Optical mapping of biological water in single live cells by stimulated Raman excited fluorescence microscopy

Abstract

Water is arguably the most common and yet least understood material on Earth. Indeed, the biophysical behavior of water in crowded intracellular milieu is a long-debated issue. Understanding of the spatial and compositional heterogeneity of water inside cells remains elusive, largely due to a lack of proper water-sensing tools with high sensitivity and spatial resolution. Recently, stimulated Raman excited fluorescence (SREF) microscopy was reported as the most sensitive vibrational imaging in the optical far field. Herein we develop SREF into a water-sensing tool by coupling it with vibrational solvatochromism. This technique allows us to directly visualize spatially-resolved distribution of water states inside single mammalian cells. Qualitatively, our result supports the concept of biological water and reveals intracellular water heterogeneity between nucleus and cytoplasm. Quantitatively, we unveil a compositional map of the water pool inside living cells. Hence we hope SREF will be a promising tool to study intracellular water and its relationship with cellular activities.

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

“…The distinct solvation variance between the cytoplasm and nucleus observed here is in substantial agreement with prior studies (both SREF 47 and NMR microscopy 10 ). Taken together, these results provide new insights into the solvation heterogeneity in different cellular compartments.…”

“…At the time of the submission of this work, Min and co-workers reported the use of stimulated Raman enhanced fluorescence (SREF) as a method to apply the "nitrile shift" idea to probe the intracellular water heterogeneity. 47 SREF uses a fluorescence readout rather than directly measuring the SRS signal, but the principle is the same. They measured a distinct difference in the solvation between the cytosol and nucleus [36% (cytosol) vs. 65% (nucleus), compared to 29.5 ± 1.8 % (cytosol) free water content in the cytoplasm and 57.3 ± 1.0 % (nucleus) in the current work].…”

“…46 In this paper, we demonstrate that the combination of SRS and a near-resonant probe can extend the sensing capability of nitriles to probe solvation and Hbonding characteristics in live cells with sub-micron resolution. Recent work by Min and coworkers 47 have shown that a SRS-modulated fluorescence technique can be used to measure solvation heterogeneity in living cells. Here we demonstrate that a direct vibrational measurement can be similarly applied, potentially reducing the reliance on a fluorescent label and dramatically increasing the application scope of this novel approach.…”

Super-resolution vibrational microscopy by stimulated Raman excited fluorescence

“…Most recently, we have developed a ultrasensitive vibrational spectroscopy called stimulated Raman excited fluorescence (SREF) 20 , 21 , which enables all-far-field Raman spectroscopy with sensitivity down to the single-molecule level for the first time. SREF spectroscopy has found exciting applications in biophotonics, biophysics, and materials science where the exquisite vibrational specificity and sensitivity are harnessed for advanced sensing or super-multiplexed imaging 20 , 22 , 23 . In the current work, we leveraged the high chemical specificity and detection sensitivity of SREF spectroscopy and exploited its potential for high-contrast super-resolution vibrational imaging.…”

Label‐Free Imaging of Intracellular Temperature by Using the O−H Stretching Raman Band of Water

“…In this study, we propose a label-free method for evaluating and visualizing both intracellular and extracellular temperatures simultaneously using the Raman imaging of water inside a cell. Raman microscopy is one of the most powerful methods for observing living systems, [13][14][15][16][17][18][19][20][21][22][23] and we pay attention to the fact the shape of the O À H stretching Raman band of water changes in accordance with temperature. [24][25][26] We expected that a calibration curve obtained from the shape of the OÀH stretching band and the temperature at each intracellular or extracellular region would enable temperature measurements of various regions.…”