Raman imaging for high sensitivity detection of intracellular molecules in cryofixed biological specimens

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.