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Hyperspectral stimulated Raman scattering (SRS) imaging has rapidly become an emerging tool for high content analyses of cell and tissue systems. The label-free nature of SRS imaging combined with its chemical specificity allows in situ and in vivo biochemical quantification at submicrometer resolution without sectioning and staining. Current hyperspectral SRS data analysis methods are based on either linear unmixing or multivariate analysis, which are not sensitive to small spectral variations and often provide obscure information on the cell composition. Here, we demonstrate a spectral phasor analysis method that allows fast and reliable cellular organelle segmentation of mammalian cells, without any a priori knowledge of their composition or basis spectra. We further show that, in combination with a branch-bound algorithm for optimal selection of a few wavenumbers, spectral phasor analysis provides a robust solution to label-free single cell analysis., Chemistry and Chemical Biology

We introduce a two-dimensional (2D) multisurface reaction free energy description of the catalytic cycle that explicitly connects the recently observed multi-time-scale conformational dynamics as well as dispersed enzymatic kinetics to the classical Michaelis-Menten equation. A slow conformational motion on a collective enzyme coordinate Q facilitates the catalytic reaction along the intrinsic reaction coordinate X, providing a dynamic realization of Pauling’s well-known idea of transition-state stabilization. The catalytic cycle is modeled as transitions between multiple displaced harmonic wells in the XQ space representing different states of the cycle, which is constructed according to the free energy driving force of the cycle. Subsequent to substrate association with the enzyme, the enzyme-substrate complex under strain exhibits a nonequilibrium relaxation toward a new conformation that lowers the activation energy of the reaction, as first proposed by Haldane. The chemical reaction in X is thus enslaved to the down hill slow motion on the Q surface. One consequence of the present theory is that, in spite of the existence of dispersive kinetics, the Michaelis-Menten expression of the catalysis rate remains valid under certain conditions, as observed in recent single-molecule experiments. This dynamic theory builds the relationship between the protein conformational dynamics and the enzymatic reaction kinetics and offers a unified description of enzyme fluctuation-assisted catalysis., Chemistry and Chemical Biology