Real-time visualization of intracellular hydrodynamics in single living cells

Abstract

Intracellular water concentrations in single living cells were visualized by nonlinear coherent anti-Stokes Raman scattering (CARS) microscopy. In combination with isotopic exchange measurements, CARS microscopy allowed the real-time observation of transient intracellular hydrodynamics at a high spatial resolution. Studies of the hydrodynamics in the microorganism Dictyostelium discoideum indicated the presence of a microscopic region near the plasma membrane where the mobility of water molecules is severely restricted. Modeling the transient hydrodynamics eventuated in the determination of cell-specific cytosolic diffusion and plasma membrane permeability constants. Our experiments demonstrate that CARS microscopy offers an invaluable tool for probing single-cell water dynamics.

Biological imaging with coherent Raman scattering microscopy: a tutorial

“…9). This approach has been widely used in high-speed CRS microscopy to rapidly identify the compound of interest in tissues and cells, including flowing water, 28 phospholipids, 101,102 neutral lipids, 103 and drug compounds. 100,104 Recently, the unique vibrational contrast of isotope-labeled compounds has also been employed to visualize de novo synthesis of macromolecules in cells by feeding them small deuterated precursor molecules that become permanently incorporated in newly synthesized biomolecular components.…”

“…Examples include studying the health of the nervous system through CRS imaging of myelin, [16][17][18][19][20] following lipid metabolism in living organisms, 21,22 mapping cholesterol content in atherosclerotic plaques, [23][24][25][26][27] and recording water diffusion in cells and tissues. 28,29 In this tutorial, we highlight some of the most important imaging properties of CRS microscopy. We explain the basics of CRS in a simple yet intuitive way to emphasize the unique character of this technique compared to related imaging methods.…”

CARS imaging with a single laser pulse

“…5,6 For that reason it is desirable to generate the CARS signal of Raman active structures within the sample whereby the signal from the solvent remains suppressed. Unfortunately the solvent always generates a non-resonant CARS background which often overwhelms the CARS signal generated from small structures within the solvent.…”

New methods in femtosecond multiphoton microscopy

“…This relatively large size poses a problem when attempting specific labeling with nanoparticles, since labeling specificity decreases with particle size (due to steric hindrance of the nanoparticle to the conjugated antibody). This problem can be addressed by aggregating [10][11][12][13][14][15][16][17][18][19][20] or by chemical enlargement of small particles by silver enhancement, where silver ions in solution nucleate around gold particles and precipitate as silver metal. In this way, few nanometer sized particles may be enlarged up to lOOnm in size.…”