Liquid Dynamics - American Chemical Society

Chapter 5

Translational Dynamics of Fluorescently Labeled Species by Fourier Imaging Correlation Spectroscopy Downloaded by UNIV OF GUELPH LIBRARY on September 14, 2012 | http://pubs.acs.org Publication Date: June 3, 2002 | doi: 10.1021/bk-2002-0820.ch005

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Michelle K. Knowles , Daciana Margineantu , Roderick A. Capaldi , and Andrew H. Marcus 1,*

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Department of Chemistry and Materials Science Institute, University of Oregon, Eugene, OR 97403 Department of Biology and Institute of Molecular Biology, University of Oregon, Eugene, OR 97403

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Time-dependent spatial distributions of density fluctuations in synthetic and biological systems are determined from purely incoherent fluorescence signals. This is accomplished using a new method, Fourier imaging correlation spectroscopy (FICS), that is based on the detection of modulated fluorescence signals and measures temporal fluctuations of a spatial Fourier component of the sample particle number density. The information contained by FICS measurements provides details about the spatial relationship between fluorescent species, usually only obtained by direct imaging single-particle experiments. The FICS approach offers significant advantages in signal-to-noise detection efficiency, allowing a broader dynamic range to be accessed experimentally.

The dynamics of complex fluids is an area of fundamental importance and technological relevance. Macromolecular or mesoscopic translational motion is important in many different kinds of soft-matter systems ranging from self-

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© 2002 American Chemical Society

In Liquid Dynamics; Fourkas, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

Downloaded by UNIV OF GUELPH LIBRARY on September 14, 2012 | http://pubs.acs.org Publication Date: June 3, 2002 | doi: 10.1021/bk-2002-0820.ch005

59 organized block-copolymer films to protein transport in live biological cells. Traditionally, experimental information about the structure of complex fluids is obtained by light scattering from ordered arrangements of atoms, molecules or larger scattering centers (/). Dynamics are studied by performing dynamic light scattering (DLS) measurements of the fluctuations of scattered light intensity (2). Such experiments reveal the existence of multi-exponential relaxations that arise from the complex interactions between fluid components occurring over a range of spatial and temporal scales (3). Nevertheless, many intriguing soft materials have been left unexplored due to a lack of sufficient light scattering contrast. In this chapter we present an overview of a new method called Fourier imaging correlation spectroscopy (FICS) that retains the wave number selectivity of DLS while overcoming many of its limitations in sensitivity (4,5).

Fourier Imaging Correlation Spectroscopy In FICS experiments, modulated fluorescence signals are detected from the intersection of an excitation fringe pattern (an optically generated grating) with a microscopically heterogeneous configuration of chromophores, C(r,t). The basic principle is illustrated in Figure 1A for the case of a two-dimensional fluid. A system of Ν uniformly labeled fluorescent particles (shown as discs with diameter σ) is illuminated by an excitation grating having a fringe spacing dû.

Figure I. (A) Schematic of the FICS experimental geometry. Fluorescent particles are represented as circles and the excitation grating as gray bars. (B) A static particle configuration is uniquely described by a sequence of vectors in the complex plane whose superposition is C. The grating is characterized by a wavevector, k - 2πdG" i, that is directed along the x-axis. The time-dependent microscopic density is given by 1

G


60 th

C(r,t) = -^X^ A 5[r-r (t)], where A is the area of the n particle, proportional to that particle s fluorescence intensity, and V is the total area of the illuminated system. The phase of the grating is modulated at the angular frequency (ûc so that its position is swept across the system at a velocity, υ = COG / k , much greater than the average speed that a particle travels the interfringe distance. The resulting fluorescence intensity has the functional form {4,5\ =l

n

n

n

0

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I (t) = (^)l {c(0)+|c(k ,t)|cos[eo t + a(k ,t)]}, Downloaded by UNIV OF GUELPH LIBRARY on September 14, 2012 | http://pubs.acs.org Publication Date: June 3, 2002 | doi: 10.1021/bk-2002-0820.ch005

G

0

G

G

G

= (l/t Jl

( 4 )

A(t )A(t + T),

m

0

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to where A = ReC, ImC. The decay time of the autocorrelation function is a meaure of the time required for a fluorescent particle to move the distance k ". The upper limit to the temporal resolution of FICS measurements is determined by the modulation frequency. Essentially, 10 cycles of the reference oscillator are necessary to determine a single data point. For the experiments presented here, a modulation frequency of 50 KHz is used, corresponding to an instrumental time resolution of 200 μ8βα The FICS apparatus can also be run in direct visualization mode. In this way, microscopic information is obtained by recording sequenced images of the sample plane via digital video fluorescence microscopy (DVFM). This allows direct comparison to be made between the time-correlation functions measured by the FICS method and the same quantities calculated from the microscopic particle trajectories via statistical mechanics (5,7). The experimental procedure to perform DVFM measurements is described in detail by Crocker and Grier (#), who developed the original particle tracking algorithms. G

Dynamics of Monolayer Colloid Suspensions In Figure 3A is shown the results of FICS measurements performed on a dilute monolayer suspension of Rhodamine labeled poly(styrene) spheres (diameter, σ = 1 μηι) with ρ* = 0.02 (Ν ~ 160 particles contained by an area A « 7850 μτη ). At low density, the mean inter-particle separation is large, [L = (ρ7σ2)~!/2 ~ 7.1 μηι], such that the system behaves like a superposition of noninteracting Brownian particles for d < L. Under these conditions, the dynamic structure function takes the Gaussian form (2) 2

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F (k,r) = exp[-k D r] s

()

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where F (k,t) is called the self dynamic structure function and Ds is the selfdiffusion coefficient. The data shown! in Figure 3A decays in time as a single exponential and scales with wave-number as a Gaussian, in precise agreement with the theoretical prediction. The solid lines correspond to eq 5 with D = 3 x s

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τ (sec)

τ (sec)

Figure 3. (A) Results of F/CS measurements performed on a dilute monolayer colloid suspension and (B) a semi-dilute suspension. (Reproduced from reference 5. Copyright American institute of Physics.) }

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\0' cm sec". This value for the self-diffusion coefficient is in good agreement with that of the free diffusion coefficient, D = 0.707k T / 6πν\α^ 3.1 χ 10" cm sec", calculated from the Stokes-Einstein equation with a correction to account for the hydrodynamic friction due to the effect of the cell walls (5,7). In Figure 3B is shown plots of F(k,x)/S(k) as a function of time for a semidilute monolayer poly(styrene) suspension (p* = 0.31). For this system, the dynamics is complicated by multi-exponential decays that vary with k. The mean inter-particle separation at this density is L - 1.8 μνη. The data indicates that the particle dynamics exhibit multi-exponential behavior when the system is probed at do = 2.23 μηι > L , but remains single-exponential for all fringe spacings d = 0.49, 0.53, 0.67, 0.87, 1.28 μηι < L. For d = 2.23 μηι, there is an apparent transition of the effective diffusion coefficient from short- to long-time behavior, which occurs at τ = 150 msec. When the system is probed on length scales less than L, particles appear to diffuse freely without hindrance from neighboring particles. When the system is probed on length scales greater than I, the aparent diffusion coefficient is dressed by collisions between nearest neighbors, effectively decreasing the diffusion coefficient. The observed time scale of this kinetic transition is slightly smaller than the average collision time between particles (x = [(2π IL) Do]" = 250 msec). When measurements are performed on dense crowded systems, the wave number and time-dependence of the dynamics becomes more complex. Figure 4A displays a direct comparison of F(k,x)/S(k) determined from F1CS data (solid 9

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B

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65 curves) and from DVFM data (open circles) as a function of time for a monolayer poly(styrene) suspension with p* = 0.51. The inset shows a fluorescence micrograph of a static particle configuration. Decays are shown for three different wave numbers. The FICS data were constructed according to eq 3, while the microscopy data were calculated by Fourier inversion of the microscopic particle trajectories as described in reference 6. Agreement between the two independent measurements of the intermediate scattering function is excellent. For the highest value of k shown (d - 0.86 μηι), the decay is nearly single-exponential. As the wave number is decreased (or the fringe spacing is increased) the decays begin to exhibit multi-exponential character. Our interpretation of this result is that the high-k measurement is primarily sensitive to pre-collisional free motion, while the low-k measurements are sensitive to particle-particle interactions. An analogous comparison is shown for S(k), where the FICS data (filled circles) have been scaled by an arbitrary factor along the vertical axis. The agreement to microscopy data (solid curve) is very good, indicating that the FICS method is capable of measuring structural correlations of the fluid. In this example, the temporal resolution of the FICS measurement is


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Figure 4. Comparison between FICS and DVFM measurements for a dense monolayer colloid suspension. Plots are shown for (A) the intermediate scatteringfunction, and (B) the static structure factor. (Reproducedfrom reference 5. Copyright 2000 American Institute of Physics.) 2.0 msec, 5 times faster than current digital video technology. We expect future variations of the technique to access microsecond time scales.


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Dynamics of Mitochondria in Live Cells The ability to determine F(k,i)/S(k) from weakly fluorescent biological samples is demonstrated in Figure 5. FICS experiments were performed on live human osteosarcoma cells (143B) treated with aqueous JC-1 solution (Molecular Probes, 0.25 μΜ, 5 minute exposure), a fluorescent dye (k = 590 nm) that accumulates inside the mitochondrial compartment (9). The cells were cultured using HG-DMEM medium supplemented with 10% fetal calf serum, and incubated in 5% C 0 atmosphere. The inset shows a contrast enhanced fluorescence micrograph, taken with an intensified CCD camera (Princeton Instruments Pentamax), of a similarly treated cell. In the samples we studied, the mitochondrion exists as a network of flexible filaments that constantly undergo rearrangements in position. Little is known about the details of this motion. The average power level used during a -45 minute data run was 0.1 mW. No laser induced pathological effects in cell behavior was observed. Figure 5A shows that the dynamics of the mitochondrion is complicated by multiexponential relaxations; a full analysis is presented in reference 9. Comparison between FICS (solid curves) and microscopy (open circles) measurements of F(k,i)/S(k) show excellent agreement between the two methods. cm


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Figure 5. (A) Comparison between FICS and microscopy data for reticulated mitochondria in live cells. (B) The effective self-diffusion is plotted as a function of time. (Reproducedfrom reference 9. Copyright 2000 Biophysical Society.) The kinetic behavior of mitochondrial filaments can be studied through the time- and wave number-dependent self-diffusion coefficient, D (k,r). We s


67 calculate D (k,r) from our FICS data according to D (k,r) = - l n f F ( k , T ) / k r ] , a generalization of eq 5. In Figure 5B are shown plots of D (k,r) corresponding to the data shown in Figure 5A. For intermediate times (1 sec < τ < 60 sec) and fixed k, D (k,r) decays on the time scale Xi — 15 sec. This relaxation indicates a kinetic transition from short-timefilamentmotion to a dressed collective long-time behavior. The values for £> (k,t) lie in the range 3.5 - 0.5 x 10" cm sec", consistent with velocities observed for mitochondria undergoing cytoskeletal-assisted directed motion (-50 nm sec"). Examination of the k-dependence of D (k,r) atfixedτ reveals that the effective diffusion coefficient at all times is consistently smaller for do ~ 0.55 μηι than it is for d ~ 0.82, 1.0 μηι. Our observations suggest that the kinetic transition from short- to long-time behavior is the result of a structural rearrangement of the local mitochondrialfilamentenvironment on the length scale di - 0.8 μτη. To examine the effects of metabolic activity we used FICS to study JC-1 labeled cells after incubation with drugs known to alter metabolism. In Plate 1 are shown fluorescence micrographs of JC-1 labeled cells after exposure to various drugs. JC-1 is a positively charged carbocyanine dye that is a quantitativefluorescenceindicator of membrane potential, ΔΨ. Local regions of the membrane that are energized promote an uptake of JC-1 into the mitochondrial matrix with subsequent formation of a J-aggregate of JC-1 that emits yellow fluorescence (-590 nm). The monomer form of JC-1 emits green fluorescence. As shown in Plate 1 A, the spatial distribution of ΔΨ in reticulate mitochondria appears heterogeneous under physiological conditions. This heterogeneity is sensitive to the metabolic state of the cell. In Plate IB, we show the effects of treatment with Nigericin, an ionophore that exchanges K" and H across the mitochondrial inner membrane resulting in uncoupling of respiration from ATP production. The net effect of Nigericin treatment is the hyperpolarization of the mitochondrial inner membrane. The spatial distribution of Δ Ψ becomes uniformly large throughout the reticulum after -30 minutes incubation with Nigericin. In Plate 1C, we show the effects of inhibition of respiration. Antimycin A inhibits the activity of mitochondrial respiratory chain complex III. Cells treated with Antimycin A (-10 minutes incubation) show a progressive decrease in local membrane regions with high ΔΨ. In Plate ID we show the effects of Staurosporine, a protein kinase inhibitor that induces apoptosis (programmed cell death). Dramatic changes in mitochondrial membrane morphology are observed in cells that have been treated with Staurosporine (-4 hours incubation). Staurosporine has the initial effects of hyperpolarizing the mitochondrial membrane, membrane swelling, and the disruption of the reticulum structure with the formation of giant mitochondrial vescicles. We used FICS to study the motion of hyperpolarized regions of the mitochondrial membrane by detecting emission only at 590 ± 5 nm. In Figure 6, is shown direct comparisons between D (k,T) r = -ln[F(k,T)/k"] for control s

s

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s

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68 cells and for those treated with the drugs described above. For these measurements the fringe spacing was set to do = 0.8 μπι, the length scale associated with filament reorganization. In Figure 6A are shown results for Nigericin treated cells in which mitochondrial ATP synthesis has been uncoupled from respiration. For short times (τ < 15 sec) £> (k,t)r is indistinguishable from the corresponding control cell measurement. For long times (τ > 15 sec), the slope, D (k,r), is a factor of 1.5 smaller for Nigericin in comparison to control cells. The transition time (~ 15 sec) is the same as the interaction time scale, Τι, obtained from our kdependent study. The effects of Antimycin A, shown in Figure 6B, are almost identical to those of Nigericin. Similar to Nigericin, cells treated with Antimycin A do not produce mitochondrial ATP. Both types of treated cells show a decreased rate of long-range filament motion, suggesting that this motion is due to the action of s


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Figure 6. Comparison between FICS measurements on normal cells and cells treated with drugs that (A) uncouple respiration, (B) inhibit respiration, (C) depolymerize cytoskeletal actin, and (D) induce apoptosis. (Reproducedfrom reference 9. Copyright 2000 Biophysical Society.) ATP-driven cytoskeletal filaments. The short-time short-range motion, however, is independent of metabolic activity. Figure 6C shows results for cells treated with Latrunculin A that depolymerizes actin filaments, a major component of the cytoskeleton. For short-


69 times, D (k,r)r is indistinguishable from control measurements, while for τ > 50 sec, the long-range motion is completely turned off. This suggests that the short time motion is a consequence of the mechanical properties of the membrane. The effects of apoptosis induced by Staurosporine are shown in Figure 6D. In this case, short- and long-time motions are dramatically reduced, consistent with the expected behavior of a swelled membrane. The absence of motion at long-times is consistent with the fact that ATP synthesis is shut down early in the apoptotic process.


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Comparison to Other Methods In comparison to microscopy, FICS has both important advantages and limitations. In essence, direct imaging experiments carry out many singleparticle measurements in parallel. FICS experiments probe the time-course of collective fluctuations from an /V-body system. While real-space trajectories contain all of the dynamical information that characterizes the system, this information must be statistically averaged to construct physically meaningful distribution functions. FICS data provide a direct route to the same relevant twopoint distributions yielded by microscopy. Variations of FICS in which spatial information is simultaneously determined at more than one wave number at a time should provide the necessary information to compute higher-order spatial and temporal distributions. Such information may easily identify the signatures of non-uniform dynamics, without the necessity of measuring the full spatial distribution at once.

Acknowledgements This work was supported by grants from the National Science Foundation (CHE-9876334 and CHE-9808049), the M. J. Murdock Charitable Trust (No. 98181), and the American Chemical Society Petroleum Research Foundation (No. 34285-G7).

References 1. 2. 3.

Chaikin, P. M.; Lubensky, T. C. In Principles of Condensed Matter Physics; Cambridge University Press: Cambridge,1995;pp 353-354. Berne, B. J.; Pecora, R. In Dynamic Light Scattering; Krieger: Malabar, 1976. Boon, J. P.; Yip, S. In Molecular Hydrodynamics; Dover: New York, 1991.


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7. 8. 9.

Knowles,M.K.;Grassman, T. J.; Marcus, A. H. Phys. Rev. Lett. 2000, 85, 2837-2840. Grassman, T. J.; Knowles, M. K.; Marcus, A. H. Phys. Rev. Ε 2000, 60, 5725 — 5736. Fleming, G. In Chemical Applications of Ultrafast Spectroscopy; Oxford University Press: New York, 1986. Marcus,A.H.;Schofield,J.;Rice, S. A. Phys. Rev. Ε 1999, 60, 5725 - 5736 Crocker,J.C.;Grier,D.G.J.Colloid Interface Sci. 1996, 179, 298. Margineantu, D.; Capaldi,R.Α.;Marcus, A. H. Biophys. J. 2000, 79, 18331843.


Liquid Dynamics - American Chemical Society

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