Dynamics Of Live Cells

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Figure 3. Representative plots of the number of cells with SEGNPs versus incubation time: (A) AABM, (B) nalB-1, (C) WT [8]. Samples were prepared by incubating the cell solution (absorbance at 600 nm = 0.1) with the nanoparticle solutions at 0.26 (A), 0.52 (□), 1.30 (♦), and (*) pM in a vial while the timer was simultaneously activated. At each corresponding time, a sample of 40 pL was transferred to the microcham-ber and imaged directly by dark-field microscopy using a CCD camera and color digital camera (Fig. 2). Ten images taken from each sample similar to those shown in Figure 2 were analyzed by counting the number of cells with and without nanoparticles. The total number of cells analyzed for each cell type was 400. Reprinted with permission from [8], X.-H. Xu et al., Nano Lett. 2, 175 (2002). © 2002, American Chemical Society.

The study of the dynamics of live cellular membranes using single-particle tracking (SPT) has been described in several excellent review articles [36-39]. The use of nanopar-ticles as probes for the study of the real-time dynamics of subcellular events can be tracked back to the 1980s, when Nuydens and co-workers demonstrated the detection of microtubule-dependent intracellular motility using 20-to 40-nm Au nanoparticles through NANOVID (nanoparticle video ultramicroscopy) [40]. Nuydens and co-workers explored the application of Au nanoparticles for the study of molecular biology by the conjugation of Au nanoparticles with specific ligands. They demonstrated that Au nanopar-ticles conjugated with ligands could be used to determine the intracellular motility, cell membrane dynamics, receptor translocation, internalization, and intracellular routing [41]. These pioneering efforts realized the potential of nanoparti-cles as an essential tool for the study of live cells. Nuydens and co-workers provided a new and improved automatic technique for the study of intracellular mobility of live cells using the latest tracking algorithms and image processing hardware of that time [42]. They found the results collected from live-cell experiments in astonishingly close agreement with the experiments in a buffer solution containing micro-tubules and a kinesin-like protein extract. The development of this technique led to the successful determination of distributions of jump time, jump velocity, stop time, and orientation of the Au nanoparticles in PTK-2 cells [42]. They demonstrated that it was possible to monitor the lateral diffusion and the rearward movements of cellular protein toward the boundary between lamelloplasm and perinuclear cytoplasm in live cells using Au nanoparticles with a velocity of 0.5-1 ^m/min and a diffusion coefficient of 0.1-0.2 ^m2/s. Inspired by this work, Sheetz and co-workers developed the techniques to study the motion of motor molecules and membrane proteins in live cells using nanoparticles [43-45]. By the 1990s, these techniques have been advanced to record the reorganization of individual components on live cells [45].

Since then, an array of nanoparticles, including noble metal nanoparticles and polymer-based nanoparticles incorporated with dyes, has been developed for the study of the dynamics of biochemical events in live cells. Kopel-man and co-workers studied the kinetics of calcium release in live cells using nanoparticles prepared by the incorporation of the polymer matrix with fluorescent indicators (PEBBLEs) [14, 15]. They also developed Au nanoparti-cles attached with fluorescent dyes (fluorescein derivative) and then used these labeled nanoparticles to measure NO release in macrophages [13]. Recently, Xu and co-workers (our research group) applied the new approaches for the study of the dynamics in live cells using the unique properties of Ag and Au nanoparticles [8, 9]. We directly measured the real-time transformation of the size and dynamics of single-living-membrane transporters using nanoparticle optics and single-live-cell imaging [8]. The unique optical properties of the Ag nanoparticles allow us to monitor in real time the size transformation of the single-membrane transporters of live cells at a nanometer resolution and a millisecond temporal resolution using dark-field microscopy and spectroscopy. We also studied the ligand-receptor interaction on a single live cell in real time using SEGNPs [22].

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Figure 4. Direct observation of real-time size transformation of membrane pores (permeability) in WT cells [9]. (A) Representative optical images (a-c) of single cells (WT) recorded by the CCD and digital color camera through a dark-field optical microscope for 2 h. The single cell was selected from approximately 60 cells in the full-frame images. The solutions containing the WT cells (^600nm = 0.1), 1.3 pM Ag nanoparticles, and (a) 0 jUg/mL, (b) 25 jUg/mL, (c) 250 ^g/mL chlorampheni-col, were prepared in a vial and imaged in a fresh microchannel every 15 min. (B) Representative histograms of Ag nanoparticles in WT cells versus sizes of nanoparticles acquired from 70 images (~4200 cells), recorded for 2 h, showing a great number of larger nanoparticles were presented in the membrane at the higher concentration of chloram-phenicol. The solutions were the same as those in (A). (C) Representative plots of percentage of WT cells with Ag nanoparticles versus time from solutions in (A). Images for each solution were acquired at each particular time and about 600 cells were analyzed for each point.

Figure 5. Real-time images of single IgG molecules binding with single ZZ-T (ZZ is a short derivative of protein A) anchored on single live fibroblast cells (L929) cultured directly on a microscope coverslip [22, 23]. The interaction of single IgG with ZZ-T on live-cell surfaces was monitored in real time. (a) Binding IgG (b) unbound IgG (3S$),

Nanoparticles have been developed as delivery systems and imaging probes at a brisk pace in recent years. Many researchers have described the development of nanoparticles for the study of microtubules [39, 46-48]. The relative motions of the microtubules have been measured using Au nanoparticles labeled with monoclonal tubulin antibodies [47]. These tools have been extended to study the motor-driven microtubule transport, measure the forces required for ligand-receptor interactions, and monitor the kinetics of ligand-receptor interactions on the surface of the micro-tubules [47, 48]. Semiconductor QDs have also been used to study ligand-receptor interactions in buffer solution and have shown a great potential for use as probes for the study of the kinetics of interactions in live cells [10, 11].

In summary, the study of the dynamics of subcellular events in live cells using nanoparticles has drawn much attention and appears to have gained momentum in recent years as research in nanoscience and nanotechnology progresses.

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