Info

Figure 3.7. Dependence of optical properties (LSPRS, color) of nanoparticles on its size. Correlation of color index and size index of Ag nanoparticles is determined using cumulative histograms of the number of Ag nanoparticles versus nanoparticle sizes. The color index of Ag nanoparticles obtained from 100 nanoparticles determined by the optical microscope and spectroscopy is compared with the size index of the nanoparticles measured by TEM, showing that color index of Ag nanoparticles with violet, blue, green, and red is correlated with the size index of 30 ± 10, 50 ± 10, 70 ± 10, and 90 ± 10 nm, respectively. [Reprinted from Biochemistry 43:140-147, with permission of American Chemical Society.] See insert for color representation of this figure.

Therefore, the colors (LSPRS) of Ag nanoparticles are correlated with the sizes of nanoparticles, while the shapes of nanoparticles and their embedded medium remain unchanged (Figure 3.7) (Xu et al., 2002, 2004; Kyriacou et al., 2004). Such unique size-dependent optical properties allow us to use the color index of nanoparticles (violet, blue, green, red) as the nanometer size index probes (30 ± 10, 50 ± 10, 70 ± 10, 90 ± 10 nm) for real-time sizing the change of living cellular membrane permeability and porosity at the nanometer scale.

Individual Ag nanoparticles are extremely bright under dark-field optical microscope and can be directly imaged by a digital or CCD camera through the dark-field microscope. Unlike fluorescence dyes, these nanoparticles do not suffer photodecomposition and can be used as a probe to continuously monitor dynamics and kinetics of membrane transport in living cells for an extended period of time (hours). In addition, these nanoparticles can be used as nanometer probes to determine the sizes of substrates that are transporting in and out of the living cellular membrane at the nanometer scale in real time.

As described above, the quantum yield of Rayleigh scattering of 10 nm of Ag nanopar-ticles is about six orders of magnitude higher than the fluorescent quantum yield of a single fluorescence dye molecule (R6G). The scattering intensity of Ag nanoparticles increases proportionally as the volume of nanoparticles increases. Unlike fluorescence dyes, Ag nanoparticles resist photobleaching. Therefore, these Ag nanoparticles are extremely bright under the dark-field microscope and can be used for real-time monitoring of membrane transport in living cells for an extended time.

These new tools have been used to study the function of aztreonam (AZT) at the nanometer scale and single living bacteria (P. aeruginosa) resolution (Kyriacou et al., 2004). AZT is a monocyclic j-lactam antibiotic. It is well known that the mode of action of AZT is the disruption of the cell wall (Greenwood, 1997). Thus, AZT is used to validate Ag nanopar-ticles as nanometer assays for real-time sizing membrane porosity and permeability as AZT disrupts the cell walls (Kyriacou et al., 2004). The complete disruption of the cell wall by a high concentration of AZT (31.3 Mg/mL) causes the overflowing of intracellular nanoparticles, leading to the aggregation of intracellular Ag nanoparticles.

The validated nanoparticle assay is then used to study the membrane transport mechanisms of living microbial cells (P. aeruginosa) in the absence of antibiotics and in the presence of an antibiotic (chloramphenicol), which is neither a i-lactam nor aminoglyco-side antibiotic. The primary target of chloramphenicol in microbial cells is to inhibit the ribosomal peptidyl transferase rather than disrupt cell wall (Greenwood, 1997). The dependence of membrane transport upon the dosages of chloramphenicol is investigated using single-nanoparticle optics and single-living-cell imaging. An efflux pump (MexAB-OprM) of living microbial cells (P. aeruginosa) is selected as a working model for probing the role of the efflux pump in controlling the accumulation of substrates (nanoparticles, EtBr, chloramphenicol) in P. aeruginosa.

As these nanoparticles transport through the living microbial cells, their sizes have been measured at the nanometer scale in real-time using single-nanoparticle optics, demonstrating that single-Ag-nanoparticle optics and single-living-cell imaging can be used to monitor the modes of action of antibiotics in real time and probe the new function of antibiotics at single-living-cell resolution with temporal (ms) and size (nm) information (Xu et al., 2004). TEM has also been used to determine the size and location of the intracellular Ag nanoparticles at subnanometer resolution, confirming the size measured using single-nanoparticle optics. Such new tools offer an important new opportunity to advance our understanding of membrane transport kinetics and MDR mechanism in living cells in real time.

3.3.2.2. Real-Time Sizing Membrane Permeability Using Single-Nanoparticle Optics

Silver nanoparticles with diameters 10-100 nm are prepared by reducing AgNO3 aqueous solution with freshly prepared sodium citrate aqueous solution as described previously (Lee and Meisel, 1982; Kyriacou et al., 2004; Xu et al., 2004). Ag nanoparticle concentration is determined by dividing the moles of nanoparticles by the volume of the solution. The moles of Ag nanoparticles are determined by dividing the number of Ag nanoparticles with Avogadro's constant (6.02 x 1023). These Ag nanoparticles are characterized using UV-vis spectroscopy, dark-field optical microscopy and spectroscopy (LSPRS), and TEM (Kyriacou et al., 2004; Xu et al., 2004).

The color distribution of single nanoparticles in 0.4 nM solution measured using dark-field microscopy and spectroscopy is used to compare with the size distribution of single nanoparticles from the same solution determined by TEM. The results indicate that the color index of violet, blue, green, and red is correlated with size index of 30 ± 10, 50 ± 10, 70 ± 10, and 90 ± 10 nm, respectively, as shown in Figure 3.7 (Kyriacou et al., 2004). The solution contains approximately 23% of violet (30 ± 10 nm), 53% of blue (50 ± 10 nm), 16% of green (70 ± 10 nm), and 8% of red (90 ± 10 nm) nanoparticles.

These multicolored nanoparticles are then used as nanometer-sized probes to directly measure the sizes of substrates that can transport through living microbial membrane, aiming to determine the change of membrane permeability and pore sizes in real time. To minimize the possible effects of competitive transports of nanoparticles with the substrates of interest (e.g., chloramphenicol) and prevent the possible aggregation of nanoparticles, a low concentration of nanoparticles (1.3 pM) is used to incubate with the living cells (OD600 nm = 0.1) for real-time monitoring of membrane transport at the nanometer scale.

Many living individual cells and single nanoparticles in the microchannel are monitored simultaneously using a CCD camera through a dark-field microscope, showing that more nanoparticles are observed in the cells as chloramphenicol concentration increases. Representative images of single cells selected from the full images illustrate that the cells contain nanoparticles in detail, indicating that more nanoparticles are present in the cells as chloramphenicol concentration increases (Figure 3.8A). Normalized histograms of the number of nanoparticles with the cells versus sizes of the nanoparticles (Figure 3.8B) indicate that a greater number of larger Ag nanoparticles are with the cells as chloramphenicol concentration increases, suggesting that the permeability and porosity of the cellular membrane increase as chloramphenicol concentration increases. Note that a higher percent of 50 ± 10-nm nanoparticles (53% of total nanoparticles) are present in the solution. Therefore, more 50 ± 10-nm nanoparticles are observed in the cells. The relative numbers of specific sizes of intracellular nanoparticles in the presence of 0, 25, and 250 ^g/mL chloramphenicol are compared to determine the change of membrane permeability and porosity as chloramphenicol concentration increases.

The integrated scattering intensity of selected individual nanoparticles in and out of cells is measured and subtracted from the background in the same image, indicating that the intensity of intracellular nanoparticles is about 10% less than extracellular nanoparticles in the solution and about 20% less than those extracellular nanoparticles on the membrane. The scattering intensity of nanoparticles decreases slightly (~10%) as nanoparticles enter the cellular membrane because the cellular membrane and matrix absorb the microscope illuminator light and reduce its intensity. The decreased intensity of intracellular nanoparticles appears to depend upon the location of nanoparticles inside the cells, such as the depth below the cellular membrane. Quantitatively, study of the dependence of scattering intensity of intracellular nanoparticles upon their locations inside the cells become impossible because optical diffraction limit defines the spatial resolution (~200 nm) and makes the nanoparticles appear larger than their actual sizes. Nevertheless, intracellular and extracellular nanoparticles can be qualitatively determined based upon their intensity changes (Xu et al., 2004).

The quantum yield of Rayleigh scattering of intracellular Ag nanoparticles is smaller than extracellular Ag nanoparticles because intracellular Ag nanoparticles are surrounded by biomolecules (e.g., proteins, lipid) that reduce the reflection coefficient of Ag nanoparticles (Mie, 1908; Bohren and Huffman, 1983, Kreibig and Vollmer, 1995). Therefore, the nanoparticles outside the cellular membrane appear to radiate more sharply and brightly, whereas nanoparticles inside the cells look blurry and dim as shown in Figure 3.9. This feature allows us to determine whether the nanoparticles are inside or outside the cells using optical microscopy. The size of the nanoparticles (<100 nm) and the thickness of cell membrane (36 nm including 8-nm inner or outer membrane and ~20 nm between inner and outer membrane of gram-negative bacteria) are below the optical diffraction limit (~200 nm). The scattering intensity of nanoparticles is much higher than that of the cellular membrane because of the higher intrinsic optical dielectric constant of Ag nanoparticles. Therefore, the nanoparticles are much brighter than the membrane, appearing to be accumulated on the membrane (Figure 3.9A). However, these nanoparticles are blurry and are dimmer than the extracellular nanoparticles (Figure 3.9B), indicating that these blurry nanoparticles are inside the cells. Taken together, the results indicate that, in the absence of chloramphenicol, the majority of red nanoparticles (> 80 nm) appear to stay outside the cells, whereas violet, blue, and green nanoparticles (< 80 nm) can enter the cells.

Figure 3.8. Real-time sizing membrane permeability and pore sizes in single living cells (WT), induced by chloramphenicol in a dose-dependent manner, using single nanoparticle optics: (A) Representative optical images (a-c) of single cells selected from ~60 cells in the full-frame images. The solutions contain the same concentration of cells (OD600nm = 0.1) and Ag nanoparticles (1.3 pM), but different concentration of chloramphenicol: (a) 0, (b) 25, and (c) 250 |g/mL chloramphenicol. Each solution is prepared in a vial and imaged in the microchannel at 15-min intervals for 2 h. The scale bar represents 2 |im. (B) Representative normalized histograms of the number of Ag nanoparticles with the cells versus sizes of nanoparticles from the solutions in (A), showing that more larger nanoparticles are accumulated inside the cells as chloramphenicol concentration increases. [Reprinted from Biochemistry 43:10400-10413, with permission of American Chemical Society.] See insert for color representation of this figure.

Figure 3.8. Real-time sizing membrane permeability and pore sizes in single living cells (WT), induced by chloramphenicol in a dose-dependent manner, using single nanoparticle optics: (A) Representative optical images (a-c) of single cells selected from ~60 cells in the full-frame images. The solutions contain the same concentration of cells (OD600nm = 0.1) and Ag nanoparticles (1.3 pM), but different concentration of chloramphenicol: (a) 0, (b) 25, and (c) 250 |g/mL chloramphenicol. Each solution is prepared in a vial and imaged in the microchannel at 15-min intervals for 2 h. The scale bar represents 2 |im. (B) Representative normalized histograms of the number of Ag nanoparticles with the cells versus sizes of nanoparticles from the solutions in (A), showing that more larger nanoparticles are accumulated inside the cells as chloramphenicol concentration increases. [Reprinted from Biochemistry 43:10400-10413, with permission of American Chemical Society.] See insert for color representation of this figure.

Figure 3.9. Determination of intracellular and extracellular Ag nanoparticles using intensity of nanoparticles: optical images of Ag nanoparticles accumulated (A) on membrane (outside the cell) and (B) inside single live cells (P. aeruginosa) recorded by the CCD with 100-ms exposure time and digital color camera through the dark-field optical microscope. Extracellular nanoparticles appear to radiate more sharply and brightly than intracellular nanoparticles because the scattering intensity of nanoparticles decreases about 10% as nanoparticles enter the cells, owing to light absorption of cellular membrane and decreased of reflection coefficient of intracellular Ag nanoparticles. The scale bar represents 2 |im. [Reprinted from Biochemistry 43:10400-10413, with permission of American Chemical Society.] See insert for color representation of this figure.

Figure 3.9. Determination of intracellular and extracellular Ag nanoparticles using intensity of nanoparticles: optical images of Ag nanoparticles accumulated (A) on membrane (outside the cell) and (B) inside single live cells (P. aeruginosa) recorded by the CCD with 100-ms exposure time and digital color camera through the dark-field optical microscope. Extracellular nanoparticles appear to radiate more sharply and brightly than intracellular nanoparticles because the scattering intensity of nanoparticles decreases about 10% as nanoparticles enter the cells, owing to light absorption of cellular membrane and decreased of reflection coefficient of intracellular Ag nanoparticles. The scale bar represents 2 |im. [Reprinted from Biochemistry 43:10400-10413, with permission of American Chemical Society.] See insert for color representation of this figure.

To determine the sizes and locations of intracellular nanoparticles at the subnanometer (A) level, ultrathin sections (70-80 nm) of the fixed cells that have been incubated with 1.3 pM nanoparticles and chloramphenicol (0,25, and 250 |g/mL) for 2 hours are prepared and imaged using TEM as described above. TEM images in Figure 3.10 unambiguously demonstrate that Ag nanoparticles with a variety of sizes (20-80 nm in diameter) are inside the cells in the absence of chloramphenicol. The majority of the nanoparticles are located in the cytoplasmic space of the cells, whereas a few nanoparticles are just underneath the cellular membrane. The cells with embedded triangular nanoparticles (Figure 3.10) are particularly selected for easy identification of Ag nanoparticles. The TEM images show that the nanoparticles with sizes ranging up to 80 nm are embedded inside the cells in the absence of chloramphenicol, which agrees well with those measured using single-nanoparticle optics, suggesting that, even in the absence of chloramphenicol, the nanoparticles with size

Figure 3.10. TEM images show sizes and locations of Ag nanoparticles inside the cells (WT), which are acquired from ultra-thin sections (70-80 nm) of fixed WT cells sliced using a diamond knife. The cells (OD600 nm = 0.1) have been incubated with 1.3 pM Ag nanoparticles in the absence of chloramphenicol for 2 h before fixation. The scale bars represent 220 nm, and the circles are used to highlight the nanoparticles. [Reprinted from Biochemistry 43:10400-10413, with permission of American Chemical Society.]

Figure 3.10. TEM images show sizes and locations of Ag nanoparticles inside the cells (WT), which are acquired from ultra-thin sections (70-80 nm) of fixed WT cells sliced using a diamond knife. The cells (OD600 nm = 0.1) have been incubated with 1.3 pM Ag nanoparticles in the absence of chloramphenicol for 2 h before fixation. The scale bars represent 220 nm, and the circles are used to highlight the nanoparticles. [Reprinted from Biochemistry 43:10400-10413, with permission of American Chemical Society.]

a 40

0 0

Post a comment