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 nuclear magnetic resonance 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 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 between the cytoplasm (29.5%, S.E. 1.8%) and the nucleus (57.3%, S.E. 1.0%), which is in good agreement with previous studies. This work sheds light on heterogeneous solvation in live systems using coherent Raman microscopy and opens up new avenues to explore environmental variance in complex systems with high spatiotemporal resolution.

Bringing Vibrational Imaging to Chemical Biology with Molecular Probes

“…A well-known example is that hydrogen-bounding and electrostatics can shift the peak frequencies of triple bonds, especially nitriles, an effect known as vibrational solvatochromism. By specifically mapping the peak frequency of a nitrile-bearing MARS Raman dye in live cells, the bound-water percentage in cytoplasm was revealed to be around 60%, while that in nucleus was about 30%. , Recently, the SRS peak of voltage-sensitive rhodopsin has also been shown to shift upon voltage changes …”

“…By specifically mapping the peak frequency of a nitrile-bearing MARS Raman dye in live cells, the boundwater percentage in cytoplasm was revealed to be around 60%, while that in nucleus was about 30%. 113,114 Recently, the SRS peak of voltage-sensitive rhodopsin has also been shown to shift upon voltage changes. 115 Intensity enhancement represents another less explored category of design principles for SRS sensors (Figure 8d).…”

Coherent Raman scattering microscopy: capable solution in search of a larger audience

“…Nitriles have the added benefit of a resonance frequency that is sensitive to the local electrostatics of its immediate chemical environment, allowing, for instance, local probing of water content. 51 The use of Raman tags is not unique to CRS microscopy per se. In fact, the concept was introduced to improve the molecular specificity in conventional Raman microscopy, and many of the tag-bearing small molecules now used in CRS imaging were first developed for linear Raman applications.…”

High-Resolution Low-Power Hyperspectral Line-Scan Imaging of Fast Cellular Dynamics Using Azo-Enhanced Raman Scattering Probes

“…Small-molecule Raman probes present narrow spectral bands reflecting their vibrational fingerprints and provide an attractive solution to enable dynamic Raman imaging. They are generally cell-permeable, target-specific, and with low toxicity for biomolecular imaging in living cells. − Based on the full-spectrum information, Raman probes feature greater ability for multiplex imaging that indicates more specific responses to the local chemical environment. − Since the first demonstration of Raman cellular imaging based upon 5-ethynyl-2′-deoxyuridine (EdU), many alkyne-tagged Raman probes have been developed as bio-orthogonal reporters in the cellular Raman-silent region (1800–2800 cm –1 ). ,− Additionally, some resonance Raman probes enhance the sensitivity of spontaneous Raman imaging , and stimulated Raman imaging ,− by coupling electronic and vibrational transitions. However, these Raman probes are not sensitive enough to perform high-resolution imaging of fast cellular dynamics without the requirement of coherent optics and high excitation power.…”