Mapping Solvation Heterogeneity in Live Cells by Hyperspectral Stimulated Raman Scattering Microscopy

Abstract

Water provides a dynamic matrix in which all biochemical processes occur in living organisms. The structure and dynamics of intracellular water constitute the cornerstone for understanding all aspects of cellular function. Fundamentally, direct visualization of subcellular solvation heterogeneity is essential but remains challenging with commonly used NMR methods due to poor spatial resolution. To explore this question, we demonstrate a vibrational-shift imaging approach by combining the spectral-focusing hyperspectral stimulated Raman scattering (hsSRS) technique with an environmentally-sensitive nitrile probe. The sensing ability of a near-infrared nitrile-containing molecule is validated in the solution phase, microscopic droplets and cellular environments. Finally, we quantitatively measure the subcellular solvation variance in the degree of free water content between the cytoplasm (29.7%, S.E. 1.1%) and the nucleus (56.4%, S.E. 1.3%), which is in good agreement with previous studies. This work sheds light on mapping the heterogenous solvation in live systems using coherent Raman microscopy and opens up new avenues to explore environmental variance in complex systems with high spatiotemporal resolution.

Analysis on Transmission Characteristics of Stimulated Raman Scattering Based on the Multi-Sensor Signal Enhancement Technique

“…In addition, studies have shown that ASE can also act as Raman seed light to cause Raman amplification, leading to a reduction in the SRS threshold. Lang et al [33] investigated the effect of Raman scattering noise in the seed light on the SRS and gave a formula for calculating the Raman threshold of high-power amplifier when considering Raman scattering noise in the seed light, showing that when the Raman scattering noise in the seed light is greater than 10-8 W, it plays an important role in the Raman threshold of the high-power amplifier.…”

Visualization of a Limonene Synthesis Metabolon Inside Living Bacteria by Hyperspectral SRS Microscopy

“…Compared to previous study of limonene biosynthesis detection by single-color SRS (25,26), hSRS allows simultaneous quantification of multiple chemicals inside one specimen by recording a Raman spectrum at each pixel. Intracellular compositions of various biological specimens have been studied using hSRS, ranging from tissues (27,28), to cancerous cells (29,30), to bacteria (31,32). Many of these applications employ the C-H stretching region (2800 -3100 cm -1 ) for its strong SRS signal or the C-D stretching region (2070 -2300 cm -1 ) for selective imaging of Raman tags.…”