High-Resolution Intracellular Viscosity Measurements using Time-Dependent Fluorescence Anisotropy

Abstract

A characteristic of living cells is that they continuously respond to changes in their environment. Our ability to observe and measure these responses on micro- and nanoscale levels gives us insight into the internal organization of the cell, and allows us to formulate a more complete model of cell physiology. We have developed a technique for makinghigh-resolution, sub-micron measurements of intracellular viscosity in vivo. A low-cost pulsed laser is used in conjunction with a homebuilt confocal laser-scanning epifluorescence microscope with submicron lateral and axial spatial resolution to measure fluorescence anisotropy at specific locations within a mouse J774 microphage cell. Global deconvolution techniques are used to determine rotational correlation times for fluorophores in those locations. In order to effectively determine the quantitative viscosity of the selected intracellular region, we first measure molecular rotational correlation times of our chosen fluorophore (HPTS, or pyranine) in known viscosity solutions of trehalose in water. We then construct a calibration curve relating the rotational behavior of the fluorophore to viscosity. This calibration curve is used to generate quantitative viscosity measurements for the measured intracellular rotational correlation times. The data show that local viscosities within the cell are not uniform. In the cytoplasmic areas measured, rotational correlation times of HPTS ranged from 0.144 ns to 0.320 ns, and viscosities ranged from 1.00 to 2.21 cP. We will compare the use of time-dependent fluorescence anisotropy with fluorescence correlation spectroscopy techniques used to determine intercellular viscosity, and identify the conditions under which each technique is most beneficial.