Direct Measurement of Sizes and Dynamics of Single Living

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NANO LETTERS

Direct Measurement of Sizes and Dynamics of Single Living Membrane Transporters Using Nanooptics

2002 Vol. 2, No. 3 175-182

Xiao-Hong Nancy Xu,* Jun Chen, Robert B. Jeffers, and Sophia Kyriacou Department of Chemistry & Biochemistry, Old Dominion UniVersity, Norfolk, Virginia 23529 Received November 28, 2001; Revised Manuscript Received January 8, 2002

ABSTRACT Sizes and dynamics of single membrane transporters on single living cells, Pseudomonas aeruginosa, were directly measured showing transported substrates up to 80 nm in diameter, which is one order magnitude larger than the reported exclusion limit of the outer membrane, over times of seconds to hours. The uptake and efflux dynamics depend on size and concentration of substrates, incubation time, and mutants. The strikingly active efflux of substrates by mutants devoid of pump proteins was observed, suggesting that substrates trigger the assembly of a new extrusive system. This new observation is in excellent agreement with the recent results of genome project analysis showing that P. aeruginosa encode more than a dozen unidentified efflux pump proteins. An extraordinary heterogeneity of size and dynamics of membrane transport mechanisms was observed, demonstrating real-time transformation of membrane permeability and an active extrusion system. This constitutes the first direct observation of living membrane transportation triggered by substrates with a variety of sizes at the single membrane pump level and opens up the new possibility of direct measurements of membrane active extrusion mechanisms for advancing the understanding of multidrug resistance and living molecular pumps.

Multidrug extrusion systems are being reported for both prokaryotes and eukaryotes.1-3 Membrane permeability and active extrusion systems may play a crucial role in universal cellular defense mechanisms. For instance, the efflux pump of Pseudomonas aeruginosa can extrude a variety of structurally and functionally diverse antibiotics.1 It is very likely that membrane proteins are triggered by substrates to assemble membrane transporters that are optimized for the extrusion of encountered substrates. Therefore, direct measurements of the sizes and dynamics of membrane permeability and membrane transporters at the molecular level are crucial to gain a better understanding of such universal extrusion cellular defense mechanisms for a variety of fundamental research and practical applications. Currently, learning the sizes of membrane transporters relies upon X-ray crystallography measurements, which are limited by the difficulties of crystallization of membrane proteins and are impossible for real-time dynamics study in-vivo.3-7 Despite extensive study over decades,8-14 the structure and mechanism of cellular transport remains unclear. We report here on a new platform for real-time measurement of sizes and dynamics of single living membrane transporters using single nanoparticles and molecular dynamics microscopy for advancing the understanding of multidrug resistance and living molecular pumps. Optical properties * Corresponding author. E-mail: [email protected]; www.odu.edu/sci/xu/ xu.htm. Tel: (757) 683-5698. Fax: (757) 683-4628. 10.1021/nl015682i CCC: $22.00 Published on Web 02/20/2002

© 2002 American Chemical Society

(colors) of silver-enhanced gold nanoparticles (SEGNP) are contributed by surface plasmon resonance of nanoparticles and depend on the size and shape of nanoparticles and the dielectric constant of the embedding medium of the particles, as described by the Mei theory with a quasi-static regime.15-17 Unlike the bulk plasmon, the surface plasmon of nanoparticles can be directly excited by propagating light waves (electromagnetic wave). This leads to spectrally selective light adsorption and scattering of nanoparticles. Therefore, we could tune the color of nanoparticles by carefully selecting the particle sizes and by maintaining particle-shape (sphere) and embedding medium as constant. We calibrated a wide spectrum of nanoparticle probes using plasmon resonance spectra and transmission electron microscopy (TEM) and then used these multicolor probes as a nanometersize index to directly measure sizes and extrusion dynamics of single membrane pumps on living microbial cells. We demonstrated that dark-field microscopy and spectroscopy were able to measure the sizes of many single nanoparticles inside and outside of single living cells simultaneously by direct observation of the colors of single particles, and hence we measured many single membrane transporters on single living cells at the nanometer spatial resolution and millisecond temporal resolution. This unique system is superior to TEM because TEM cannot provide real-time measurement of dynamics events (e.g., transformation of membrane pump) of living cells, even though TEM may offer high spatial

Figure 1. Schematic of a three membrane protein (MexA, Mex B, and OprM) assembly of efflux pump in Pseudomonas aeruginosa. MexA functions as a hollow, connecting MexB and OprM to form the path for substrates and then MexA interacts with MexB while MexB pumps out noxious compounds using the transmembrane proton gradient. MexA transmits this energy to OprM and opens the gate while it blocks the OprM channel.

resolution. This system is also superior to scanning-probe microscopy (SPM) (e.g., AFM, STM, or NSOM) because SPM offers low temporal resolution and is unable to monitor many single nanoparticles simultaneously. Furthermore, SPM is unable to perform noninvasive and nondestructive analysis of nanoparticles inside living cells. The MexAB-OprM pump of P. aeruginosa consists of two inner membrane proteins (MexA and MexB) and one outer membrane protein (OprM) (Figure 1).9,18 The MexB protein consists of 1046 amino acid residues and is assumed to extrude the xenobiotics utilizing proton motive force as the energy source.19-20 Typically, the extrusion pumps are assembled by several units of membrane proteins that can reorganize or transform themselves into a variety of conformations to meet the demands of diverse and complex environments.11,18 Studies show that it seems most likely that the inner membrane subunits specify the substrates to be transported. However, many questions remain unanswered. For example, (i) how do the cells sense such a wide range of diverse substrates? (ii) how do these membrane protein molecules assemble and recognize these diverse substrates and extrude them selectively out of cells? (iii) do unidentified extrusion pump proteins exist? Such efflux pump machinery is an excellent model for the design and fabrication of intelligent pumps that are able to “sense” and “act” on their own. Three strains of P. aeruginosa,20-22 wild-type (PAO4290, a wild-type cell level expression of MexAB-OprM), ∆ABM (TNP076, deletion of MexAB-OprM, derivative of PAO4290), and nalB-1 (TNP030#1, overexpression of all MexA, MexB, and OprM, derivative of PAO4290), were constructed. We used this MexAB-OprM membrane pump as a working model and investigated the roles of these membrane proteins on extrusion systems by measuring the sizes and dynamics of substrates (nanoparticles) transported by mutants of overexpression or deletion of these subunits. We measured the sizes of single substrates (nanoparticles) transported through living cell membranes by directly visualizing the color of individual particles, and hence we measured the size of the extrusion pump. This work demonstrates the possibility of real-time tracking of membrane permeability and observation of pump machinery of single living cells at the single-extrusion-pump level and may lead to an advanced understanding of multidrug resistance and designing of pump inhibitors. Furthermore, alternative molecular pump designs may be explored in hopes that 176

pumps may one day be engineered to selectively transport specific molecules into and out of living cells for a variety of applications (e.g., intelligent drug delivery). Nonbleaching, multicolor, silver-enhanced gold nanoparticles (40-100 nm in diameter) were synthesized and characterized using dark-field microscopy and spectroscopy and TEM.23-24 Both the surface plasmon-resonance wavelength (color) and light scattering intensity of the nanoparticles demonstrated a function of the particle size (Figure 2). As the diameter of the particle increases from 50 to 100 nm, the scattering spectra shift to the longer wavelength region, which is in excellent agreement with the fact that the resonance wavelength shifts to the red with increasing diameter of the nanoparticle. Careful selection of particle sizes permitted creation of a vast spectrum of particle probes (Figure 2). Furthermore, the scattering intensity increases with increasing diameter of the particle because of the increasing volume of the particle.15-17 These particles scattered light elastically with extremely high efficiency, and single particles were directly observed using a dark-field microscope equipped with a CCD camera, a digital camera, or even with the naked eye. The optical images of nanoparticles appeared larger than their actual sizes because of the optical diffraction limit (∼200 nm). These multicolor single nanoparticles were incapable of being photobleached or blinking as do more conventional fluorescent dyes or semiconductor nanoparticle probes. Thus, these particles served as ideal nanoprobes that permitted measurement of the size of membrane transporters and efflux dynamics as a function of particle size in living cells over a broad range of time scales (ms-hour). Three strains of P. aeruginosa,20-22 wild-type (PAO4290), ∆ABM (TNP076), and nalB-1 (TNP030#1), were selected and incubated with the nanoparticles. Single particles in solution, on the surface of membrane and inside living cells, were monitored using dark-field microscopy equipped with a CCD camera with millisecond accuracy (100 ms exposure time with readout time at 70 ms and delay time of sequencing images at 0 or 2 s). We observed the following. (i) Single, free-diffusion particles in buffer solution showed the larger diffusion distance and hence spread the intensity over a larger number of pixels during 100 ms exposure time. Thus, freediffusion particles in the solution appeared dim. (ii) Single particles remained on the surface of the membrane, and the diffusion distance of single particles shrank to the resolution of the CCD camera (2×2 pixels) and hence appeared brighter than single free-diffusion particles in solution. In addition, scattering by the cell membrane may also enhance the intensity of particles on the membrane. (iii) Single particles inside living cells showed smaller diffusion distance than that in buffer solution because the viscosity of cellular fluid is higher than the buffer solution. This is in a good agreement with random-walk theory.25 Single particles inside living cells also showed the lowest scattering intensity because it appeared that the cell membrane reduced the illumination intensity inside the cells due to the absorption and scattering properties of the cellular membrane and hence decreased the scattering intensity of the particles inside the cells. RepreNano Lett., Vol. 2, No. 3, 2002

Figure 2. Representative plasmon resonance spectra (A) and close-up real-color images (B) of single particles with sizes of 52 nm (blue), 74 nm (green), and 97 nm (red). The sizes of particles were characterized using TEM (JEOL). The spectra (A) were recorded using darkfield microscopy equipped with LN back-illuminated CCD camera coupled with SpectraPro-150 and (B) acquired by a Coolpix 990 digital camera. These data demonstrate that it is possible to simultaneously size the nanoparticles using their colors while following the membrane transport in real time. The optical images of nanoparticles look larger than their actual sizes because of the optical diffraction limit (∼200 nm).

sentative snapshots of real-time videos of single particles actively transported across living cell membranes are shown in Figures 3 and 4 (see also Supporting Information for the real-time videos), which show a blue nanoparticle (∼50 nm in diameter) participating in several uptake/efflux events in the ∆ABM cell (Figure 3A): first uptake at 7.170 min with efflux at 14.313 min; second uptake at 25.050 min and efflux at 25.386-min; third uptake at 25.601 min and efflux at 25.855 min; forth uptake at 26.948 min and efflux at 27.085 min; and fifth uptake at 27.182 min and efflux at 27.241 min. The bright yellow particle was taken up at 13.182 min and extruded at 28.317 min by the same ∆ABM cell. This yellow particle (∼80 nm) is bigger than the blue particle (∼50 nm) and therefore appears brighter. The larger size made it harder for the particle to enter the cell. Similar uptake and efflux phenomena were observed for WT and nalB-1 (Figure 3B), showing the blue particle taken up and extruded by a nalB-1 cell at 7.596 min and 8.503 min, respectively. We performed dozens of experiments for each cell type and recorded the influx and efflux of multicolor SEGNPs by single living cells, showing that all three strains of P. aeruginosa could uptake and efflux blue (∼50 ( 5 nm in diameter), green (∼70 ( 5 nm in diameter), and occasionally yellow (∼75 ( 5 nm in diameter) silver nanoparticles. The red particles (∼95 ( 5 nm) tended to remain outside the cells on the cell membranes and were rarely observed inside the cells (Figures 3-4). These results suggest that the membrane of the three mutants of P. aeruginosa permeate Nano Lett., Vol. 2, No. 3, 2002

and efflux substrates of size up to 80 nm in diameter. This is about one order magnitude larger than the reported exclusion limit of the outer membrane.10,20-22 The structure and size of the MexAB-OprM pump has not yet been characterized by X-ray crystallography or TEM. One research article described the sizes of silver particles accumulated inside Pseudomonas stutzeri AG259 (a different strain of Pseudomonas) as being up to 200 nm.26 However, direct evidence of particles transporting through the membrane was not reported in this paper. The particles remained positioned for periods of seconds to hours, either on the cell membranes or inside the cells. The particles were also uptaken and extruded across the cell membrane within 2.34 s (next sequencing image) as measured by dark-field dynamics microscopy with exposure time at 100 ms and readout time at 70 ms per frame and interval time at 2 s. This was determined based upon the observation of free-diffusion particles in the solution and inside living cells that took place within the interval frame at 2.34 s in total. The striking uptake and efflux of nanoparticles by ∆ABM (Figures 3A-5A) (devoid of MexAB-OprM pump) imply that ∆ABM may yet reassemble another type of membrane efflux pump that is capable of extruding invading substrates. This new finding echoes the recent results of genome project analyses that show that P. aeruginosa encode more than a dozen unidentified efflux pump proteins.27 Similar efflux phenomena were observed for WT and nalB-1 cells. Blank control experiments were performed by incubat177

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Figure 3 (cont’d). Representative snapshots of (A) ∆ABM and (B) nalB-1 cells uptaking and effluxing silver-enhanced gold nanoparticles (SEGNPs). The images were taken through a cover slip using dark-field microscopy with a 100× objective, equipped with a PID 1030 × 1300 pixel CCD camera (Micromax, 5 MHz Interline) at 100 ms exposure time with temporal resolution at 1.17 s. The time shown in each image represents real-time with one minute as a unit. (A) A blue nanoparticle (∼50 nm in diameter) participated in several uptake/efflux events in the ∆ABM cell: first uptake at 7.170 min with efflux at 14.313 min; second uptake at 25.050 min and efflux at 25.386 min; third uptake at 25.601 min and efflux at 25.855 min; forth uptake at 26.948 min and efflux at 27.085 min; and fifth uptake at 27.182 min and efflux at 27.241 min. The bright yellow particle was taken up at 13.182 min and effluxed at 28.317 min by the same ∆ABM cell. This yellow particle (∼80 nm) is bigger than the blue particle (∼50 nm) and therefore appears brighter. The larger size made it harder for the particle to enter the cell. (B) The blue particle taken up and extruded by a nalB-1 cell at 7.596 min and 8.503 min, respectively. The gray scale was set at 262-500 electron counts. The color of the nanoparticles was directly observed and confirmed with spectroscopy. Samples were prepared by directly mixing 20 µL of the cell solution (10× dilution of absorbance at 600 nm ) 0.1) with 20 µL of a 2.60 pM silver particle solution. The timer was started at the point of mixing.

ing nanoparticles at the corresponding concentrations with mouse fibroblast cells (L929) for 2 h, showing no extrusion of nanoparticles by the fibroblast cells. Representative plots of the number of P. aeruginosa cells with particles versus incubation time at particle concentrations of 0.26, 0.52, 1.30, and 2.60 pM demonstrated that Nano Lett., Vol. 2, No. 3, 2002

particle uptake by cells is proportional to incubation time and particle concentration prior to and at 100 min, respectively (Figure 5). The uptake and efflux dynamics also depend on mutants. At a particle concentration of 2.6 pM, a greater number of nalB-1 cells uptake and retain particles during the first 100 minutes compared to the ∆ABM and 179

Figure 4. Representative real color images of (A) ∆ABM, (B) nalB-1, and (C) WT cells, with silver-enhanced gold nanoparticles (Figure 2) taken from the solution in Figure 3 using a dark-field microscope equipped with a color digital camera (Nikon Coolpix 990). The silver particles are either inside the cells or attached onto the cellular membranes. There are green, blue, and red nanoparticles. Blue nanoparticles were observed most often inside the cell; whereas red nanoparticles were rarely observed inside the cells.

WT cell types. Interestingly, at 100 min, the nalB-1 cell type seems to begin extruding particles and the number of cells with particles decreases, whereas the number of ∆ABM and WT cells with particles remained almost constant, suggesting the uptake and efflux dynamics reached equilibrium. The total number of cells contained with particles represents the net gain of uptake and efflux dynamics, and hence the number of cells with particles remains constant at the equilibrium of uptake and efflux. At particle concentrations of 0.26-1.30 pM, the extrusion pumps of ∆ABM act to equilibrate the uptake and efflux of particles at 100 min; therefore, the number of cells with particles remain constant after 100 minutes of incubation time. Similar phenomena were observed for nalB-1 and WT showing the equilibration of nanoparticle uptake and efflux after 100 minutes. The observation of astonishing extrusion of particles of size up to 80 nm in diameter by all three strains of P. aeruginosa, especially by ∆ABM, implies that substrates (nanoparticles) may trigger the assembly of membrane transporters optimized for the extrusion of encountered substrates (nanoparticles). It is also important to note that cells with single nanoparticles could still grow and divide (Figure 4), with each individual particle remaining well isolated and unchanged in color, suggesting that single nanoparticles may not create a significant disturbance in cellular physiological environments. 180

Figure 5. Representative plots of the number of cells with silverenhanced gold nanoparticles (SEGNPs) versus incubation time: (A) ∆ABM, (B) nalB-1, (C) WT. Samples were prepared by incubating the cell solution (absorbance at 600 nm ) 0.1) with the particle solutions at 0.26, 0.52, 1.30, 2.60 pM in a vial while the timer was simultaneously activated. At each corresponding time, a sample of 40 µL was transferred to the microchamber and imaged directly by dark-field microscopy using a CCD camera and color digital camera (Figures 3, 4). Ten images taken from each sample similar to those shown in Figure 4 were analyzed by counting the number of the cells with and without particles. The total number of cells analyzed for each cell type was 400.

The heterogeneous distribution of number of particles within individual cells was also noteworthy (Figures 5-6). More than 90% of cells (∼360 out of ∼400 cells) were not uptake nanoparticles. Among the cells involved in uptake, there was a strikingly diverse distribution of nanoparticles in cells at a concentration 5.6 pM (Figure 6). Overall, most cells contained no particles (Figure 6a), 10% contained one Nano Lett., Vol. 2, No. 3, 2002

now use these nanoparticle probes to study the size and dynamics of cellular membrane transporters and subcellular (e.g., nuclear membrane) permeability and transporters. The uptake and efflux dynamics of nanoparticles by P. aeruginosa cells was extraordinarily heterogeneous. Aggregation of nanoparticles on the cell membrane was occasionally observed at the higher concentration, which may provide a new opportunity to study the mechanism of nucleation at a living interface and may present a new platform for the study of interfaces between nanomaterials and living systems for advancing the understanding of biocompatibility of nanomaterials. Furthermore, the nanoparticles at the high concentration may serve as an unconventional drug to inhibit cell growth by clogging the membrane transport.28 Figure 6. Images recorded from the same solution of nalB-1 (absorbance at 600 nm ) 0.1) incubated with 5.19 pM of silverenhanced gold nanoparticles in the microchamber for 3.5 h using dark-field microscopy. An astonishingly heterogeneous distribution of particles in the cells was observed: (a) most cells with no particles, (b) one cell with two particles (one blue color and the other orange color), (c) one cell with four particles (two particles with blue color and two particles with green color), (d) one cell with six particles, which appear to aggregate on the cell membrane, (e)-(j) particles aggregate on the cell membranes and hence lead to cell death.

or two particles (Figure 6b), and some cells (∼1%) contained three to six nanoparticles (Figure 6c-d). Those cells with three to six nanoparticles on the membrane were subject to nanoparticle aggregation and hence lead to cell death. In the latter case, nanoparticles may have lost their colloidal nature and then served as points of nucleation for growth of other nanoparticles. Additionally, the increasing numbers of nanoparticles on the cell membrane may have made it impossible for the particles to remain sufficiently separated and hence isolated. The aggregation process took place gradually and the nanoparticles naturally changed color as the size increased. In conclusion, multicolor silver-enhanced gold nanoparticles at low concentrations served as ideal nonbleaching molecular probes for real-time measurement of sizes and dynamics of membrane permeability and membrane transporters in living cells. It was evident that P. aeruginosa transported substrates up to 80 nm in diameter, which is one order magnitude larger than the reported exclusion limit of the outer membrane, suggesting that substrates might trigger the assembly of membrane transporters optimized for the extrusion of encountered substrates. Particles either remained in and on the living cells for seconds to hours or crossed the membrane within 2.34 s. Direct observation of extrusion of blue and green SEGNPs by ∆ABM (deletion of MexABOprM) (Figures 3-5) demonstrated that substrates (nanoparticles) triggered the assembly of new extrusion pumps. Taken together, we demonstrated the first direct measurement of living cellular membrane transportation triggered by sizes of substrates at the single membrane pump level. This opens up the new possibility of identification of unknown membrane pumps and direct measurements of active extrusion pump machinery for gaining a better understanding of multidrug resistance and living molecular pumps. One can Nano Lett., Vol. 2, No. 3, 2002

Acknowledgment. We thank Hiroshi Yoneyama (Tohoku University, Japan) and Taiji Nakae (Tokai University School of Medicine, Japan) for three strains of Pseudomonas aeruginosa and helpful discussion. The support of this work in part by the NIH (R21-RR15057-01), Old Dominion University, in the form of start-up, and Dominion Scholar Fellowship (R.B.J.) is gratefully acknowledged. Appendix A: Materials. P. aeruginosa bacteria cell lines,20-22 wild-type (PAO4290, a wild-type cell level expression of MexAB-OprM), ∆ABM (TNP076, deletion of MexAB-OprM, derivative of PAO4290), and nalB-1 (TNP030#1, overexpression of all MexA, MexB, and OprM, derivative of PAO4290), were constructed and received as a gift from Taiji Nakae. Cells were grown in L-broth medium containing 1% tryptone, 0.5% yeast extract, and 0.5% NaCl at pH 7.2 while rotating at 230 rpm and 37 °C. Cells were first precultured in the medium to ensure full growth and then cultured for an additional 8 h. Cells were harvested by centrifugation at 7500-rpm and 23 °C for 10 min, washed three times using 50 mM phosphate buffer (pH ) 7.0) and then suspended again in the same buffer at A600 ) 0.1. The cell solution was prepared at 10× dilution of A600 ) 0.1 before single-particle imaging experiments. Various sized silver-enhanced gold nanoparticles were prepared using 6.5 nm colloidal gold nucleating cores and a commercially available silver enhancement kit.23-24 The gold particles at 6.5 nm were prepared using the synthesis procedure as described in previous studies.24 Glassware was cleaned with royal water (3:1 of HCl to HNO3), rinsed with nanopore ultrapure water, and then dried prior to use. An aqueous solution of HAuCl4 (1 mM, 500 mL) was brought to reflux while stirring, and 10 mL of 1% (w/v) of trisodium citrate and 2.25 mL of 1% of tannic acid were added rapidly, which resulted in a color change from yellow to deep red. After the color change, the solution was refluxed for another 5 min and allowed to cool to room temperature, then subsequently filtered through a 0.22 micrometer filter. The gold particles were characterized by TEM showing the sizes of particles at 6.5 ( 0.3 nm with the sphere shape. Gold particles have their unique surface plasmon resonance characteristics and show a size dependence of absorption bands.15-17,24 This 6.5 nm colloidal gold solution was characterized by UV-vis spectroscopy indicating absorbance 181

of 0.7742 at 520 nm. Three silver-enhanced gold nanoparticles (SEGNP) with plasmon resonance peak wavelengths at 458 nm (blue), 539 nm (green), and 663 nm (red) (Figure 2) were prepared by adding 100 µL of initiator from the silver enhancement kit into 20 mL ultrapure water containing 6.5 nm gold particles at 5.2 pM, following addition of 10, 30, and 50 µL of silver enhancer into the solution, respectively. The solutions were continuously stirred at room temperature during the entire process. The reaction was completed after 2 min. These three types of SEGNPs were then characterized by TEM, UV-vis spectroscopy, and dark-field microscopy. Appendix B: Single-Particle and Single-Cell Dynamics Microscopy. The dark-field microscope (Nikon-E600) was equipped with an oil dark-field condenser, a 100× objective (Nikon Plan fluor 100x oil, iris, SL. N. A. 0.5-1.3, W. D. 0.20 mm), a PID 1030 × 1300 pixel CCD camera (Roper Scientific, Micromax, 5 MHz Interline, pixel size at 0.067 µm via 100× objective) for high-resolution cell imaging, a LN back-illuminated CCD camera (Roper Scientific, pixel size at 0.24 µm via 100× objective) coupled with a SpectraPro-150 (Roper Scientific) for simultaneous spectroscopic measurements, and a Coolpix 990 digital camera (Nikon) for real-color imaging. This microscopy system is capable of real-time single-cell imaging and single-particle microscopy and spectroscopy measurements. Sample solutions were sandwiched in a microchamber between a glass microscope slide (VWR, 1-mm thickness) and a cover slip (0.08-mm thickness) with double-stick tape as a spacer (0.05mm thickness) and directly imaged using dark-field microscopy equipped with CCD cameras. The glass slides and cover slip were cleaned by methanol and dried with nitrogen. Supporting Information Available: Real-time videos of simultaneous measurement of size and dynamics of a single living membrane pump on single living bacteria cells, Pseudomonas aeruginosa, using nanoparticles. This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Nakae, T. Microbiologia 1997, 13(3), 273-284, and reference therein. (2) Cole, S. P.; Bhardwaj, G.; Gerlach, J. H.; Mackie, J. E.; Grant, C. E.; Almquist, K. C.; S. A. J.; Kurz E. U.; Duncan, A. M.; Deeley, R. G. Science 1992, 258, 1650.

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(3) Rosenberg, M. F.; Mao, Q.; Holzenburg, A.; Ford, R. C.; Deeley, R. G.; Cole, S. P. C. J. Biol. Chem. 2001, 276, 16076. (4) For review: Tate, C. G. FEBS Lett. 2001, 504, 94, and reference therein. (5) For review: Scarborough, G. A. Cell Mol. Life Sci. 2000, 57(6), 871, and reference therein. (6) Mishima, Y.; Momma, K.; Hashimoto, W.; Mikami, B.; Murata, K. Acta Crystallogr. 2001, D57, 884. (7) Tate, C. G.; Kunji, E. R. S.; Lebendiker, M.; Schuldiner, S. EMBO J. 2001, 20, 77. (8) Juliano, R. L.; Ling, U. Biochim. Biophys. Acta 1976, 455(1), 152. (9) Poole, K.; Krebes, K.; McNally, C.; Neshat, S. J. Bacteriol. 1993, 175(22), 7363. (10) For review: Nakae, T. Microbiol. Immunol. 1995, 39(4), 221, and reference therein. (11) Yoneyama, H.; Maseda, H.; Kamiguchi, H.; Nakae, T. J. Biol. Chem. 2000, 275, 4628. (12) Nikaido, H. Science 1994, 264, 382. (13) Ma, D.; Cook, D. N.; Hearst, J. E.; Nikaido, H. Trends Microbiol. 1994, 2(12), 489. (14) Maseda, H.; Yoneyama, H.; Nakae, T. Antimicrob. Agents Chemother. 2000, 44(3), 658. (15) Mei, G. Ann. Phys. 1908, 25, 377. (16) (a) Bohren, C. F.; Huffman, D. R. Absorption and Scattering of Light by Small Particles; Wiley: New York, 1983; pp 287-380, and reference therein. (b) Lamprecht, B. Dissertation, Karl-FranzensUniversity of Graz., 2000; pp 1-24, and reference therein. (17) Kreibig, U.; Vollmer, M. Optical Properties of Metal Clusters; Springer: Berlin, 1995; pp 14-123, and reference therein. (18) Morshed, S. R.; Lei, Y.; Yoneyama, H.; Nakae, T. Biochem. Biophys. Res. Commun. 1995, 210(2), 356. (19) Lei, Y.; Sato, K.; Nakae, T. Biochem. Biophys. Res. Commun. 1991, 178(3), 1043. (20) Ocaktan, A.; Yoneyama, H.; Nakae, T. J. Biol. Chem. 1997, 272(35), 21964. (21) Guan, L.; Ehrmann, M.; Yoneyama, H.; Nakae, T. J. Biol. Chem. 1999, 274(15), 10517. (22) Saito, K.; Yoneyama H.; Nakae, T. FEMS Microbiol. Lett. 1999, 179(1), 67-72. (23) Schultz, S.; Smith, D.; Mock, J.; Schultz, D. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 996. (24) Handley, D. A. Methods for synthesis of colloidal gold. In Colloid Gold: Principles, Methods and Applications; Hayat, M. A., Ed.; Academic Press: New York, 1989; Vol. 1, pp 15-27. (25) (a) Atkins, P. W. Physical Chemistry; Freeman: San Francisco, 1982; pp 823-905. (b) Bard, A. J.; Faulkner, L. R. Electrochemical Methods Fundamentals and Applications: Wiley: New York, 1980; pp 488-510. (26) Klaus, T.; Joerger, R.; Olsson, E.; Granqvist, C. Proc. Natl. Acad. Sci. U.S.A. 1999, 96 (24), 13611. (27) (a) Stover, C. K. et al. Nature 2000, 406, 959. (b) Paulsen, I. T.; Sliwinski, M. K.; Saier, M. H., Jr. J. Mol. Biol. 1998, 277, 573. (28) Xu, X.-H.; Jeffers, R.; Kyriacou, S. U.S. Patent (pending).

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