-resolved SHG measurements probes the earlier stages of degradation. This approach is first validated by comparing NLO quantitative parameters to thermal measurements by differential scanning calorimetry on artificially altered contemporary parchments. We then analyze invaluable parchments from the Middle Ages.
Large-scale microscopy approaches are transforming brain imaging, but currently lack efficient multicolor contrast modalities. We address this issue by introducing chromatic multiphoton serial (ChroMS) microscopy, a method combining multicolor multiphoton excitation through wavelength mixing and microtome-assisted serial block-face image acquisition. This approach delivers large-scale micrometric imaging of spectrally distinct fluorescent proteins with constant micrometer-scale resolution and sub-micron channel registration over the entire imaged volume. We achieve multicolor 3D imaging over several cubic millimeters and brain-wide serial 2D multicolor imaging. We illustrate the potential of this method for several novel types of measurements interesting for region-scale or whole brain studies: (i) color-based analysis of astrocyte morphology and spatial interactions in the mouse cerebral cortex, (ii) tracing of densely labeled neurons, and (iii) brain-wide mapping of axonal projections labeled with distinct tracers.
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