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Keywords:

  • nanoparticles;
  • side scatter;
  • titanium dioxide;
  • flow cytometry;
  • darkfield microscopy

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Evaluation of the potential hazard of man-made nanomaterials has been hampered by a limited ability to observe and measure nanoparticles in cells. In this study, different concentrations of TiO2 nanoparticles were suspended in cell culture medium. The suspension was then sonicated and characterized by dynamic light scattering and microscopy. Cultured human-derived retinal pigment epithelial cells (ARPE-19) were incubated with TiO2 nanoparticles at 0, 0.1, 0.3, 1, 3, 10, and 30 μg/ml for 24 hours. Cellular reactions to nanoparticles were evaluated using flow cytometry and dark field microscopy. A FACSCalibur™ flow cytometer was used to measure changes in light scatter after nanoparticle incubation. Both the side scatter and forward scatter changed substantially in response to the TiO2. From 0.1 to 30 μg/ml TiO2, the side scatter increased sequentially while the forward scatter decreased, presumably due to substantial light reflection by the TiO2 particles. Based on the parameters of morphology and the calcein-AM/propidium iodide viability assay, TiO2 concentrations below 30 μg/ml TiO2 caused minimal cytotoxicity. Microscopic analysis was done on the same cells using an E-800 Nikon microscope containing a xenon light source and special dark field objectives. At the lowest concentrations of TiO2 (0.1–0.3 μg/ml), the flow cytometer could detect as few as 5–10 nanoparticles per cell due to intense light scattering by TiO2. Rings of concentrated nanoparticles were observed around the nuclei in the vicinity of the endoplasmic reticulum at higher concentrations. These data suggest that the uptake of nanoparticles within cells can be monitored with flow cytometry and confirmed by dark field microscopy. This approach may help fulfill a critical need for the scientific community to assess the relationship between nanoparticle dose and cellular toxicity Such experiments could potentially be performed more quickly and easily using the flow cytometer to measure both nanoparticle uptake and cellular health. Published 2010 Wiley-Liss, Inc.

The rapid development and commercialization of man-made nanomaterials have outpaced information regarding the potential hazards of these materials to the environment, humans, or other organisms (1). This situation has prompted the US federal government to form a National Nanotechnology Initiative, which is focused on promoting the safe and responsible development of these promising new technologies (2). The US Environmental Protection Agency (EPA) has developed an overall approach for addressing man-made nanomaterials and a research strategy for the EPA Office of Research and Development (3, 4). Nanoparticles may pose unique health risks beyond those posed by larger particles of the same material due to their compositions, reactivity, small sizes, and increased surface areas (3, 5, 6). Research on potential hazards of manufactured nanomaterials presents many technical challenges, one of which is a limited ability to detect and quantify nanoparticles in environmental media, tissues, or cells that may have been exposed to nanomaterials. The development of techniques to identify and characterize nanoparticles in cells and various media would be an asset for evaluating potential risks of manufactured nanomaterials.

Key to evaluating the potential hazard of man-made nanomaterials is the observation and measurement of nanoparticles in cells. A number of research groups have used a variety of techniques to detect the presence of these particles in the environment, including transmission electron microscopy (7), scanning electron microscopy (8), and atomic force microscopy (9–11). Suspensions of nanoparticles can be assessed using dynamic light scattering (DLS) to yield a hydrodynamic diameter. While these techniques have been useful in determining the physical properties of nanoparticles, they provide limited quantitative information regarding cellular interactions with nanoparticles.

Conventional titanium dioxide (TiO2), with a particle size greater than 100 nanometers, has been used commercially for over a 100 years as a white pigment in numerous commercial and consumer products, including paints and other coating products, foods, cosmetics, and skin care products such as topical sunscreens (12). Several newer technologies have incorporated the use of nanosized TiO2. Nanosized TiO2 like many other metallic nanomaterials, scatters light with relatively high intensities for its size. This is in contrast to Rayleigh scattering, in which the intensity of light scatter in microsized particles is proportional to the particle size (the 6th power of the particle diameter) (13, 14).

These particles are available mainly in two crystalline structures, anatase and rutile, both of which efficiently reflect visible light and UVA radiation. Anatase crystals are photoreactive and serve as photocatalysts for numerous chemical reactions. This photocatalytic activity has been exploited in some products to introduce self-cleaning and disinfection upon UV exposure. In other applications, such as cosmetics or sunscreens, it may be important to limit photocatalytic activity by coating particles (15). The anatase crystal structure of TiO2 is thought to be more photoactive than the rutile (16). Rutile crystals are considered less photoreactive, but they scatter more light than anatase crystals due to a higher refractive index (anatase: 2.493, rutile: 2.903) (17). This property of rutile TiO2 has been used by the sunscreen industry to produce effective UV blockers (1, 6) and makes it possible to examine the particles through light-scattering techniques. In this experiment, two light-scattering methods were used to assess the quantity and status of nanoparticles in solution and within cells: dark field microscopy and flow cytometry.

Uncoated TiO2 nanoparticles, like other insoluble nanomaterials, tend to clump together in aqueous media (18). The terminology commonly used to describe this clumping varies. For the purposes of this experiment, the individual TiO2 particles (as characterized when dry) will be referred to as “primary particles.” Masses consisting of particulate subunits will be described as fine and coarse “aggregates” (19), with coarse aggregates appearing more loosely associated under microscopic examination.

In this experiment, two light-scattering methods were used to assess the quantity and status of nanoparticles in solution and within cells: dark field microscopy and flow cytometry. Dark field microscopy was used to confirm that particles had entered the cells and to estimate their size and number. By selectively staining cell components, the general location of the particles, changes in cell morphology, and possible interference with mitosis could also be observed.

Flow cytometers have been used in many diverse applications to measure both light scattering and fluorescence from particles or biological cells. Most flow cytometers measure the small angle forward scatter (FSC) intensity with a photodiode and side scatter (SSC) intensity with a photomultiplier tube. Generally, it is considered that the FSC provides information on the overall size of cells while the SSC provides information on internal structures and organelles (20–24).

Forward light scatter is routinely used as a measure for cellular size comparisons (12, 13, 21, 22), but it can be influenced by strongly absorbing or reflecting materials, the cell nucleus, other organelles, irregular cell shape, or damage to the cell membrane. The presence of particles inside a cell can change its refractive index (RI), which will ultimately change the FSC intensity. Therefore, FSC is often not a monotonic function of cell size (21).

The SSC intensity is mainly thought to be determined by the internal structure of the cell. It is also related to cellular mass and possibly protein content as well (12, 21–23). The SSC signal is affected by the RI of the cytoplasm and the number and type of organelles present in the cell. SSC has been used to show differences in the physical state of the cell, including mitosis, particle uptake, and sperm decondensation (20–32). Cell populations can be defined by their specific light-scattering clusters in a cytogram (plot of FSC vs. SSC), and then the amount of fluorescence from specific probes in various channels can be correlated to the scatter patterns of the specific cell populations.

The rationale behind this experiment is based on the premise that morphologically different blood cells will scatter 488-nm laser light differently in the forward and side directions. The scatter is believed to be based on the cell size, cell surface, and intercellular refractive index (12, 21, 22). When flow cytometry was in its infancy, it was discovered that lymphocytes, monocytes, and granulocytes could be differentiated by their unique FSC and SSC properties. Salzmann showed that larger monocytes scattered more light than smaller lymphocytes (13). Granulocytes scattered the most light in the 90-degree direction, due to granules located in the cytoplasm. We report in this manuscript that, in a similar manner to granules in granulocytes, nanoparticles located inside cells will increase the scatter of light in the 90-degree direction in a dose-dependent manner.

In this study, dark field microscopy and flow cytometry were used to monitor the effects of TiO2 nanoparticles on a human-derived line of retinal pigment epithelial cells (ARPE-19). These cells were used in a related project to evaluate potential photoactive mechanisms of nanomaterial toxicity because the retinal pigment epithelium is an important site in mediating ocular phototoxicity (33). Microscopy is used to visually confirm the uptake and intracellular distribution of nanoparticles. In flow cytometry, the addition of nanoparticles to the cytoplasm can cause increased SSC and decreased FSC (12, 21, 22). These technologies could also provide a quantitative basis to estimate cytotoxicity.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Cell Culture

For microscopic observations, ARPE-19 cells (ATCC, Manassas, Virginia) were plated in T75 culture flasks in a 1:1 mixture of Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient Mixture (DMEM/F-12) with 10% fetal bovine serum (FBS). Cells were grown to confluency, trypsinized, and plated on chambered glass tissue culture slides (1 ml cell suspension per chamber, 2 × 105 cells/ml). After plating, cells were incubated for 24 h (37°C, 5% CO2) without TiO2, then treated with different concentrations of TiO2 nanoparticles for a further 24 h before staining and fixation.

For flow cytometry, ARPE-19 cells were plated in seven T75 culture flasks (5 × 104 cells/ml) in DMEM/F-12 with 10% FBS. Cells in flasks were incubated for 24 h without TiO2, then treated with different concentrations of TiO2 nanoparticles for 24 h. They were then trypsinized while still in proliferative phase growth, centrifuged, brought up in 2 ml media, and placed on ice before analysis.

Nanoparticle Suspension and Treatment

TiO2 primary particles [nominally rutile, 30–40 nm, Nanostructured and Amorphous Materials original source (NanoAmor), Houston, TX Cat#5485HT] were obtained. The dry materials were characterized under a contract (EPA contract #EP-D-08-074, University of Kentucky) that included analysis of surface area/porosity using a Brunauer Emmett Teller (BET) test, primary and aggregate particle size by transmission electron microscopy (TEM), dynamic light scattering (DLS), crystal structure by X-ray diffraction, and particle shape and morphology by TEM and BET. These analyses determined that the primary material was a mixture of rutile and anatase crystalline structures. In addition, the material had a BET surface area of 22.2 m2/g, corresponding to a primary particle size of about 70.1 nm. By TEM and DLS, the average particle size was about 57 nm, with 90% of the sample distribution between 36 and 97 nm. Most of the primary particles were associated with compact aggregates 400–800 nm. By SEM most of the mass was in large aggregates 5–15 μm.

To prepare dosing solutions, the dry TiO2 particles were weighed and suspended in DMEM/F-12 with 10% FBS. The suspension was then sonicated (until suspension appeared homogeneous to the naked eye) and characterized by dynamic light scattering (DLS, Malvern Zetasizer Nano) and microscopy. FBS in the dispersant was found to provide a relatively stable, monodispersed particle suspension on DLS evaluation. At a concentration of 3 μg/ml, an average hydrodynamic diameter of ∼800 nm was observed, with a polydispersity index of 0.523, indicating a fairly disperse solution containing a range of nanosized particles and larger aggregates.

ARPE-19 cells were treated with nanoTiO2 at 0, 0.1, 0.3, 1, 3, 10, and 30 μg/ml. Cellular reactions to nanoparticles were evaluated using a flow cytometer and an E-800 Nikon microscope containing a xenon light source and special dark field objectives. Preliminary results suggested that 24-h incubation allowed most of the particles to settle onto the cells or onto the slide bottom, with larger coarse aggregates appearing under microscopy to form during the incubation and sediment more quickly than smaller particles. Flow cytometry experiments and most microscopic observations were done at 24 h of treatment, with some microscopy done at both 4 and 72 h.

Staining, Fixation, and Mounting

To determine viability, cell cultures were incubated with calcein-AM and propidium iodide (PI), which stain cells with intact or compromised membrane integrity, respectively. After flow cytometry analysis, the remaining cells were fixed with an equal amount of 4% paraformaldehyde (PF). In addition, about 0.2 ml cells that were used for flow cytometry were immediately fixed with an equal amount of 4% PF for later observation by microscopy and flow cytometry. The fixed cells were stained with DNA probes including DAPI, HO3342, YoPro1, SYTOX green, and SYTOX orange to visualize the nuclei and nanoparticles within the cells.

Microscopy

A Nikon E-800 microscope was used to observe dark field and fluorescence images. This microscope had space for five filter cubes. The fluorescence excitation cubes were for DAPI, FITC, TRITC, and Cyan GFP. The fifth space was intentionally left without a cube to acquire a clean dark field image without any distortion from filters. However, the dark field image was so bright that it could be observed through any of the four filter cubes. The dark field was about 100 times brighter than the fluorescence image as measured by exposure times. The combination of fluorescence and dark field images was made sequentially with Nikon Elements software. Colocalization was measured with 0.5-μm Tetra spec beads and 1 and 15-μm multiwavelength ring beads (Molecular Probes). Xenon light supply was used to optimize the shorter wavelength excitation that provided for better resolution. A GG420 filter was put in the eyepieces to protect the user's eyes from possible UV damage from this light source.

The most suitable Nikon lens when using a Nikon infinity-corrected microscope was a 60× Plan Fluor with an iris diaphragm to control the numerical aperture (NA) between 0.55 and 1.25. The lens had a sufficiently large magnification to observe cellular details while the background scatter could be controlled by adjusting the diaphragm. The lower NA yielded good depth of field for bright nanoparticles, and the higher NA (1.25) yielded bright fluorescence images of the cellular components. By balancing the fluorescence and dark field signals, a sequential image could be acquired with the same NA setting (∼0.8 NA). During the course of this study, the dark field images were as obtained using the following dry lenses: Plan Apo 20×, (NA 0.75), Plan Apo 40× (NA 0.95), and Achromat 60× (NA 0.8) and the following oil lenses: 20× multi-immersion (NA 0.75), Achromat 100× with iris (NA 1.25–0.55), and 60× Plan Fluor with iris (NA 1.25–0.55). NA values had to be below 1.0 with an oil darkfield condenser to provide proper illumination for good dark field images of nanoparticles. Most of the work was done by observing the field first with a 20× MI oil lens and then taking the picture with the 60× Plan Fluor oil lens with variable NA to optimize both the fluorescence and dark field images.

Flow Cytometry

A BD FACSCalibur™ (BD Biosciences, San Jose, CA) flow cytometer containing a 488-nm laser, FSC diode detector, and photomultiplier tube SSC detector was used in this study. This instrument was checked daily for fluidic alignment using Molecular Probes 2.5-μm and Duke 3.0-μm alignment beads. The coefficient of variation on the Molecular Probes beads was under 2% for the fluorescent channels and under 3% for the scatter channels. Because the flow rate affects these measurements, they were always performed at low flow rates. If the coefficient of variation was higher than 2.0% for the fluorescence channels, the cytometer was cleaned with 10% Clorox bleach followed by 10% Contron 70 (34, 35). The cytometer was set up to measure SSC logarithmically and FSC linearly. Calcein-AM and PI were measured using log amplifiers. The dynamic ranges of the PMTs were optimized to show the maximum changes for the TiO2 doses used. The highest dose of nanoparticles was run first to set the range for the maximum SSC signal and the minimum FSC signal.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Microscopy

Nanoparticles gradually sedimented onto ARPE-19 cells and passed through the cell membrane. Once the nanoparticles entered a cell, they appeared to migrate to the endoplasmic reticulum (ER) and surround the nucleus (Zucker et al., in preparation). In many cells, the nucleus was seen as a distinct object with nanoparticles outlining it. (Fig. 1) The particles did not appear to penetrate into the nucleus, though some aggregates may have been in the cytoplasm between the nuclear envelope and the nucleus. This nanoTiO2 distribution was seen in both live and fixed cells. The particles also appeared to aggregate within the cytoplasm (Fig. 1). This aggregation was dependent in part on the concentration of particles added to the growing cells.

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Figure 1. Combined dark field and fluorescent image. ARPE-19 cells incubated in slide chambers for 24 h with 10 μg/ml nano-TiO2. Cells were fixed with PF and mounted with Prolong (Molecular Probes) containing 10 μg/ml DAPI. Images of cell nuclei (blue) and nanoparticles (white) were acquired sequentially. Cells and nanoparticles are pseudo colored with cell nuclei appearing blue, and TiO2 appearing as white dots or larger coarse aggregates. Magnification 600×. Plan Fluor 60× lens with a NA between 1.25 and 0.55 was adjusted to about 0.70 NA to reduce background for dark field imaging. Images of fluorescent cells and dark field nanoparticles were acquired sequentially and then combined using Nikon Elements software.

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When examined by dark field microscopy, control cells contained no particles. At the lowest doses (0.1 and 0.3 μg/ml), only a few aggregates (∼5–10) were observed in the cytoplasm of each cell (Fig. 2A). More aggregates were discernable at 3μg/ml (Fig. 2B) and 10μg/ml (Fig. 2C). At the highest dose (30 μg/ml), the cytoplasm was completely filled with TiO2 material (Fig. 2D). Representative images shown in Figure 2.

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Figure 2. Combined dark field and fluorescent image. Nanoparticles (A: 0.1 μg/ml; B: 3 μg/ml; C: 10 μg/ml; D: 30 μg/ml) were incorporated into ARPE-19 cells for 24 h in T-Flasks Cells in suspension that were trypsinized and used for flow cytometry were fixed in suspension with 4% PF for a final concentration of 2%. Nanoparticles are shown in white and nuclei were stained with DAPI and shown in blue. Magnification 600×. Plan Fluor 60× lens with a NA between 1.25 and 0.55 was adjusted to about 0.70 NA to reduce background for dark field imaging Images of fluorescent cells and dark field nanoparticles were acquired sequentially and then combined using Nikon Elements software.

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Flow Cytometry

At the highest concentration tested (30 μg/ml), TiO2 showed a minimal decrease in cell viability (∼2%) as measured by PI membrane permeability. NanoTiO2 had a dose-dependent effect on the cells that was measurable using the FSC and SSC parameters. From the cytogram and histogram analysis, there were overlapping distributions with the different doses of nanoparticles used on the cells. The histogram and cytogram distribution between 0.3 and 30 μg/ml showed a monotonic increase in mean intensity of SSC with increasing dose (Figs. 3 and 4). The 0.1 μg/ml dose also showed greater SSC than the untreated control cells (data not shown). There was a 10-fold increase in SSC between control and 30 μg/ml (Fig. 4). Corresponding to this increase in SSC was a dose-dependent decrease in the FSC between 3 and 30 μg/ml (Fig. 5). The FSC was not as sensitive to TiO2 as the SSC. As previously shown with human granulocytes, this FSC value decreased if the SSC signal was great, due to light scattering in all directions and not being transmitted to the FSC detector (21, 22). To get a better perspective on the magnitude of the changing light scatter distribution, the mean data of cells treated with TiO2 concentrations of 0.1 to 30 μg/ml are shown in Fig. 6 and Table 1. This linear relationship suggests that the amount of nanomaterials inside the cells is related to the dose applied to the cells. Since the SSC parameter can be measured using a flow cytometer, it gives us the possibility of correlating cellular nanoparticle content with observable cellular toxicity and viability effects in future experiments.

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Figure 3. A cytogram of FSC (linear scale) and SSC (log scale) showing that SSC increased as FSC decreased. Panel A shows control and 10 μg/ml, while Panel B shows control, 3, 10, and 30 μg/ml, which have overlapping distributions. Doses of 1 μg/ml and below, which have distributions overlapping with those of control cells are not shown for clarity of the figure.

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Figure 4. The SSC (log scale) increased as the dose increased from 0.3 to 30 μg/ml. The dose 0.1 μg/ml is not shown for figure clarity, but there was an observable increase compared to control at that level.

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Figure 5. A decrease in FSC was observed as the SSC values increased. The high level of light scattered in the 10 and 30 μg/ml doses decreased the light going to the forward detector.

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Figure 6. Relative changes in SSC vs. the TiO2 dose added to control cells. These changes correlated to the histogram values in Figure 4. Data correlated the amount of nanoparticles that were added to cell cultures to the amount that was detected in the cell. Future studies will correlate this data to the measurable health of the cell.

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Table 1. Unfixed cells: Mean side scatter changes
SAMPLE (μg/ml)RATIO (TREATED/CONTROL)
Control1
0.11.09
0.31.17
11.44
32.33
104.83
3010.49

The dose-dependent effects of a decreasing FSC and increasing SSC were mostly maintained in the fixed cells (Fig. 7, Table 2), with only a slight decrease in SSC and population resolution compared to that of unfixed cells. This suggests that the nanoparticles contained within cells scattered a sufficient amount of light and the laser interaction with nanoparticles was not inhibited by the 2% PF cellular fixation process. This is significant as it indicates samples can be acquired, fixed, and measured at a later date maintaining the scatter parameter changes after fixation.

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Figure 7. The SSC (log scale) increased as the dose increased from 1 to 30 μg/ml. Cells fixed with 2% paraformaldehyde showed a similar increase as in Figure 4 in SSC as the dose was increased. Fixed cells, scale expanded.

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Table 2. Fixed cells: Mean side scatter changes
 RATIO (TREATED/CONTROL)
SAMPLE (μg/ml)EXP 1EXP 2
Control11
11.51.6
32.12.2
104.14.0
307.98.7

These fixed cells were also used for dark-field microscopy. A representative image of four doses of cells treated with TiO2 is shown in Figure 2. The cellular morphology shown in Figure 2 was correlated with the flow cytometry light scatter data as shown in Figures 3 and 4. The same fresh cells that were used for flow cytometry measurements (shown in Figs. 3–5) were also fixed immediately in 2% PF for future flow cytometry or microscopic analysis observations. The dark field microscope representative images shown in Figure 2 correlate with the flow cytometry data shown in Figures 4 (unfixed cells) and 7 (fixed cells). X axis had been expanded using FCS Express.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The uptake of TiO2 by ARPE-19 cells was observed in a dose-dependent manner with both dark field microscopy and flow cytometry. Low concentrations of TiO2 resulted in visible individual aggregates, while cells treated with high concentrations showed fine and coarse aggregates throughout the cytoplasm. In all cases, particles tended to be present in perinuclear rings, collocating with the ER and the Golgi apparatus. When these TiO2-treated cells were examined by flow cytometry, the SSC increased and FSC decreased with increasing TiO2 concentrations. The results from these two techniques were concordant, with more particles visible in cells that showed increased flow cytometry SSC. The light scattering properties of TiO2 allowed detection and observation by both flow cytometry and dark field microscopy.

NanoTiO2 scattered more short-wavelength light than long-wavelength light, so a shorter-wavelength light source (xenon light source with 400-nm blue emission) was used to yield increased scatter and resolution compared to a standard halogen light source that emits predominantly longer wavelength light. Because the TiO2 nanoparticles scattered a large amount of light, they appeared extremely bright under dark-field microscopy. The exposure times from nanoparticle images using darkfield microscopy were about 100 times faster than exposure times of cellular constituents using fluorescent imaging. The scattering detected by the microscope's dark field optics was so efficient that aggregates appeared in the cell as larger sized submicron- or micronsized objects. The apparent particle size was optically increased because the microscope detected the amount of light scattered by the particle, not the actual size of the particle.

Salzmann observed high SSC in granulocytes due to the reflections of the granules (13). This observation was key to the ability to classify white blood cells by their scatter differences. In a similar manner, the amount of light scattering of TiO2 nanoparticles in cells was detected by flow cytometry. In another study, 40-nm gold nanoparticles attached to antibodies were shown to have an over tenfold increase in SSC compared to control (32). While light scattering is typically proportional to a particle's size, it can also be dependent upon the particle's material. For example, gold scatters more light than silver (12, 32). It is conceivable that metal ions like gold, silver, or titanium will exhibit surface plasmon resonance, resulting in the scattering of a large amount of laser light (12, 20, 36–41). It may be possible to detect using flow cytometry other nanoparticles that scatter less light than TiO2 by increasing the detection optics of the scatter signal and using a lower wavelength laser (405 or 372 nm) to excite the particles instead of the standard 488-nm signals.

It is an oversimplification to accept the premise that forward scatter is always a direct measure of cellular size. Forward light scatter is usually, but not always proportional to the particle volume. The forward scatter parameter can be influenced by a number of variables. For example, Shapiro gave an example from Kevin Becker's research in which 8 u beads appear smaller in forward scatter than 7u beads (12). The presence of strongly absorbing or reflecting materials in cells also tends to decrease the amplitude of the forward scatter light. Forward scatter is also affected by the wavelength of light and the range of angles in which the light is collected. The possibility of the absorption of light at the illumination wavelength by nanoparticles may indeed decrease in the FSC signals. The presence of internal structures, such as granules in granulocytes may also decrease the FSC signals. Another factor that can influence the FCS parameter include the differences between the RI of the cells and the suspending medium. In fact as a cell dies the RI of the interior of the cell decreases and the FSC also decreases. In this study, TIO2 was shown to be located inside the cells by darkfield microscopy and there was a corresponding decrease in the FSC signals. Possible explanations for the decrease in FSC and increase in SS include the absorption of light by the particles or reflection of light by the particles reducing the amount of light reaching the forward scatter detector.

Side scatter is generally thought to be related to both the granularity of the cell and the cell mass. Side scatter parameter has been shown to change if the RI of cells increases or decreases (28). In the case of gold Nano particles bound to the surface of the cell, the side scatter increases, presumably due to surface plasmonic resonance of the gold (32). Smaller mitotic nuclei show decreased side scatter relative to larger interphase nuclei (24, 25), presumably due to smaller size. In the sperm decondensation process, the sperm become gradually larger with the RI inside the cell decreasing and becoming similar to the suspending medium. This process shows a decrease in both the side scatter and the forward scatter signals (28). It is clear, that there are many factors that can influence light scatter in the forward and side directions.

Different flow cytometers, cell types, concentrations, and types of nanoparticles have been previously assessed by flow cytometry (26, 27). Stringer et al. showed that diesel particles, TiO2 and quartz could be observed in alveolar macrophages (26). Suzuki et al. found that TiO2 moved into the cytoplasm, but not the nucleus of Chinese hamster ovary cells (27). Both of these authors used very high concentrations of TiO2 to observe side scattering effects and toxicity. The current study observed a fourfold increase in SSC at 10 μg/ml relative to control. In contrast, Suzuki saw only a minimal increase in SSC at this concentration, with most of the changes occurring at 200 μg/ml and above. However, the nanoparticle suspensions applied to the cells in different studies may not be equivalent with regard to aggregation in the cells and thus the studies may not be directly comparable. Another advance in the current experiment is the correlation of dark field microscopy observations with flow cytometry patterns. At doses of 1 μg/ml and below, dark field microscopy could be used to detect about 5–10 nanoparticle aggregates per cell, which were correlated to a small increase in SSC compared to control. Our work clearly shows an increased resolution and sensitivity of our flow cytometer ability to detection nanomaterials compared to previous studies of Suzuki and Stinger (27, 28).

This manuscript describes a new technique to study TiO2 nanoparticles in cells using light scatter detection by flow cytometry. The effect is confirmed with darkfield microscopy. Other new techniques and approaches to detect, count, and measure small particles are continually being developed in the scientific community. Investigators have tried to push the limits of detection by using bright fluorescent stains to observe particles or membrane-bound vesicles with a flow cytometer or confocal microscope (42–44). A Raman spectral flow cytometer has been used to measure particles in the far red range (45, 46). Although light scatter is not the primary focus of most flow cytometry research that usually deals with surface antigens or DNA staining, it is apparent that light scatter continues to be a very interesting and useful parameter for the scientific community. Investigators are continually finding new and useful data in the measurement of cellular size, refractive indices and internal structures (47, 48). Recently the light scatter parameter has been proposed as a method to monitor changes in intercellular structures in cells that have responded to chemicals and disease (42, 48).

The light-scattering of nanoparticles occurs from gold nanoparticles and quantum dots (Q-dots) (32, 43). These materials serve as excellent markers that have high quantum yields and are detectable by both flow cytometry and microscopy. They can be injected into cells to specially deliver chemicals or stain specific parts of the cells. Both gold nanoparticles and Q-dots have been proposed to help view organelles within the cells (43, 49). The unique properties of these small particles increase the signal-to-noise ratio of the staining reaction (42, 43, 49–52).

The cell can responds to the chemicals and microparticles by undergoing apoptosis, necrosis, or other cellular changes (53). There is a need to understand these cellular processes that are stimulated by nanoparticles, microparticles, and chemicals. We have found in this study that darkfield microscopy can be used to observe cellular morphological changes induced by nanoparticles and flow cytometry light scatter can be used to measure the relative amount of nanomaterials that is absorbed by the cell. Light-scattering techniques, such as dark-field microscopy and flow cytometry enable the quantification of light-reflective nanomaterials within cells (54). In the future, we plan to correlate changes in the functional cellular processes with the amount of light scattered (flow cytometry) and the cellular morphological changes (darkfield and fluorescent microscope). Microscopic and scatter measurements could indeed be combined with specific fluorescent probes to measure viability, membrane potential, reactive oxygen species, or other biochemical characteristics (54, 55). This combination will enable evaluation of potential harmful effects of nanoparticles in a more quantitative and efficient manner.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Thanks are extended to John Rogers, Chris Lau, Julian Preston, and Kevin Dreher for their helpful comments and encouragement for many aspects of the article and to Jim Birk of BD for restoration of a flow cytometer that was inactive for three years after a hurricane damaged an EPA Laboratory

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED