A novel fluorescence ratiometric method confirms the low solvent viscosity of the cytoplasm

Abstract

Two homologous indocyanine dyes, Cy3.18 and Cy5.18, can be used as a ratio pair for fluorometric determination of solvent viscosity. Succinimidyl ester derivatives of these dyes can be attached to inert carrier macromolecules, such as Ficoll 70, for measurement of intracellular or intravesicular solvent viscosity. When the viscosity of the solvent was varied by various methods, the fluorescence intensity ratio (Cy3/Cy5) in a mixture of Cy3.18-Ficoll 70 (Cy3F70) and Cy5.18-Ficoll 70 (Cy5F70) in solution was found to be solely a function of solvent viscosity and was insensitive to other solvent parameters such as dielectric constant, temperature, and the ability of the solvent to form hydrogen bonds. Most important, it was insensitive to the presence of large macromolecules, such as proteins, which increase the shear viscosity but have little effect on solvent viscosity. Following microinjection into the cytoplasm of living tissue culture cells, no binding of Cy3F70 or Cy5F70 to intracellular components was detected by fluorescence recovery after photobleaching. Fluorescence intensity ratio imaging of Cy3F70 and Cy5F70 in non-motile interphase CV1 and PtK1 cells showed that the solvent viscosity of cytoplasm was not significantly different from water and showed no spatial variation.

Brownian motion of polyphosphate complexes in yeast vacuoles: characterization by fluorescence microscopy with image analysis

“…Most methods for intracellular viscosity measurements are based on the use of fluorescent (Luby-Phelps et al, 1993;Bicknese et al, 1993) or spin probes (Morse, 1986). Loading of live (unfixed) yeast cells, and specifically their vacuoles, with the probes remains a problem as yet.…”

Imaging intracellular viscosity of a single cell during photoinduced cell death

“…8 demonstrated that the local microviscosity can be as high as 140 cP, while the microviscosity in the aqueous phase of the cellular cytoplasm is several cP, similar to that of pure water. [9][10][11] Such large viscosity variations within a cell can influence diffusion and bimolecular reaction rates, 12 and need to be considered, for example, while developing strategies for drug delivery and cancer therapy. Fluorescent molecular rotors, in which the non-radiative decay of an excited state can be altered by the ambient viscosity, have emerged as a new modality for measuring the microviscosity in a biological environment.…”

Size-dependent DNA Mobility in Cytoplasm and Nucleus

“…Analysis of the individual factors slowing solute diffusion, including fluid-phase viscosity, binding, and collisional interactions, indicated that the principal barrier for diffusion of small solutes was collisional interactions due to macromolecular crowding (8). The "fluid-phase" viscosity of cytoplasm and nucleus, defined as the viscosity sensed by a small probe that does not interact with cellular components, was determined by time-resolved anisotropy (10) and ratio imaging of a viscosity-sensitive fluorescent probe (11) to be only 1.2-1.4 times greater than the viscosity of water. The translational diffusion of larger, macromolecule-sized solutes (FITC 1 -labeled dextrans and Ficolls) in cytoplasm and nucleus was only 3-4-fold slower than in water for solutes Ͻ500 -750 kDa (12) but was markedly slowed for larger solutes (11,12).…”

“…The "fluid-phase" viscosity of cytoplasm and nucleus, defined as the viscosity sensed by a small probe that does not interact with cellular components, was determined by time-resolved anisotropy (10) and ratio imaging of a viscosity-sensitive fluorescent probe (11) to be only 1.2-1.4 times greater than the viscosity of water. The translational diffusion of larger, macromolecule-sized solutes (FITC 1 -labeled dextrans and Ficolls) in cytoplasm and nucleus was only 3-4-fold slower than in water for solutes Ͻ500 -750 kDa (12) but was markedly slowed for larger solutes (11,12). The diffusional mobilities of targeted green fluorescent protein chimeras have been measured recently in the aqueous phase of cytoplasm (13), mitochondria (14), and endoplasmic reticulum (15).…”