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.
Raman microscopy provides a variety of insights into molecular composition, chemical state, and environmental conditions in biological samples. However, biological imaging with Raman microscopy have faced challenges such a low signal-to-noise ratio, mainly due to the low scattering efficiency of Raman scattering. To overcome this limitation, we developed a cryo-Raman microscope integrated with a cryostat capable of rapid freezing of biological samples and low-temperature Raman imaging. The spatiotemporal cryofixation of biological samples allows long exposure measurements to accumulate signals without photodamage. We observed both reduction of photobleaching in resonant Raman scattering of cytochromes in cryofixed HeLa cells, and the preservation of redox states of cytochromes in rat heart tissue by cryofixation.
Raman microscopy enables us to obtain molecular information in biological samples but has suffered from low signal-to-noise ratio (SNR) due to low of Raman scattering cross-section. Here we developed a cryo-stat equipped Raman microscope for low temperature measurement, allowing long time accumulation of Raman signals. We confirmed the SNR improvement in Raman imaging of cryofixed HeLa cells without photodamage under long time observation at low temperature. The reduction of photobleaching in resonant Raman scattering of carotenoid and cytochrome significantly increases the SNR, demonstrated by 7-color high SNR Raman imaging with multiple Raman tags, including EdU, MitoBADY, and alkyne-tagged Coenzyme Q (AltQ2). AltQ2 is a mobile small molecule that cannot be fixed by chemical fixation.
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