Article |
Address correspondence to Watt W. Webb, 212 Clark Hall, Cornell University School of Applied and Engineering Physics, Ithaca, NY 14853. Tel.: (607) 255-3331. Fax: (607) 255-7658. email: www2{at}cornell.edu
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Abstract |
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Key Words: Rous sarcoma virus; two photon; fluorescence resonance energy transfer; proteinprotein transfer
Abbreviations used in this paper: 2PE, 2-photon excitation; CA, capsid domain; FCS, fluorescence correlation spectroscopy; FRET, fluorescence resonance energy transfer; MA, matrix domain; NC, nucleocapsid domain; PR, protease domain; RSV, Rous sarcoma virus; VLP, virus-like particle.
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Introduction |
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Here, we use two fluorescence-based methods using 2-photon excitation (2PE; Denk et al., 1990) to address the extent and spatial location of interactions between Gag molecules in living cells. The first is fluorescence resonance energy transfer (FRET), between donor and acceptor fluorophores tagged to two different molecules of Gag. The rate of energy transfer depends on the inverse sixth power of the distance between donor and acceptor, so FRET can be used to probe distances on the order of 5 nm (Periasamy, 2001; Sekar and Periasamy, 2003). The second method is fluorescence correlation spectroscopy (FCS; Magde et al., 1972; Elson and Magde, 1974). FCS provides dynamic information on fluorescent fluctuations and can be used to measure diffusion and concentration of fluorescent species (Thompson, 1991; Hess et al., 2002). Diffusion coefficients measured by FCS are used to calculate approximate sizes of Gag-containing complexes in cells, and quantification of the fluorescence intensity of the diffusing complex is used to estimate the number of Gag molecules in the complex. FRET and FCS are complementary techniques in that FRET is sensitive to the distance between donor and acceptor, and FCS provides information about the size of complexes and their interaction with the cellular environment.
We describe the implementation of 2PE FCS and FRET for studying Rous sarcoma virus (RSV) GagGag interactions in living cells. FRET measurements indicate that direct GagGag interactions occur not only at the plasma membrane but also in the cytoplasm, suggesting that the plasma membrane is not necessary for the initial steps of RSV assembly. FCS measurements reveal that most Gag molecules exist in the cytoplasm as part of large complexes. Deletion of the NC abolishes all GagGag interactions. These results indicate that cytosolic GagGag interactions are necessary for membrane binding and budding and are mediated by NC-RNA binding. The methods developed here should have utility for studying a wide array of cell biological problems involving assembly of complex supermolecular structures.
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Results |
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The second pair of mutant Gag constructs, GagNC-CFP and Gag
NC-YFP, lack NC. When coexpressed these constructs gave rise to diffuse homogeneous staining (Fig. 3 D), similar to that observed in cells expressing free CFP and YFP. No punctate features were evident and the plasma membrane showed no enhanced staining. Moreover, unlike the wild-type Gag and the mutant Gag
MBD chimeric proteins, Gag
NC-CFP/Gag
NC-YFP fluorescence was not excluded from the nucleus.
Cytosolic GagGag interactions measured by FRET
The VLPs in the process of budding from the plasma membrane provide a positive control for FRET. The particles, when completely formed, are expected to contain 1,500 copies of chimeric Gag (Vogt and Simon, 1999) in a sphere with an external diameter of 125 nm (Kingston et al., 2001), with the CFP or YFP domains located in the central portion of the VLP, in a volume of
105 nm3 (Yu et al., 2001). Thus the CFP and YFP domains approach closest packing and extensive energy transfer is expected. The negative control for FRET comes from the cells coexpressing free CFP and YFP (Fig. 3 A), which accounts both for CFP emission bleedthrough into the yellow channel and for direct excitation of the YFP.
FRET analysis was applied to stacks of images acquired at 0.5-µm intervals throughout the cells. The vertical dimension of the focal volume in which 2PE occurs is 1.2 µm, and, thus, molecules excited in this volume will be scored in at most three z sections. Control cells expressing either free CFP or Gag-CFP (Fig. 4 A, black circles) or coexpressing free CFP and YFP (Fig. 4 A, gray circles) showed similar apparent FRET ratios, implying that CFP bleedthrough into the YFP channel is dominant. In all plots, negative x axis values correspond to z positions below the coverslip, and positive values are above the coverslip. The apparent FRET ratios increased for z sections moving from below the coverslip into the plane of the cells. This slow rise is due primarily to the thresholding procedure of the algorithm. Because only nonzero values of the cyan counts are valid in the denominator, when there is little fluorescence in both channels, as would be true below and above the cell, there are more pixels with cyan counts above threshold than pixels with yellow counts above threshold, leading to an apparent low FRET ratio. In addition, due to low light levels and mechanical and biological differences in the positions of the ventral and dorsal surfaces of multiple cells, the regions below and above the cell layer tend to be noisy, as reflected in the error bars. In sections through the cell (0 µm < z < 5 µm), there were many more counts, thresholding levels were unimportant, and the ratio was much more robust.
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The FRET ratios in cells coexpressing Gag-CFP and -YFP were dramatically higher than the ratios in cells expressing the control proteins (Fig. 4 B, green circles compared with black circles). The FRET ratio rose to a maximum at 0 µm, the position corresponding to the ventral surface. This peak reflects the putative sites of assembly and budding at the plasma membrane. The FRET ratio decreased in z sections through the middle of the cell but remained well above the control levels, implying energy transfer in the cytosolic regions of the cell that are dominated by cytoplasm instead of plasma membrane. As the z distance increased, the FRET ratio reached a second maximum, corresponding to the dorsal surface. Above the dorsal surface, the data became noisier due to low levels of fluorescence, and the ratio approached control levels. In conclusion, the height of the central trough over the control curve strongly suggests interactions between Gag-CFP and -YFP molecules in the cytoplasm.
Cytosolic GagGag interactions are abolished by NC but not by
MBD
We also performed the whole cell FRET analysis on cells expressing either of the two chimeric deletion mutants. For the pair of proteins GagMBD-CFP and Gag
MBD-YFP, which lack the membrane-binding domain, the bimodal FRET ratio was abolished and instead a single broad peak was evident (Fig. 4 C). These proteins become trapped inside the cell as large aggregates (Fig. 3 C) because they cannot undergo membrane association and budding. However, GagGag interactions still occur, as evidenced by a high FRET ratio. Because Gag
MBD assembles into VLPs in vitro, it is possible that the macroscopic fluorescent aggregates represent clusters of such particles.
For the pair of proteins GagNC-CFP and Gag
NC-YFP, the FRET ratio was indistinguishable from control levels (Fig. 4 D), implying that GagGag interactions were so sparse as to be below the limit of detectability. This result is consistent with previous reports that at least part of the NC is crucial for Gag assembly, and it also corroborates the finding that the NC-nucleic acid binding is necessary for Gag dimerization in an in vitro assembly system with purified proteins (Ma and Vogt, 2002).
GagGag interactions in regions lacking sites of assembly
The FRET ratios in Fig. 4 B are the result of whole-cell analyses in which the image information was killed to obtain a robust measurement of FRET at each z position in the cell. The interpretation of significant cytosolic FRET relies on the expectation that the cytosolic signal should dominate in the equatorial region of the cell. To confirm this expectation, we visually selected small centrally located regions in cells that appeared to be devoid of punctate staining (Fig. 5, red square). In the example shown, budding sites were clearly visible on the plasma membrane on the ventral surface (Fig. 5, z = 0.0 µm), but as the z distance increased these spots disappeared in the cytoplasm (Fig. 5, z = 2.0 µm), and then new spots appeared on the dorsal surface (Fig. 5, z = 3.5 µm).
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FCS reveals that Gag is part of large complexes in the cytosol
The second method that we have used to study GagGag interactions in vivo is FCS. The GFP chimeras were first imaged, and then the beam was positioned for the FCS measurement in a region of the cell without obvious punctate features. Normalized representative FCS curves from four fluorescent samples are shown in Fig. 6.
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Gag-GFP (Fig. 6, yellow triangles) displayed diffusion properties intermediate between those of cytosolic (GFP) and membrane-bound (PM-GFP) proteins. The autocorrelation curve was fitted with a diffusion component corresponding to cytoplasmic diffusion (D = 0.023 µm 2/ms, 41%) and with a diffusion component, which is intermediate between cytoplasmic and membrane diffusion (D = 3.2 x 10-3 µm2/ms, 59%). This component is an order of magnitude larger than membrane diffusion coefficients (D 10-4 µm2/ms) and an order of magnitude smaller than cytosolic diffusion coefficients (D
10-2 µm2/ms). Furthermore, using a two-component diffusion model, we observed this intermediate component alternately present with cytoplasmic and membrane diffusion components. We interpret this intermediate diffusion coefficient to represent Gag-containing complexes in the cytosol (see Discussion). The size of such complexes can be gauged only approximately from these data because the diffusion coefficient is relatively insensitive to molecular mass, being proportional to the inverse cube root of the mass for a fixed density. But in approximate terms for spherical particles, the observed 10-fold decrease in diffusion coefficient compared with free GFP would correspond to a particle of
10100 MD.
The appearance of the intermediate diffusing Gag-GFP component was dependent on presence of NC. Gag NC-GFP showed a diffusion coefficient nearly identical to that of free GFP (D = 0.021 µm2/ms, 96%) (Fig. 6, red diamonds). Thus, the NC is necessary for the formation of the large Gag-containing complexes, as well as for significant membrane association.
We generated a frequency plot of the diffusion coefficient distribution, compiled from many cells from separate experiments (Fig. 7). The x axis is the log of the diffusion coefficient (log[D]), and the y axis is the frequency of occurrence, weighted by the relative fraction measured in the FCS curve. The left side of the histograms corresponds to slow diffusion (coefficients with large negative exponents) and the right side to fast diffusion (coefficients with exponents approaching zero). GFP (Fig. 7 A) showed a sharp peak with the expected value for GFP diffusion in the cytoplasm. The lesser peaks are due to the small fraction of slow diffusing components (e.g., the 5% component of GFP in Table I) and also to the error inherent in diffusion measurements in living cells. PM-GFP (Fig. 7 B) showed a clearly bimodal distribution, with well-resolved peaks corresponding to the membrane and the cytosolic fractions. By contrast, Gag-GFP showed a broad distribution of diffusion coefficients with some values typical of the cytosol and of the membrane-bound components, but also a significant fraction with intermediate values (Fig. 7 C). Deletion of the NC collapsed this broad distribution to one more similar to that of free GFP (Fig. 7 D). Very little membrane diffusion was observed for GagNC-GFP, indicating that NC is required for Gag to become stably attached to the membrane under these conditions. One model to explain this result is that stable membrane association requires cooperative binding, which is mediated by NC. Formation of the large putative cytosolic complexes also was drastically diminished for Gag
NC-GFP, in agreement with the FRET result that no significant GagGag interaction occurs for this mutant.
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The values for counts per diffusing species were calculated for the cytosolic fractions of GFP, PM-GFP, Gag-GFP, and GagNC-GFP (Table II). This quantity is calculated using the fraction of species with diffusion coefficient of 10-3 µm2/ms and greater, thereby explicitly rejecting the dominant diffusion peak associated with the membrane, as seen for example in PM-GFP (Fig. 7). The most significant feature of the normalized count rates is the absence of an overwhelming difference between Gag-GFP and the other moieties. If the large cytosolic diffusion coefficient were due entirely to aggregates of Gag in the cytoplasm, one would expect to see a 1030-fold increase in brightness. Fluorescence quenching is likely to reduce the overall brightness of such a Gag aggregate, but the absence of any substantial increase in normalized count rates for Gag-GFP over the other fusion proteins suggests that these cytosolic aggregates do not consist of labeled Gag alone. The intermediate diffusing fluorescent species appear to represent at most a few Gag-GFP molecules bound to larger cellular complexes.
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Discussion |
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Several biochemical studies previously have implicated large Gag-containing complexes in assembly of HIV-1. One report provided evidence for two distinct complexes with density similar to authentic virus particles, one resistant to and one disrupted by detergents (Lee and Yu, 1998; Lee et al., 1999). NC was required for both. In another report, several putative assembly intermediates were identified in a coupled cell-free translation-assembly system, as well as in cells expressing HIV-1 Gag (Lingappa et al., 1997). The analysis used in both studies has several limitations. First, it is difficult to demonstrate that the Gag complexes are true assembly intermediates, because 80% of newly synthesized HIV Gag protein is reported to be degraded by proteasomes (Tritel and Resh, 2000). We found that all of the pulse-labeled RSV Gag protein eventually assembles and buds as VLPs, obviating this problem. Second, in subcellular fractionation, it is difficult to exclude formation of irrelevant complexes in the crude extract. Third, the reports on HIV-1 complexes do not distinguish between GagGag interactions and Gagcell protein interactions. Here, we avoid these problems by using two nonperturbative fluorescence techniques, which demonstrate direct GagGag interaction with minimum manipulation of the cell.
FRET and FCS are complementary techniques for measuring intracellular Gag multimerization and trafficking. Both the whole cell FRET analysis and the inset analysis revealed strong FRET between donor and acceptor in regions of the cytoplasm where no punctate staining was visible. However, it is not possible to determine the extent of multimerization of Gag in the cytosol using FRET alone. FCS provides complementary information about multimerization of Gag: diffusion coefficients are a measure of aggregate size, and brightness provides an additional indication of multimerization. Furthermore, FCS can differentiate between membrane (including internal membranes) and cytosol, a distinction which is often difficult to make with imaging. Gag-GFP showed a diffusion peak intermediate between slow membrane bound diffusion and fast cytosolic diffusion. For this 310-fold change in diffusion, the mass would have to change by a factor of 301,000, according to the Stokes-Einstein equation. Although the Gag-GFP did display increased brightness in comparison to the GagNC-GFP, this increase was <50%. Together, the FRET and FCS measurements show that there is molecular interaction between Gag molecules in the cytosol, and that there are cellular complexes composed in part but not entirely of Gag.
There are several caveats for the interpretation of Gag diffusion coefficients in cells. The decrease of macromolecular mobility in the cytosol due to crowding is highly nonlinear with respect to the mass of the macromolecule (Verkman, 2002). Luby-Phelps and co-workers showed that when the mass of dye-labeled dextrans exceeds 500 kD, there is a sharp drop in cytosolic mobility (Luby-Phelps et al., 1986). Given the mass of Gag-GFP (97 kD), a Gag multimer containing 5 or more Gag monomers would be sufficient to give the observed diffusion coefficient by this criteria. Although a "5mer" might not be sufficiently bright in the cytosol to observe with imaging, we would expect to observe close to a fivefold increase in the counts/molecule measured using FCS. The fact that we did not observe this dramatic increase argues that this intermediate diffusion coefficient is not due to aggregates of Gag alone. Other explanations for diffusion coefficients that are intermediate between those characteristic of the cytosol and of the membrane might be membrane flow or perhaps active transport along microtubules or microfilaments. However, the observation that labeled Gag molecules appeared to sediment with discrete S-values in sucrose gradients would argue against these possibilities.
The interaction between Gag and RNA appears to be the driving force for assembly of retroviral Gag proteins. Studies using in vitro systems have demonstrated that nucleic acid promotes formation of Gag dimers, which are inferred to be a key assembly intermediate (Yu et al., 2001; Ma and Vogt, 2002). Gag dimerization is also an essential step in vivo, as shown for Gag proteins with artificial dimerization domains substituted for NC (Zhang et al., 1998; Accola et al., 2000; Johnson et al., 2002). It seems likely that in vivo NC-RNA binding occurs first, leading to GagGag interaction in cytoplasm, which then enables efficient membrane binding. The composition of the large cytosolic Gag complex inferred from FCS and from sedimentation analysis remains unknown.
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Materials and methods |
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Cell culture and transfection
The permanent chicken fibroblast cell line DF-1 was maintained in DME supplemented with 10% FBS and 1% vitamins. Transient transfections were performed by mixing plasmid DNA and Fugene reagent (Roche) as suggested by the manufacturer. For fluorescence studies, the cells were transferred to a glass bottom 35-mm dish (MatTek) 24 h after transfection and maintained in Leibovitz L-15 medium (GIBCO BRL) with the same supplementation for 12 h before microscopic analysis on a temperature controlled stage at 37°C. For FRET studies, 1 µg/dish each of the plasmid DNAs encoding Gag-CFP and Gag-YFP were cotransfected. The plasmid encoding PM-GFP, a GFP derivative engineered to contain the myristoylated and palmitylated NH2-terminal sequence from the Lyn tyrosine kinase (Pyenta et al., 2001), was a gift from B. Baird (Cornell University, Ithaca, NY).
Metabolic labeling, immunoprecipitation and rate zonal gradient
DF-1 cells in 60-mm plates were labeled 24 h after transfection with 100 µCi of [35S]methionine for 10 min in methionine-free medium (pulse) and were incubated further with unlabeled complete medium for various lengths of time (chase). To estimate budding efficiency, culture medium was collected and precleared by centrifugation at 1,500 g for 5 min. Virus particles were collected by centrifugation through a cushion of 15% sucrose in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 1 mM EDTA at 70,000 rpm in a TLA100.4 rotor (Beckman Coulter) for 20 min. The virus pellet was dissolved in SDS-PAGE sample buffer. To compare protein expression levels, transfected cells were lysed in ice-cold RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, and 1 mM PMSF). Cell debris and nuclei were removed by centrifugation at 10,000 g for 5 min and immunoprecipitation was performed with anti-CA antiserum or anti-GFP antiserum (CLONTECH Laboratories, Inc.) as described previously in Schatz et al. (1997).
For rate zonal sedimentation analysis, DF-1 cells transfected with GagD37S were labeled with 300 µCi [35S]methionine for 10 min and chased for various amounts of time, or continuously labeled for 1 h. The cells were then washed twice with ice-cold lysis buffer (1 mM Tris-HCl, pH 7.5, and 0.1 mM MgCl2), scraped off from the plate in 250 µl lysis buffer and transferred to a microfuge tube followed by 1 h incubation on ice. The swollen cells were then lysed in a tissue homogenizer with a tight-fitting Teflon pestle. Nuclei, cell debris, and membranes were pelleted at 10,000 g for 10 min. The supernatant was loaded on a 4-ml 1030% (wt/vol) gradient, which was made in buffer containing 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 0.15 or 0.5 M NaCl. The gradient was centrifuged for 75 min at 50,000 rpm in a SW60 rotor (Beckman Coulter). Fractions were collected from the top of the gradient, diluted in 800 µl RIPA buffer, and immunoprecipitated with a mixture of antiMA-p2 and anti-CA antibodies.
Two-photon microscopy and FCS
Imaging and FCS experiments were performed on a custom two-photon laser scanning microscope capable of laser scanning microscopy and stationary beam FCS. The excitation source was an ultrafast Ti:Sapphire Tsunami (Spectra-Physics). The beam was raster scanned at the sample with a MRC 600 scan box (Bio-Rad Laboratories) modified for near infrared excitation and coupled to an inverted microscope (model Axiovert 35; Carl Zeiss MicroImaging, Inc.) with a 63x C-Apochromat water objective (NA = 1.2; Carl Zeiss MicroImaging, Inc.). After the tube lens of the microscope, a 50-mm lens was used to collimate the emission. The emission was separated from the excitation with a 670 DCLP dichroic followed by an E800sp IR blocker. For FRET, the emission was then further separated with a 510 DCLP dichroic into a cyan channel (emission filter, HQ480/40 m) and a yellow channel (emission filter, HQ535/30 m). FCS measurements were performed using a single channel optimized for GFP (emission filter, HQ 575/150 m). All filters and dichroics were from Chroma Technology Corp. The detectors in both channels were GaAsP photon counting photomultipliers (Hamamatsu). Because 2PE provides intrinsic three dimensional resolution, no pinholes or apertures were used in the detection pathway. Correlation measurements were done with a 6010 Multiple Tau digital correlator (ALV).
The 830-nm excitation wavelength for the FRET experiment was chosen to maximize CFP absorption and minimize YFP absorption (Blab et al., 2001; Tsai et al., 2002). All FCS measurements were done with GFP, at an excitation wavelength of 910 nm. In general, it is preferable to use long wavelengths (>900 nm) in cellular studies to reduce damage and minimize autofluorescence (Chen et al., 2002), and this wavelength is also near the peak of the GFP two-photon cross section (Heikal et al., 2001). Each curve was the average of five 10-s runs, resulting in a 50-s exposure to the cell. Only one curve was taken from each cell, with the error bars being the measured SD from the five runs. The average power in these FCS measurements was <2.5 mW.
FRET analysis
Due to the broad emission profiles of fluorescent proteins (Miyawaki et al., 1997; Tsien, 1998), there is significant overlap between donor emission and acceptor emission, resulting in spectral bleedthrough between fluorescence channels. To account for this bleedthrough, several correction schemes and analysis methods have been proposed (Miyawaki et al., 1997; Gordon et al., 1998; Xia and Liu, 2001; Erickson et al., 2001; Hoppe et al., 2002; Elangovan et al., 2003). The common aim of these methods is to generate a FRET image that is corrected for bleedthrough, expression levels, collection efficiency, background, and photobleaching, where each pixel is proportional to FRET efficiency.
We have implemented a methodology specifically designed to address the question of whether FRET occurs in the cytoplasm or at the plasma membrane. This method is based on z-series scans of cells in a culture dish and calculation of a FRET ratio at each z position. There is a greater fraction of plasma membrane at the ventral surface of the cell (in contact with the coverslip) and at the dorsal surface than in the equatorial regions of the cell. Therefore, a z-dependent FRET measurement can better distinguish between FRET contributions from the plasma membrane and the cytoplasm. Furthermore, this method does not attempt to generate a FRET image based on ratiometric approaches, which are inherently noisy when applied on a pixel-by-pixel basis. The approach described here is more robust, particularly in a cellular environment, but does not provide laterally resolved information.
All image analysis was done with IDL 5.4 (Research Systems, Inc.). The FRET ratio was calculated as the ratio of yellow intensity to cyan intensity within the cell for each plane of the z series. Thus, two images (cyan and yellow) at a single z position were reduced to one number, which reflects the amount of FRET, according to the following algorithm. First, the median of the image was subtracted to correct for the black level. Then, a binary mask was created by smoothing, choosing all points above a threshold, and finally using a gray scale closing operation. Once the binary mask was obtained, the original background-subtracted image was multiplied by the mask and summed. The net result of these manipulations was simply to define a region that constitutes the cell and to total the counts in that region. The ratio was obtained by dividing the value in the yellow channel by the value in the cyan channel. The same mask was used in both numerator and denominator. In a z-series acquisition, this operation was repeated at every plain of the z series for each set of images, yielding one ratio for each z step.
This method uses only a single excitation wavelength, namely the excitation maximum of the donor. We used this single excitation approach to eliminate the chromatic aberration effects and alignment artifacts, which are introduced by changing excitation wavelengths. The disadvantage of this method is that one cannot directly measure the acceptor concentration or account for direct excitation bleedthrough of the acceptor. However, the experimental results indicated that the acceptor was minimally excited at these wavelengths and that the donor fluorescence was the primary source of fluorescence bleedthrough (Results).
FCS analysis
FCS measures the fluorescence fluctuations resulting from diffusion of fluorescent entities into and out of an optically defined volume as a function of time. In this case, the volume is that in which 2PE occurs: 1.2 µm in height and 0.5 µm in width. The concentration and the diffusion coefficient of the fluorescent species can be calculated from these data. The advantage of 2PE is the excellent registry between the recorded image and the positioning of the laser beam, enabling one to position the beam in the cell using the two-photon image as a guide. The autocorrelation function G(
) is defined as:
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Acknowledgments |
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Submitted: 31 March 2003
Accepted: 5 August 2003
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