Title 2-PHOTON FLUORESCENCE CORRELATION SPECTROSCOPY-METHOD AND APPLICATION TO THE INTRACELLULAR ENVIRONMENT Permalink

We report on the application of two photon molecular excitation to fluorescence correlation spectroscopy. We demonstrate the first fluorescence correlation spectroscopy measurements of translational mobility in the cytoplasm of living cells. Two-photon excitation inherently excites small sample volumes in three dimensions, providing depth discrimination similar to confocal microscopy, without emission pinholes. We demonstrated accurate measurements of the diffusion constant, D, for particles of several different known sizes, in bulk solutions of different viscosity. We then showed measurements of translational diffusion for 7and 15-nm radius latex beads in the cytoplasm of mouse fibroblast cells. We measured time-dependent diffusion coefficients. When first injected in the cells, the spheres moved from two to five times slower than in water, with average rates of 18 x 10-8 cm2/s for the 7 nm and 5 x 10-8 cm2/s for the 15 nm radius spheres. After a few hours, spheres stick to the cells, and the motion slows down 10 to 100 times. INTRODUCTION Since its introduction more than 20 years ago, fluorescence correlation spectroscopy (FCS) has had proposed applications in many fields of biophysical investigation (Elson and Magde, 1974; Elson et al., 1974). Fluorescence fluctuation is an attractive method because of the high sensitivity and spectral selectivity one can achieve with fluorescence measurements. With optimized optical systems, one can detect single molecules (Eigen and Rigler, 1994; Rigler et al., 1992). FCS has been applied to measuring translational diffusion, rotational diffusion, flow, and chemical reactions (Elson and Magde, 1974; Elson et al., 1974; Magde et al., 1978; Elson, 1985; Icenogle and Elson, 1983a, b). Applications have been proposed in membrane dynamics, polymer dynamics, and many other areas (Icenogle and Elson, 1983; Fahey and Webb, 1978; Borejdo, 1979). Much work has also been done to realize the FCS potential of monitoring aggregation, with both scanning correlation methods and high order correlation analysis (Weissman et al., 1976; Palmer and Thompson, 1987; Petersen, 1986; Meyer and Schindler, 1988; Petersen, 1984; Qian and Elson, 1990; Koppel et al., 1994). Due to various experimental difficulties, however, it is not a widely used technique. The basic problem is that experimentally measurable fluctuations in fluorescence intensity occur only for very small sample volumes. For onephoton molecular excitation, lasers can be focused to submicron beam waists in the radial dimensions, but there is no confinement along the beam axis (z axis). Thus, most early applications were limited to two-dimensional systems, such as diffusion of labeled proteins or lipid probes in membranes Receivedfor publication 8 September 1994 and in finalform 11 November 1994. Address reprint requests to Dr. Keith M. Berland, Department of Physics, University of Illinois, Loomis Laboratory of Physics, Laboratory for Fluorescence Dynamics, 1110 West Green St., Urbana, IL 61801. Tel. : 217244-5620; Fax: 217-244-7187; E-mail: lfdwmeg@uxl.cso. uiuc.edu. C) 1995 by the Biophysical Society 0006-3495/95/02/694/08 $2.00 (see above references). Another possibility was to use very thin sample holders in order to confine the sample (volume) along the z axis. In any case, there was no possibility to do FCS measurements inside of live, intact cells. In recent years, the application of confocal microscopy techniques to FCS has successfully solved the volume problem and made possible measurements of diffusion in bulk (three dimensional, 3D) samples (Qian and Elson, 1991; Rigler et al., 1993; Koppel et al., 1994). However, confocal FCS has not been applied to the study of diffusion inside cells. An alternative method to solve the 3D volume problem is to use two-photon excitation, which excites only a small defined sub-volume of a bulk sample. This allows us to do FCS in bulk solutions, as in confocal techniques. There are additional benefits associated with two-photon excitation which will be discussed below, where we demonstrate the possibility to perform fluctuation measurements inside of live cells. Research on the transport properties and hydrodynamic conditions of cellular environments is a very active area of biophysical research (Luby-Phelps, 1994). FCS and photobleaching techniques and, more recently, single particle tracking experiments have all been used to study diffusion and transport on membrane surfaces. At present, there are only a limited number of techniques capable of probing intracellular diffusion. Fluorescence photobleaching (FPR/ FRAP) is the most widely used, and many authors have investigated transport properties in the cytoplasm in some detail. Single particle tracking is also becoming a very popular method, and has recently been introduced as a 3D method (Kao and Verkman, 1994). Our interest is in providing an alternative method to probe the environment inside of living cells. FCS and FRAP experiments can complement each other well. There are some important differences in the methods, and thus comparing results may prove valuable. First, FCS is generally useful for very low probe concentrations, while FRAP experiments can be done with higher probe concentrations. Also, FRAP experiments are done by perturbing 694 Two-Photon Fluorescence Correlation Spectroscopy the system away from equilibrium. It is not unreasonable to suppose that cellular transport may not always be linear with probe concentration, or that bleaching may in some instances introduce artifacts. Another important difference involves the 3D resolution of each method. Two-photon FCS has true 3D resolution, while most FRAP instruments use one-photon excitation. Single photon excitation bleaches through the entire cell, without z direction localization. It is possible to do two-photon photobleaching recovery to get the same 3D resolution (Piston et al., 1992). True 3D measurements may allow construction of a 3D diffusion map of a cell. Finally, others have demonstrated the FCS potential to monitor aggregation, rotations, and chemical reactions. Perhaps with some work this can be done inside of a cell. In any case, it is clear that two-photon FCS experiments may prove valuable in studying cellular interiors. TWO-PHOTON EXCITATION Two-photon excitation is the simultaneous absorption of two photons; their energies sum to the energy of the transition. Thus, for example, one can excite the ultraviolet 390-nm transition with two red 780-nm photons. The use of twophoton excitation for biological applications in microscopy was first proposed by Denk et al. (1990). For a general discussion see Friedrich (1982). Two-photon excitation is a nonlinear absorption process, and the excited fluorescence intensity is proportional to the square of the exciting laser intensity. For a system with probe concentration C(r,t) and laser intensity I(ri,t), we will observe fluorescence intensity, N I 0) -W