First demonstration: 7-color separation in mouse adipose tissue using single 800 nm multiphoton laser. Real-time spectral unmixing enables efficient, simultaneous imaging of multiple fluorophores plus collagen, simplifying multicolor microscopy; works on standard ZEISS LSM platforms.
A single track for 7 color separation using only one multiphotonic beam laser excitation Segmentation des noyaux cellulaires basée sur des méthodes conventionnelles d’analyse d’images Figure 4 - 7 colors separation using 800 nm biphotonic wave lenght excitation in mouse adiopose tissue. (A) Oneline fingers printing images, (B) Image of autofluorescence, (C) Merge images. Figure 4 - 7 colors separation using 800 nm biphotonic wave lenght excitation in mouse adiopose tissue. (A) Oneline fingers printing images, (B) Image of autofluorescence, (C) Merge images. 4. A single track for 7 color separation using only one multiphotonic beam laser excitation To ensure optimal signal quality and fluorochrome separation, staining conditions had to be carefully optimized. This was necessary for two main reasons. Although both fluorochromes could be excited at the same wavelength, their excitation efficiency varied. Furthermore, the use of a single acquisition track prevented individual adjustment of imaging parameters for each fluorochrome. We used antibodies coupled to fluorochromes illustrated in figure1 to visualize the main cellular compartments in adipose tissue in a single acquisition. Figure 4A presents each unmixed fluorescence images acquired using a single acquisition track with two-photon excitation at 800 nm. The images display the distinct signals of each fluorochrome following real-time spectral unmixing. Figure 4B shows the merged images combining the spectrally unmixed signals from each fluorochrome. This overlay allows visualization of the spatial distribution and potential interactions between the labeled cell populations, demonstrating the effectiveness of the spectral unmixing and single- wavelength two-photon excitation approach in resolving closely overlapping fluorochromes within complex tissue samples.
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