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

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 <i>Cyclotella meneghiniana</i>.

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 <i>c</i> release).