Multimodal Hyperspectral Imaging Unveils Pigment Rearrangement in the Diatom Cyclotella meneghiniana under Changing Physicochemical Conditions

Abstract Revealing the spatial organization and stress-induced rearrangements of the photosynthetic machinery in thylakoids of diatoms is crucial for understanding their metabolic responses and/or adaptation. Using a multimodal framework that combines coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence, and confocal fluorescence microscopy with machine learning, we investigated the spatial pigment distribution in thylakoids of Cyclotella meneghiniana. Under physiological conditions, we observed dense central packing of fucoxanthins (Fx’s). In contrast, the peripheral girdle lamella had a lower packing of mostly red-absorbing Fx accompanied by enhanced 710–750 nm chlorophyll (Chl) emission. Glycerol treatment induced thylakoid disintegration, which was reflected in decreased resonance-to-nonresonance CARS ratios (Fx disordering), emergence of a 690–700 nm fluorescence component, and 680 nm band narrowing (possibly fucoxanthin-chlorophyll protein (FCP) aggregation), as well as 640 nm emission (Chl c release). Our findings show that this multimodal framework provides a powerful, label-free tool for mapping the photosynthetic machinery and subcellular metabolic responses.