HS imaging of AuNPs in varying refractive indices
Glass slides were cleaned using piranha acid (7:3 mixture of sulfuric acid to hydrogen peroxide. Caution: very strong acid and oxidizer!) for 15 min followed by ethanol and DI water rinses. Slides were then immersed in 10% (3-mercaptopropyl)trimethoxysilane (MPTMS) in ethanol for at least 4 h, rinsed with ethanol, and dried with N2 gas. MPTMS-functionalized slides were baked at 80°C for 30 min to strengthen the glass-silane bond and kept in ethanol until use. A uniform submonolayer of NPs was immobilized on the slides by immersing slides in 20 mL of 1.12 × 109 NP/mL 100-nm citrate-stabilized spherical AuNPs (Ted Pella, Redding, CA, USA) for 90 min on an orbital shaker, rinsing with ethanol and ultrapure water (Milli-Q, Millipore, Billerica, MA, USA), and drying with N2 gas. HS images were collected for the same NP substrate with air (n = 1.00), water (n = 1.33), or glycerol (n = 1.47) present between the top of the substrate and the cover slip. The same field of view (defined by the coordinates of a motorized stage) was observed under each condition, and the substrate was rinsed with Milli-Q and dried with a gentle stream of N2 gas before each switch into another refractive index medium.
Mie calculations were performed using a MATLAB code adapted for light scattering from concentric spheres. Extinction, scattering, and absorption coefficients were calculated based on Mie scattering coefficients. Plane-wave illumination was assumed to obtain the Mie coefficients. This assumption is justified by our use of subdiffraction-limit sized AuNPs and the fact that they were located far from the illuminating objective compared to the wavelength of visible light. Gold properties were adapted from Johnson and Christy .
Characterizing spectral shift of NPs from water to CPRFM
To assess initial changes to the NP spectra upon incubation in complete phenol red-free cell culture media (CPRFM) prior to cellular uptake, extinction spectra of NPs before and after transfer to CPRFM was measured with an Agilent Cary 60 UV-vis spectrophotometer. CPRFM consisted of phenol red-free McCoy's 5A media (HyClone, Waltham, MA, USA) containing 10% v/v human off-the-clot type AB serum (PAA Laboratories, Pasching, Austria) and 1% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Citrate-stabilized spherical AuNPs (100 nm, Ted Pella, Redding, CA, USA) were centrifuged, redispersed in CPRFM, and incubated at 37°C in 5% CO2 in a cell culture incubator. NPs in water (negative control) and CPRFM alone were also kept under same conditions. All spectra were collected with water as the baseline. Spectra for CPRFM alone were measured at each time point and subtracted from the corresponding spectra of NPs incubated in CPRFM.
Measuring optical spectra for NPs introduced to cellular environment
Sk-Br-3 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in McCoy's 5A media supplemented with 10% v/v human off-the-clot type AB serum and 1% penicillin-streptomycin and maintained at 37°C in a 5% CO2 incubator. Sk-Br-3 cells were plated on Lab-Tek II CC2 4-well chamber slides at a density of 100,000 cells/well and grown to 70% confluence. After 24 h, culture media was removed and cells were incubated with AuNPs in CPRFM for 2, 5, 10, or 24 h in an incubator at 37°C and 5% CO2. AuNPs in CPRFM were prepared by centrifuging citrate-stabilized AuNPs (100 nm, Ted Pella, Redding, CA, USA) at 2,000 g for 20 min, redispersing in CPRFM, sonicating, passing through a 0.22-μm polyethersulfone (PES) sterile filter (Millipore, Billerica, MA, USA), and diluting in CPRFM to final concentrations of 12, 24, 48, or 96 μg/mL. Following incubation, cells were rinsed 3x with 1X Dulbecco's phosphate-buffered saline without magnesium and calcium (PBS, Invitrogen, Life Technologies, Grand Island, NY, USA), fixed using 4% formaldehyde (15 min, BD Biosciences, San Jose, CA, USA), and rinsed again 2x with PBS. Chamber slides with cells and internalized NPs were then wetted with PBS, covered with a cover slip, sealed with nail polish, and viewed under an Olympus BX-41 upright microscope (Olympus, Center Valley, PA, USA) coupled with a CytoViva high-resolution illuminator at 40x magnification (Plan Fluorite, 0.75 NA, Olympus, Center Valley, PA, USA).
Spectral data across the sample field of view were collected using the hyperspectral imaging system connected to the CytoViva microscope (CytoViva, Auburn, AL, USA). Samples were illuminated with a quartz halogen lamp with aluminum reflector (400 to 1,000 nm) and spectral data cubes were obtained by automated movement of the sample using a X-Y motorized stage (Prior, Rockland, MA, USA) across a transmission diffraction grating spectrograph with 2.8-nm resolution (Specim, Oulu, Finland). Darkfield HS images were taken at the slide plane as well as 5 μm above the slide plane (controlled by a Prior motorized z focus drive with 0.002-μm minimum step size; see Additional file 1 for more details). HS images were analyzed using the instrument's ENVI software (ITT Visual Information Solutions, Boulder, CO, USA). Cell regions of interest (ROIs) were defined by tracing the outline of cells with the polygon ROI tool. In order to objectively define ROIs without bias toward cells that appeared to have greater uptake of NPs, all cells in each HS image were identified as long as the cell boundaries could be unambiguously identified. NPC ROIs were defined by selecting 12-pixel ellipse-shaped ROIs encompassing the image pixels for NPCs (see Additional file 1 for details). Spectral data averaged across the pixels of each ROI was extracted by the ENVI software. The spectral data was calibrated for variations in lamp intensity by dividing by the normalized lamp spectrum.
Peak wavelength determination
Spectral peak wavelengths were objectively determined using the peak analyzer function of OriginPro 8.6 Data Analysis and Graphing Software (OriginLab, Northampton, MA, USA). Savitzky-Golay smoothing with a window size of 50 was first performed on spectral data followed with peak finding using a local maximum method with two local points. Calculated peak wavelengths were then manually checked against the plotted spectra to confirm accuracy. When spectra contained multiple peaks, the peak with higher intensity was analyzed.
Since peak wavelength could not be accurately identified in spectra that were dominated by cell scattering and had insufficient NP plasmonic spectra contributions, we excluded these spectra in our analysis. To objectively classify which spectra lacked sufficient signal for analysis, we inspected the spectra's normalized intensity at 500 nm. Spectra that had a normalized intensity at 500 nm of 0.95 or higher were deemed cell scattering-dominated and thus not included in the analysis.
Peak broadening measurement
A custom program was created in MATLAB to smooth and calculate the peak broadening of the spectra. Spectral data was first smoothed using a Savitzky-Golay algorithm (chosen to maximally preserve the spectral shape and intensity) of degree 2. All points in the smoothed spectrum were then shifted downward by a constant value equivalent to 95% of the spectrum's maximum intensity. The smoothed and shifted spectrum's two points of zero crossing represented the span of the spectrum at 95% of the spectrum's maximum intensity. The width of the spectrum at 95% of the maximum intensity was then calculated by taking the difference of the two points of zero crossing.
Evaluating cell viability
Cell viability after incubation with NPs was evaluated by performing a Live/Dead viability assay (Life Technologies, Grand Island, NY, USA). Following NP exposure, cells were rinsed 3x with PBS, incubated with Live/Dead reagent for 15 min at room temperature, and imaged at 20x magnification with a Zeiss Axio Observer.A1m inverted fluorescence microscope (Zeiss, Jena, Thuringia, Germany). Live cells and dead cells were visualized using filters with the following excitation and emission specifications: Ex 480 ± 20 nm/Em 535 ± 25 nm and Ex 560 ± 27.5 nm/Em 645 ± 37.5 nm (Chroma, Bellows Falls, VT, USA), respectively. Numbers of live and dead cells were counted using ImageJ (National Institutes of Health, Bethesda, MD, USA). Cell viability was determined as number of live cells/number of total cells × 100%.
Following incubation with NPs, cells were rinsed 3x with 1X PBS, fixed with 2.5% formaldehyde/2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences, Hatfield, PA, USA) at room temperature, and kept at 4°C overnight. After fixation, the samples were washed in 0.1 M cacodylate buffer and treated with 0.1% Millipore-filtered buffered tannic acid, postfixed with 1% buffered osmium tetroxide for 30 min, and stained en bloc with 1% Millipore-filtered uranyl acetate. The samples were washed several times in water, then dehydrated in increasing concentrations of ethanol, infiltrated, and embedded in Spurr's low-viscosity medium. The samples were polymerized in a 60°C oven for 2 days. Ultrathin sections were cut in a Leica Ultracut microtome (Leica, Buffalo Grove, IL, USA), stained with uranyl acetate and lead citrate in a Leica EM stainer (Leica, Buffalo Grove, IL, USA), and examined in a JEM 1010 transmission electron microscope (JEOL USA, Inc., Peabody, MA, USA) at an accelerating voltage of 80 kV. Digital images were obtained using AMT Imaging System (Advanced Microscopy Techniques Corp, Woburn, MA, USA).
Electric field simulation
The electric field was calculated using the RF module in COMSOL Multiphysics. The simulation geometry consisted of a spherical far-field scattering domain with a radius of 500 nm, a perfectly matched layer of 350-nm thickness, and clusters of AuNPs whose center coordinates are supplied in Additional file 1: Table S2. Gold properties were adapted from Johnson and Christy . The refractive index of water was set to 1.33. The scattered field was calculated with a background electric field linearly polarized in the x direction and propagating in the z direction. The definition of the E-field with respect to the AuNP cluster geometry is shown in Additional file 1: Figure S11. Direct solvers were used for the simulation. Computation was performed by a workstation equipped with dual 6-core Intel Xeon X5690 3.46 GHz processors and 144 GB of RAM. The E-field was visualized in the middle cross section (xy plane) of the cluster.
In comparisons of spectral peak wavelengths or spectral widths among different NP exposure times and doses, statistical significance was evaluated by performing one-factor analysis of variance (ANOVA) followed by a post hoc Tukey's HSD test for multiple comparisons. P values less than 0.01 were considered statistically significant.