Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging

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Raman microscopy is an emerging molecular imaging technology, yet its signal-to-noise-ratio (SNR) in measurements of biological specimens is severely limited due to the small cross-section of Raman scattering. Here, we present Raman imaging techniques of cryofixed specimens to overcome SNR limitations by enabling long exposure of specimens under highly stabilized low-temperature conditions. The observation of frozen specimens in a cryostat at a constant low temperature immediately after rapid freezing enabled the improvement of SNR and significantly enhanced spatial and spectral resolution. We also confirmed that the cryofixation can preserve physicochemical states of specimens by observing alkyne-labeled coenzyme Q in cytosol and hemeproteins in acute ischemic myocardium, which cannot be done by fixation using chemical reagents. Finally, we applied the technique for multiplex Raman imaging of label-free endogenous molecules and alkyne-tagged molecules in cryofixed HeLa cells, demonstrating its capability of high-content imaging of complex biological phenomena while maintaining physiological conditions.

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  1. Exposure times were 5 s/line and 30 s/line for imaging the live and cryofixed samples, respectively.

    How is this a fair comparison for comparing SNR between live and cryofixed conditions? I understand your point that exposure time for live Raman is limited due to photodamage, but would still be interesting to see 30s/line for live Raman (albeit with drift artifacts) for the sake of comparison.

  2. We confirmed the band width of 3.9 cm-1 at 1001 cm-1, assigned as phenylalanine, in the cryofixed conditions, whereas the same peak was observed to be 8.6 cm-1 under a practical Raman measurement condition at room temperature (Fig. 1F).

    This is a nice example of benchmarks for band widths for certain spectral peaks to differentiate between low and high SNR!

  3. high-SNR Raman imaging

    Can you more clearly define what constitutes "high-SNR" Raman? Of course it is high relative to "low-SNR" Raman, but how can someone know if they are doing high- vs low-SNR Raman? Are there thresholds that are (or can be) defined on the width of certain spectral peaks?

  4. fused silica coverslip

    Is fused silica necessary to prevent autofluorescence from surface defects in normal glass coverslips? Is it typical/recommended to used fused silica for Raman microscopy generally or only for very high sensitivity measurements?

  5. Additionally, the utility of the chemical fixation is limited in fixing biological activities in motion [24], and there are molecular species that cannot be fixed by the current chemical fixation techniques [25].

    Would be helpful to cite a couple specific examples of biological activities and/or molecular species for which fixation is insufficient to bolster motivation for cryogenic freezing.