Mapping viscosity in living cells

Abstract

Diffusion is often an important rate-determining step in chemical reactions or biological processes, and viscosity is one of the key parameters affecting transport of molecules and proteins. In biological specimen, changes in viscosity have been linked to disease and malfunction at the cellular level. Viscosity also plays a role in drug delivery and cancer therapy. While methods to measure the bulk viscosity are well developed, they require macroscopic sample quantities and use mechanical or fluid-dynamics approaches.1 However, microscale measurements remain a challenge. Until recently, viscosity maps of single cells have been hard to obtain. Optical techniques are powerful tools for studying biological samples: since they are nondestructive and minimally invasive, they can be used with living cells and tissues.2, 3 Measurements can be made in situ, thus allowing access to biological function within a true physiological context. Fluorescence-imaging techniques, in particular, are widely used because of their high contrast and easy visualization of proteins and their cellular environment. The relevant detection sensitivity extends down to the single-molecule level. Moreover, fluorescence can be characterized by multiple parameters, including intensity, wavelength, lifetime, and polarization. In particular, polarization-resolved lifetime measurements allow scientists to determine the rotational mobility of fluorophores.4 This approach depends on the viscosity of their surroundings. Thus, polarization-resolved fluorescence lifetime imaging (FLIM) or time-resolved fluorescence anisotropy imaging (TR-FAIM) can be used to map viscosity.5, 6 We recently demonstrated the feasibility of using nanosecond FLIM of molecular rotors to image microviscosity in living cells.7–9 Their structures are shown in Figure 1. Molecular rotors have a fluorescence lifetime that varies with the viscosity of their micro-environment.10 In contrast to TR-FAIM, FLIM requires no polarization-resolved detection of the fluorescence emission (or polarized excitation). The fluorescence lifetime can directly be converted into a viscosity with a calibration based on the Forster Hoffmann model.9 Figure 1. (a) and (b) Boron dipyrromethene (bodipy)9 and (c) porphyrin-based8 molecular rotors.