Using complementary optical microscopy techniques provides more detailed insight into biological samples. However, misinterpretation can occur by temporal discrepancies due to differences in temporal resolution and switching imaging modalities. Here, we demonstrated multimodal imaging of cryofixed cells using Raman and fluorescence structured illumination microscopy (SIM). Cryofixation preserves structures and chemical states of samples in their near-native states, allowing multimodal imaging without artifacts caused by temporal discrepancy. We demonstrated multimodal imaging of cryofixed HeLa cells stained with an actin probe, where Raman microscope visualized cytochromes, proteins and lipids, and SIM visualized fluorescence-labelled actin filaments.
We investigated the photophysical property of Yellow Cameleon 3.60 (YC3.60), a fluorescent calcium-ion (Ca2+) indicator based on Förster resonance energy transfer (FRET), under cryogenic conditions. By measuring the fluorescence intensity ratio of the donor and accepter at various Ca2+ concentrations under room and cryogenic temperatures, we confirmed that YC3.60 exhibits a Ca2+-dependent FRET efficiency. Although slight differences were observed in the fluorescence lifetime and spectral shape at the cryogenic temperature, which can affect the FRET efficiency, our measurement suggested that YC3.60 can be employed for quantitative Ca2+ measurement and imaging under cryogenic conditions with improved photostability and quantum yield.
We propose the use of visible-wavelength two-photon excitation (v2PE) for activation of reversibly photo-switchable fluorescent proteins (RSFPs) and successive confocal detection to achieve super-resolution imaging. In this method, three photons interact with the sample molecules in total, which provides imaging properties equivalent to using third-order nonlinearity in fluorescence response. Because this technique uses visible light, it can achieve higher spatial resolution than confocal microscopy. In this study, we performed experimental investigations to confirm the activation of negative RSFPs by v2PE and demonstrated super-resolution imaging of live cells.
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