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Nonlinear confocal microscopy using visible-wavelength two-photon activation of fluorescent proteins
Two-photon excitation microscopy is one of the key techniques used to observe three-dimensional (3-D) structures in biological samples. We utilized a visible-wavelength laser beam for two-photon excitation in a multifocus confocal scanning system to improve the spatial resolution and image contrast in 3-D live-cell imaging. Experimental and numerical analyses revealed that the axial resolution has improved for a wide range of pinhole sizes used for confocal detection. We observed the 3-D movements of the Golgi bodies in living HeLa cells with an imaging speed of 2 s per volume. We also confirmed that the time-lapse observation up to 8 min did not cause significant cell damage in two-photon excitation experiments using wavelengths in the visible light range. These results demonstrate that multifocus, two-photon excitation microscopy with the use of a visible wavelength can constitute a simple technique for 3-D visualization of living cells with high spatial resolution and image contrast.
This approach provides signals for both the morphology, related to the phenotype, and the intracellular molecular content at single-cell level, that we employed to study cell populations under different stimuli. In particular, we studied macrophage cells and their response to a simulated bacterial infection upon exposure to lipopolysaccharide, and show how this approach is able to noninvasively detect the activation state at single-cell level by coupling it with multivariate analysis and machine learning algorithms.
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