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Biomedical Optics & Medical Imaging
Mapping viscosity in living cells
Fluorescent probes enable nondestructive, in situ microscale viscosity measurements.
7 August 2009, SPIE Newsroom. DOI: 10.1117/2.1200907.1686
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 Förster Hoffmann model.9
(a) and (b) Boron dipyrromethene (bodipy)9
and (c) porphyrin-based8
The fluorescence lifetime of molecular rotors, τf, is a function of viscosity, η,
where kr is the radiative rate constant and z and α are constants.
Measurements from molecular rotors made in methanol-glycerol mixtures of different viscosities show that the fluorescence quantum yield rises dramatically with increasing solvent viscosity: see Figure 2(a). For boron dipyrromethene (bodipy)-based rotors, the fluorescence lifetime also increases with viscosity,7 while for porphyrin-based ones the fluorescence spectrum varies with the medium's viscosity.8,11 The data obtained can be used for a calibration graph using this equation, allowing conversion of fluorescence lifetime, or peak ratio, into viscosity (see Figure 3).7–9
(a) Fluorescence intensity and (b) fluorescence lifetime of bodipy-based molecular rotors7,9
and (c) fluorescence spectra of the porphyrin-based molecular rotor as a function of the medium's viscosity.8,11
Calibration graphs. (a) Logarithm of the fluorescence lifetime, τ, versus logarithm of the viscosity, η, of the medium for bodipy-based molecular rotors.7,9
(b) Logarithm of the 710/780nm emission-peak ratio versus logarithm of the medium's viscosity for porphyrin-based molecular rotors.8,11
We incubated bodipy in cells and used FLIM to determine the viscosity. The confocal fluorescence image—shown in Figure 4(a)—clearly reveals intracellular uptake of the molecular rotor with punctate-dye distribution.9 The FLIM image, obtained using excitation with a pulsed diode laser at 467nm, shows a narrow lifetime distribution between 1.4 and 1.8ns. To ensure that this high viscosity value does not result from the binding of the rotor to the intracellular targets—which could restrict the rotation of the phenyl group—we also performed TR-FRAIM of bodipy in cells. We found that the rotational correlation time, θ, was on average 1.1ns, corresponding to an intracellular viscosity of approximately 80cP (the same order of magnitude as that given by FLIM7,9).
Bodipy-based molecular rotors in cells.9
(a) The fluorescence intensity image shows a punctate and continuous distribution of the rotors. (b) The fluorescence lifetime image shows a short lifetime for the punctate distribution (yellow, 1.4–1.85ns), and a longer one for the continuous distribution (blue, 1.85–2.2ns).
Figure 5 shows the new type of ratiometric fluorescent molecular rotor based on porphyrin dyes in cells. It is suitable for imaging intracellular viscosity during cell death by photosensitized singlet oxygen. We have also demonstrated that such a viscosity increase alters diffusion-dependent cell kinetics through changes in the photosensitized production and subsequent decay of the cytotoxic-species singlet oxygen.8,12
Porphyrin-based molecular rotors monitor cell death.8
Ratiometric images demonstrate that the intracellular viscosity increases during irradiation from (a) the initial to (b) the final stage.
The bodipy dyes can reveal microviscosity through variations in their fluorescence lifetimes. Their fluorescence properties and high cellular uptake make them ideal candidates for studies in biological systems. Our measurements using fluorescent molecular rotors confirm viscosity heterogeneity in the stained regions. This result highlights the importance of spatially resolved microscopic-scale measurements in biological environments. The new type of ratiometric fluorescent molecular rotor (based on porphyrin dyes) can also be used for quantifying and imaging intracellular viscosity in live cells. The rotor enables real-time monitoring of dynamic processes. This has been illustrated by quantifying a significant increase in intracellular viscosity during photo-induced cell death. By tailoring the chemistry and delivery method of the rotor molecules, it may be possible to create microviscosity maps of a wide range of intracellular environments and targets on the basis of fluorescence lifetimes measured by FLIM, or ratiometric spectral imaging. In summary, we have developed a practical and versatile approach to measuring the microviscosity of the environment of molecular rotors in cells.
Department of Physics
Kings College London
Klaus Suhling is a reader.
Department of Chemistry
Imperial College London
Marina Kuimova is an Engineering and Physical Sciences Research Council life sciences interface fellow.
5. J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, P. M. W. French, Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): imaging the rotational mobility of a fluorophore, Rev. Sci. Instrum. 74, pp. 182-192, 2003.
6. K. Suhling, J. Siegel, P. M. P. Lanigan, S. Lévêque-Fort, S. E. D. Webb, D. Phillips, D. M. Davis, P. M. W. French, Time-resolved fluorescence anisotropy imaging applied to live cells, Opt. Lett. 29, pp. 584-586, 2004.
8. M. K. Kuimova, S. W. Botchway, A. W. Parker, M. Balaz, H. A. Collins, H. L. Anderson, K. Suhling, P. R. Ogilby, Imaging intracellular viscosity of a single cell during photoinduced cell death, Nat. Chem. 1, pp. 69-73, 2009.
11. M. K. Kuimova, H. Collins, M. Balaz, E. Dahlstedt, J. Levitt, N. Sergent, K. Suhling, M. Drobizhev, N. Makarov, A. Rebane, Photophysical properties and intracellular imaging of water-soluble porphyrin dimers for two-photon excited photodynamic therapy, Org. Biomol. Chem. 7, pp. 889-896, 2009.