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1 May 2010 Quantitative visualization of colloidal and intracellular gold nanoparticles by confocal microscopy
Author Affiliations +
Abstract
Gold nanoparticles (AuNPs) have the potential to become a versatile biomarker. For further use of AuNPs labeled with functionalized molecules, their visualization in biological systems by routine laboratory tools such as light microscopy is crucial. However, the size far below the diffraction limit affords specialized parameters for microscopical detection, which stimulated the current study, aimed to determine from which size onward AuNPs, either in dispersion or cell-associated, can be reliably detected by standard confocal microscopy. First, gold colloids of size-restricted fractions are examined in dispersion. At a minimum particle size of 60 nm, detection appears to be reliable. Particle counts in dilution series confirm these results by revealing single particle detection of 60-nm colloids. Second, AuNPs are visualized and quantified in cells, which interestingly cause a phase shift in the reflection of AuNPs. Gold mass spectroscopy confirms the number of AuNPs counted microscopically inside cells. Furthermore, it demonstrates for the first time a very high diffusion rate of 15-nm particles into the cells. In conclusion, the results back the suitability of confocal microscopy for the quantitative tracking of colloidal and intracellular gold nanoparticles sized 60 nm.

1.

Introduction

The visualization of nanomarkers has gained increasing importance in the fast developing area of biomedical imaging. As the absorption and scattering cross sections of metal nanoparticles are several orders of magnitude higher compared to fluorescing organic dyes,1, 2, 3 metal nanoparticles have the potential to become very efficient biomarkers. Among these, laser-generated colloidal gold nanoparticles appear to be biocompatible4 and are fairly easily bioconjugated for further functionalization.5 Additionally, metal nanoparticles do not suffer from photobleaching, and their extinction spectrum can be tuned by changing their size and shape.2 However, their size far below the diffraction limit affords special conditions for optical detection. Furthermore, in vivo applications in cellular systems afford detection systems not compromising the viability of the cells. Various approaches for the visualization of AuNPs have been described recently.6, 7, 8 Many of these, such as dark and near-field light microscopy and confocal microscopy (LSCM), are based on the surface plasmon resonance (SPR)-enhanced scattering of AuNPs.7, 8, 9 However, so far all reported approaches of single particle detection were performed with fixed nanoparticles. Thus, single 40-nm -sized particles8, 10, 11 were successfully visualized fixed on a glass surface, but not in a colloid. Besides SPR-enhanced scattering, SPR-enhanced absorption can be used for the visualization of AuNPs. While the scattering cross section is high for particles larger than 50nm , for particles smaller than 30nm the absorption prevails, and scattering is practically not apparent for visualization of particles with diameters of less than 20nm according to the Mie theory.3, 6 Lindfors demonstrated both detection methods again for fixed AuNPs between 5 and 100nm by applying a supercontiuum white light confocal microscopy.12 They recorded size-dependent scattering and absorption spectra using either a piezoelectric element and a charge-coupled device (CCD) camera or an imaging spectrometer. This study demonstrated surface-fixed particles larger than 50nm as suitable for detection by their scattering cross sections and confirmed previous data.1, 2

However, the visualization and quantification of AuNPs as nanomarkers in biological systems does not allow for AuNP fixation on a surface. Consequently, the visualization of nanoparticles in dispersion is required, which might have a variety of implications on imaging parameters. Additionally important for visualization in biological systems is the suspected red shift of AuNP SPR that generally occurs with increasing refractive index of the surrounding medium.13 Thus, the aim of the current study was to define parameters allowing the visualization and quantification of colloidal and intracellular laser-generated AuNPs by conventional confocal microscopy, which represents a standard laboratory tool.

AuNP colloids were excited with continuous single wavelength laser excitation, and the light microscopic limits of AuNP visualization by SPR-enhanced light scattering and photoluminescence were investigated. The size-dependent visibility of the AuNPs was studied in two different series: while the first series was adjusted to a constant mass (CM), the second series was adjusted to a constant particle number (CN). To test whether single particle detection is possible, a titration series of 60-to80-nm -sized nanoparticles was performed, of which single particle visibility was implicated by the previous experiment. Subsequently, these AuNPs were coincubated with a cultured cell line to study the intracellular visualization and quantification of laser-generated AuNPs. Quantitative mass spectrometry for the determination of gold allowed for an independent verification of the microscopic data.

2.

Methodology

2.1.

Generation and Examination of Gold Nanoparticles

The applied laser-based approach to nanoparticles consists in the ablation of a target in liquid media by intense laser radiation, leading to an ejection of its constituent and the formation of a colloidal nanoparticle solution.14, 15 In the study at hand, AuNPs were generated using a femtosecond laser system (Spitfire Pro, Spectra-Physics, Santa Clara, California) delivering 120-fs laser pulses at a wavelength of 800nm (maximum energy 400μJ per pulse, beam diameter 4mm ). The pulse energy was fixed to 200μJ at a repetition rate of 5kHz . The principle setup is described as follows. The laser beam was focused on 5×5-mm gold foil (99.99% purity), thoroughly cleaned and placed on the bottom of a Petri dish filled with 4-mL bidistilled water. The plate was placed on an axis system that moved at a constant speed of 1mms1 in a spiral with outer radius of 2mm and inner radius of 0.4mm . Time of irradiation was fixed to 12min corresponding to three spirals.

The resulting colloid was characterized by UV-visual spectroscopy using a Shimadzu 1650 (Shimadzu Europe GmbH, Duisburg, Germany) and TEM (EM 10 C electron microscope, Zeiss, Oberkochen, Germany).4, 16 500 nanoparticles were counted for the determination of the average Feret diameter (Fig. 1 ). AuNP concentration was estimated by weighing (Sartorius M3P-000V001, Sartorius AG, Göttingen, Germany) the gold foil three times prior and after laser ablation with an accuracy of 1μg .

Fig. 1

Number- and mass-weighted size distribution of laser-generated gold nanoparticles in water estimated by centrifugation. Insert: scheme of nanoparticle generation by laser ablation in liquids and representative transmission electron micrograph of nanoparticles prior to centrifugation.

036015_1_061003jbo1.jpg

2.2.

Nanoparticle Size Separation

Size separation of AuNPs was performed via successive centrifugation steps of 500μL of the colloid with a Hellma Universal 320 centrifuge Rotor 1612. The time of centrifugation (t) was fixed to 15min , and the rotation velocity (v) was calculated via Eq. 1 (for detailed values see Table 1 ).

Eq. 1

v=602πln(xxm)ts,
where x is the radius bottom, xm is the radius meniscus, and s is the sedimentation coefficient.

Table 1

Centrifugation parameters for nanoparticle size separation ( t=15min each).

Size class [nm]rpm [min−1]
> 100 1500
80 to 1001800
40 to 602500
20 to 407000
<20 supernatant 7000

The supernatant was separated from the pellet and used for the following centrifugation step. The pellet was redispersed in 100-μL H2O , and size distributions and optical properties were characterized by dynamic light scattering and UV-visual spectroscopy, respectively.

Nanoparticle mass concentrations (cm) were estimated via recorded intensities at 380nm , which were converted by interpolation from a linear standard calibration curve. The wavelength of 380nm corresponds to the interband transition of gold and is hardly affected by nanoparticle size, shape, and environment. The number concentration (cn) was subsequently calculated via Eq. 2 assuming a density of GNPs of p=19.3gcm3 , where r is the mean radius of each group.

Eq. 2

cn=3cm4πr3ρ.

Two different size-separated series were established. While one of them was adjusted to constant particle mass concentrations ( 50μgmL respective 250μM Au=CM ), a second series was adjusted to constant particle number concentrations ( 11010 AuNPmL=CN ).

2.3.

Preparation of Gold Nanoparticles in Dispersion for Laser Scanning Confocal Microscopy Analysis

Colloidal AuNPs of each of the size fractions of both series were analyzed in 0.5-μL ddH2O resting in a 5-μL silicon oil cushion. All colloids were sealed under a cover slip. Imaging was performed within two hours after preparation.

Thus the number of particles included in each size fraction within CM increased with decreasing particle size (Table 2 ). In CN, particle fractions consist of decreasing masses with smaller sizes (Table 2).

Table 2

Size-dependent reciprocal relationships of AuNP mass and particle number.

Diameter(nm)Particle mass (Pm) in fgNumber ofAuNPs in 500fg
150.0341414645.57
200.080936178.38
400.64742772.30
501.26449395.42
602.18504228.83
703.46976144.10
805.1793596.54
10010.1159249.43
20080.927366.18

2.4.

Cell Culture and Preparation for Laser Scanning Confocal Microscopy Analysis

The cells used in the coincubation studies were derived from a bovine immortalized endothelial cell line (GM7373) obtained from the Cell Culture Collection, Institute of Infectology, FLI, Riems, Germany). Cell growth conditions and postcoincubation treatment corresponded to previously described trials.4 For LSCM analyses, these prepared cells were mixed 1:4 with Vectashield (Axxora, Loerach, Germany) and mounted on a slide within a paper reinforcement ring sealed by a cover slip and nail varnish.

2.5.

Gold Nanoparticle Coincubation Assays

Coincubation assays were performed using AuNPs in a final concentration of 50μM . After seeding, cells were allowed to attach and were then exposed to the nanoparticles for 48h . The added AuNPs displayed either the complete size range with an average particle diameter of 15nm or separated size fractions of 40to60nm and 60to80nm , respectively. Cells incubated without AuNPs for 48h were used as controls. All experiments were carried out in duplicates and repeated three times.

2.6.

Imaging by Laser Scanning Confocal Microscopy

Visualization was carried out with a confocal laser scanning system LSM510 at an Axioplan 200 (Carl Zeiss MicroImaging GmbH, Jena, Germany) within the spectrum of visible light. Laser wavelengths of 514nm (argon, 15mW ), 543nm (helium neon green, 1mW ), and 633nm (helium neon red, 5mW ) were used to excite the SPR or luminescence of the colloidal AuNPs and the AuNPs in cultured bovine cells. Light scattering for each of the excitation wavelengths was recorded in multitracking mode in combination of four separate detection channels within fixed spectral bands. Complete configurations are summarized in Table 3 . The colloidal AuNPs were investigated dispersed in bidistilled water using the configurations described in Table 3. After focusing the edges of the silicone oil cushion, a volume of 10pL centered within the liquid volume, avoiding reflections at the surfaces by exclusion of 2to3μm next to each glass, was imaged. The focus of cells was adjusted using the differential interference contrast (DIC) contrast as reference.

Table 3

Configurations for surface plasmon resonance (SPR) and luminescence detection of AuNPs. Laser power is measured at the object level with 100% laser output. Laser power at 0.54 has performance at 5.9A.

Excitationwavelength(nm)DetectionLaserpower(mW)Mainbeam splitterDetectionbandpass(nm)DetectionchannelDetectiongainDetectionoffset
514ScatteredSPR0.54 8020 505 to 5453250 to 400 0.02
543ScatteredSPR0.11 8020 530 to 5502250 to 400 0.02
633ScatteredSPR0.53 8020 > 585 1250 to 500 0.05
514Lumines-cence0.54 488543 633530 to 5702700 to 800 0.03
543Lumines-cence0.11 488543 633545 to 5703700 to 800 0.05
633Lumines-cence0.53 488543 633 > 635 1700 to 800 0.1
633Differentialinterferencecontrast(DIC)0.53 488543 633NoneTransmission250 to 300 0.5

2.7.

Statistics

Statistical analyses were performed with the software SigmaStat 3.0 (StatCon, Witzenhausen, Germany). Quantification of LSCM data was performed on five repetitive measurements for the colloidal nanoparticles and ten fields of view for each of the size groups of the AuNPs in cells. Data of reflecting pixel numbers were normalized by square root transformation and were applied to one-way analysis of variance with the pair-wise multiple test by Holm-Sidak (p=0.05) .

2.8.

Mass Spectroscopic Quantification of Gold in Bovine Immortalized Cells

The summarized analysis of the gold concentration in the cells was performed by inductively coupled plasma mass spectrometry (ICPMS) by a certified laboratory (Wessling GmbH, Hannover, Germany). Samples of 200μL containing approximately 3×106 cells were analyzed. The particle numbers (Np) were calculated using the sample-specific cell numbers (nc) and the mass of gold within the sample (mau) for each group using Eq. 3. Pm refers to the mean particle masses within the groups.

Eq. 3

Np=mau106ncPm.

3.

Results and Discussion

3.1.

Physicochemical Characterization of Gold Nanoparticles

The laser-generated aqueous gold colloid is characterized by a broad size distribution, covering the range of 3to200nm with an average diameter of 15nm as determined by transmission electron microscopy (Fig. 1, insert). The size classification by centrifugation reveals that the mass-weighted distribution is centered at around 50nm , while the resulting number-weighted distribution shifts to smaller nanoparticles (Fig. 1). More than 80% of the nanoparticles seem to have a size of less than 20nm .

In accordance with the Mie theory,17 UV-visual spectra of the size fractions reveal a red shift and a broadening of the SPR with increasing mean diameter (Fig. 2 ). By normalizing the size fractions to a constant nanoparticle number of 1010AuNPmL , one additionally observes a significant intensity enhancement of the SPR for bigger nanoparticles. The SPR of nanoparticles with a diameter of less than 20nm is thus hardly visible. This fact can be explained by the higher extinction coefficient of bigger nanoparticles and is even detectable when adjusting the fractions to a constant nanoparticle mass, although the nanoparticle number is significantly lowered with increasing diameters (Table 2).

Fig. 2

UV-visual spectra of gold nanoparticle size fractions after centrifugation normalized to constant nanoparticle mass concentrations of 50μgmL (CM, left) and constant nanoparticle number concentrations ( CN=1010AuNPmL , right).

036015_1_061003jbo2.jpg

3.2.

Visualization of Size-Restricted Colloidal Gold Particles by Laser Scanning Confocal Microscopy

Representative images taken of a volume of about 10pL (31.3×31.3×10.21μm) of colloidal AuNPs of each size group of CN and CM are shown in Figs. 3, 3, 3, 3 and Figs. 3, 3, 3, 3, respectively. The number of particles contained in the visualized volume of about 10pL of each fraction of CM was calculated using the average diameter and is added in Figs. 3, 3, 3, 3. As the particles were not fixed on the glass surface, the appearing irregular arrays of pixels could reflect the Brownian motion of diffusing particles. Time series after single laser excitation, reduced field of view (2.4pL) , and short exposure times 3μspixel) verified this assumption.18 As these arrays reflect single particles, counting of the pixels provides a good indication of particles accessible to objective image analysis although the pixel numbers are higher than the number of particles contained in the sample.

Fig. 3

Representative images of size-restricted AuNPs in 10-pL volumes (31.3×31.3×10.21μm) . (a) through (e) show images from mass-constant dispersions (CM). (f) through (j) show all colloids which were set to an equal number of AuNPs independent of their size (CN). Size of AuNPs was > 100nm in (a) and (f); 60to80nm in (b) and (g); 40to60nm in (c) and (h), and 20to40nm in (d) and (i). Excitation was performed at 514, 543, and 633nm with the corresponding SPR shown in blue, green, and red, respectively. Pixel size is 80×80×700nm , and pixel time is 5.44μs . The box plots in (e) and (j) represent the median with 25 and 75% quartils of reflecting pixels in each of the size classes after excitation at 514nm , scale 1μm . (Color online only.)

036015_1_061003jbo3.jpg

Interestingly, and probably due to differences in size or arrangement, a part of single objects in the samples seem to reflect selectively one or the other excitation wavelength, while others show colocalization of reflection of the different excitation wavelengths. Thus, the quantification of the numbers of reflecting pixels was performed for each of the three detection bandpasses and in both series of size-separated colloidal AuNPs.18 The SPR was shown previously quantified for all three reflection channels of CM (see Fig. 3 of Ref. 18). Exemplarily, the data from the excitation at 514nm are shown [Figs. 3 and 3] as they accounted for the highest number of pixels due to the proximity to the reflection maximum of gold.

Dispersions with constant particle mass (500fgAu10pL) contain an increasing number of particles with decreasing diameter. Microscopic visualization reveals this number increase for the 100-to80-nm (data not shown) and 80-to60-nm samples compared to AuNPs > 100nm . However, patches of AuNPs > 100nm [Fig. 3] appear bigger than the 80-to60-nm sized particles [Fig 3], resulting in a higher pixel count for the > 100-nm fraction [Fig. 3]. This probably reflects the size difference, as the reflection of particles in the > 100-nm fraction might occupy more than one pixel. Particles 40to60nm in diameter only provide a reduced number of reflective spots [Fig. 3], and from the particles 20to40nm only single objects contained signal intensities sufficiently high for microscopic detection, despite the 2150 particles included in the visualized volume [Table 2 and Fig. 3]. From AuNPs smaller than 20nm , no reflection could be recorded (data not shown). Thus, the reduced extinction of particles smaller than 60nm measured by UV-vis spectroscopy (Fig. 2) can be correlated with a loss in scattering cross section, confirmed via limited microscopic imaging of these particles. Interestingly, all these findings result in a rather continuous decrease of pixel counts with decreasing particle diameter within CM. In contrast, a sharp drop of reflection was recorded between 60- to 80- and 40-to60-nm sized particles in all three evaluated spectral bands in CN, confirming the incomplete detection of single particles with a diameter <60nm . In the CM, this reduction of the scattering cross sections with reduced particle size is counteracted by the higher particle numbers and higher proximity of the single particles that enhanced the total reflection. However, it must be recognized that in this case, a bulk of particles adds up to one reflective spot. Furthermore, in the smaller particle groups [Figs. 3 and 3], reflection was dominant at 543-nm excitation (green), indicating preferential detection of aggregated particles having a phase shift of their SPR to longer wavelengths.1, 2, 6, 12, 19, 20 In both series, only the group of 60-to80-nm sized AuNPs [Fig. 3 and 3] shows intensive reflection in all three bandpasses. In groups of particles > 100nm [Figs. 3 and 3], the red band provides the largest clusters, although the total number of pixels reflecting in this bandpass comprises only about 10 to 20% compared to 514- and 543-nm excitation.

To conclude, visualization of colloidal AuNPs seems to be possible down to particle sizes of 60nm , while for fixed nanoparticles the visualization of single 40-to60-nm sized AuNPs has been reported. Additionally, a broadened absorption spectrum was seen for these surface-spotted AuNPs as described by others.2, 3 Doublets of single spherical AuNPs are frequently detectable in TEM images9, 21 and could be suggested from the shape of reflective spots we observed from surface-spotted particles in our study.18 This arrangement could also explain the improved imaging of fixed particles in other studies.9, 12 Furthermore, spectral luminescence of AuNPs (size 50nm±15% ), spotted on a glass slide, was previously reported by Geddes 9 However, from our data, luminescence could not be visualized from colloids of the 60-to80-nm sized group.

The missing reflection of particles sized smaller than 20nm and severe reduction of visibility of particles between 20 and 40nm is in agreement to the very low scattering previously described.1, 12 Measuring the SPR-enhanced absorption would therefore be necessary to visualize these size ranges below 40nm for quantification.12

3.3.

Quantification of Titrated 60-to80-nm Colloidal Gold Nanoparticles by Laser Scanning Confocal Microscopy

To verify whether single particles or clusters of particles were imaged, a volume of 10pL (31.4×31.4×10.2μm) of a dilution series from the AuNPs (60to80nm) was excited with laser wavelengths at 514, 543, and 633nm , and the reflecting pixels were counted. The stock dispersion of 100fg10pL was diluted down to 1fg10pL in six steps. In the 1-fg10pL dilution, only one out of ten fields of view provided a signal from four AuNPs. The detection gain was increased by 15% to allow for detection of very low reflecting spots. Therefore, this dilution step was not included in the statistical analysis; however, the data for this group are shown in Figs. 4 and 4 . However, as the particle mass of single particles sized 70nm is 3.47fg , the detection of four reflecting spots in one out of ten fields of view fits nicely with the expected appearance of one particle in about 40pL of this dilution, and proved the detection of single particles of 60-to80-nm size by confocal microscopy.

Fig. 4

(a), (b), and (c) are representative images and (d) and (e) are statistical ANOVA results of square root transformed reflecting pixel numbers in dilution series of AuNPs (60to80nm) . (a) represents a particle concentration of 100fg10pL , (b) stands for 10fg10pL , and (c) for 2fg10pL . The square root transformed pixel numbers (d) and (e) correspond to the expected reduction of pixels from separate detection of single nanoparticles only in the more diluted samples. Pixel size is 80×80×700nm , pixel time is 4.32μs , image is 25×25×10μm , and scale 1μm . Asterisks show colloids of the 1-fg dilution that provided reflection only after increased detection gains and were excluded from the statistics.

036015_1_061003jbo4.jpg

The minimum pixel size from 80×80nm up to 220×220nm applied in the current study is not suited to reflect the true size differences of AuNPs between 40to100nm . Moreover, a summary of pixels at one position might reflect agglomerates of AuNPs1, 2 or the pathway of a single particle moving freely in aqueous environment during acquisition (Fig. 3). Thus, single particle detection is most accurate for AuNPs of 60to80nm , if the interparticle distance is high enough to separate the signals of two individual AuNPs (Fig. 4).

3.4.

Visibility of Gold Nanoparticles after 48-hours of Coincubation within Bovine Immortalized Cells and Mass Spectrometric Validation

The potential of conventional confocal microscopy to quantify AuNPs was reinvestigated in bovine immortalized cells that had been coincubated with AuNPs for 48h . Reflection intensity increased in the cellular environment, and the spectral characteristics were shifted to longer wavelengths. Luminescence was not recorded after excitation at 543 or 633nm from colloidal AuNPs, but was apparent from AuNPs within cells (Fig. 5 ). Although due to the cellular background, the detection offset was raised for the discrimination of AuNPs within cells and the gain reduced, the recorded intensities of reflection increased for all bands of detection.

Fig. 5

Size restricted AuNPs ( 10-mM Au) of a mixed sample with mean particle size of 60to80nm [(a) and (d)], size selected AuNPs of 40to60nm [(b) and (e)], and of 15nm [(c) and (f)] are visualized as colloids in ddH2O [(a) and (c)] and after 48h of coincubation in GM7373 cells [(d), (e), and (f)]. Pixel size is 90×90×600nm , pixel time is 1.28μs , and image is 33×33×12μm . The figures show the overlay of four imaged channels each. The dispersed AuNPs were visualized in reflection bands after excitation at 543 and 633nm , respectively, and two luminescence bands (at 543-, and 633-nm excitation) as described in Table 3. However, exclusively SPR was visualized from the dispersed AuNPs of all size groups resulting in the green dominated false color images. The AuNPs in cells [(d), (e), and (f)] are shown as an overlay of reflection after 543-nm excitation, and luminescence after 543-nm and 633-m excitation added to the DIC transmission defining the position of the single cells. The orthogonal views at a single optical slice of the series of 900-nm -thick optical sections through a single cell (g) in comparison to the 3-D projection of this cell (h) indicate the within-cell location of several nanoparticles, scale 5μm . (Color online only.)

036015_1_061003jbo5.jpg

Imaging conditions were optimized for dispersed AuNPs achieving 100 to 200 gray values as background and avoiding intensities above the differentiation range. Typical intensity distributions are demonstrated in supplementary information (Figs. 6 and 7 ). Only pixels with intensities above 700 were included in the quantification to ensure a signal-to-noise ratio (SNR) above 3. Signals with low intensities were recorded typically above 1000, but most spots reached intensities of 2000 to 3500 from 4096 gray values.

Fig. 6

Intensity profiles of three channels from the volume of 10pL , including dispersed 60-to80-nm AuNPs of Fig. 5. The green line represents SPR after excitation at 514nm . The background values of the blue and red lines were recorded in the luminescence mode after 514- and 633-nm excitation. (Color online only.)

036015_1_061003jbo6.jpg

Fig. 7

Intensity profiles of four channels from a single optical slice of a Z-series through cells demonstrated as 3-D projection in Fig. 5. The green line represents SPR after 514-nm excitation. Within the cells, additional signals were recorded from the same AuNPs in luminescence after excitation at 514 and 633nm . The gray line corresponds to the DIC transmission. (Color online only.)

036015_1_061003jbo7.jpg

The localization of AuNPs in cells appears to inhibit the free movement of AuNPs as seen in colloids, and thus enables counting of reflecting objects instead of pixels in cells [Figs. 5, 5, 5]. This confirms previous data where the enclosure of single AuNPs in distinct electron dense structures after diffusion in the cells was demonstrated.4 Moreover, a high degree of colocalization for the different reflection bands was obvious in contrast to the dispersed particles. Although reflection bands of 505 to 545 and > 585nm were recorded for the dispersed AuNPs as well as from the cells, AuNPs were provided only from cell reflection signals and additional detectable luminescence signals from the longer wavelengths, indicating a further red shift of the SPR due to the increased refractive index of the cellular environment.

The impressive SNR and stability of the reflection compared to the fading of fluorescence dyes is further augmented if the unfixed cells, washed only once in phosphate buffered saline (PBS), were investigated (Fig. 8 ). Low excitation of autofluorescence within the reflection bandpass enabled similar clear differentiation of the AuNPs within the cells.

Fig. 8

Cells coincubated with 60-to80-nm sized AuNPs for 48h . Visualization was performed from unfixed cells washed once in PBS and mounted in Vectashield. Scale 5μm .

036015_1_061003jbo8.jpg

After the 48-h coincubation period with AuNPs sized 60to80nm , a median of 11.6 particles per cell was found in 100 cells (Table 4 ). Coincubation of cells with the mixed size sample (average particle diameter 15nm ) and AuNPs sized 40to60nm resulted in particle numbers with a median of 13.8 and 9.1 particles per cell, respectively. As an independent verification of the microscopically counted number of AuNPs inside cells, gold mass spectrometry was performed from a fraction of the same cell cultures. The number of particles recalculated from the total mass of gold found in approximately 3×106 cells confirmed that the particle counts for the size group of 60to80nm , retrieved from the microscopic evaluation, were accurate (Table 4). These data also revealed by this visualization independent measure that particles smaller than 60nm were only incompletely visualized by confocal microscopy. A mean of nine particles could be counted, but a mean 27 particles per cell had been calculated from the mass spectrometry in the sample incubated with particles of 40to60nm diameter. Especially for the size group of about 15nm , only a minute number of the AuNPs that penetrated the cells were detected microscopically by their scattering cross sections. Only 13.8 spots per cell were visualized; however, 8634 particles per cell must have been present within the cells according to the mass spectrometric data recorded from the same cell culture (Table 4).

Table 4

Mass spectrometric and microscopic quantification of gold (Au) in cells coincubated with AuNPs of different size classes. The asterisk indicates cells had been counted in individual samples after coincubation in uniform 3miocells∕ml and final processing.

AuNPsize(nm)Mass spectrometryMicroscopic cell countsparticles/cell (median of 100cells; 25 and 75% quartiles)
Cells (n∕μL)* Mass Au (ng∕μL) Mean particlemass (fg)Particles/cellcalculated
1590612.6710.03414863413.8 (9,4; 15.9)
40 to 60170050.5831.26449279.1 (4.5; 11.9)
60 to 80167500.5983.469761011.6 (9.4; 15.9)

Taking into account that the colloid AuNPs could not be detected if they had been smaller than 40nm , it is somewhat surprising that we saw any particles in the mixed AuNP group with a mean of 15nm diameter. One possible explanation is that the detected particles could be the few AuNPs of bigger size, which are to be expected in this group. On the other hand, signals detected with the microscope could also have come from agglomerated nanoparticles.

It was also surprising that ICPMS analysis found such tremendous amounts of particles with mean diameters of 15nm inside cells. Previous studies22 have indicated the best penetration efficiency for 50-nm AuNPs compared to smaller ones. However, in those cases, no visualization independent measure of AuNPs was evaluated. Nevertheless, our previous studies revealed diffusion as the most probable mechanism for cellular uptake of unconjugated, laser-generated AuNPs.4 The different surface characteristics of laser-generated AuNP in contrast to chemically synthesized ones might also be responsible for these differences in cell penetration.

Diffusion, a main pathway for cell penetration, was not discussed yet for AuNPs. Instead, endosomal pathways were presumed as uptake mechanisms of these gold nanoparticles into the cells. Taylor 4 demonstrated diffusion as the most feasible mechanism for cell penetration of the unconjugated AuNPs. As all the size-restricted samples of unconjugated laser-generated AuNPs appear in small discrete signals throughout the cells, the presence of oxidized Au+ and Au3+ providing a positive surface charge might be responsible for this unique behaviour.

4.

Conclusions

The investigation of size-separated colloids of laser-generated gold nanoparticles clearly show unrestricted visibility of AuNPs of 60nm and larger by confocal laser scanning microscopy. The scattering cross section of SPR for particles of this size can be visualized to the single particle level. A cumulative effect of scattered light from several single particles or formation of aggregates is necessary to provide detectable scattering cross sections in the groups with 40to60nm and 20-to40-nm AuNPs. Larger AuNPs are difficult to separate as colloids. Interestingly, colloidal AuNPs reflect different spectral bands within the same size group. Inside cells, the AuNPs sized 60to80nm can also be quantified in distinct compartments with a high chance of single particle separation. Within cells, a spectral shift to longer wavelengths is seen and the AuNPs gain additional luminescence that could be used for visualisation (Fig. 5).

The defined size-dependent optical detection limit of spherical AuNPs could thus be used for status-dependent visualization of AuNPs. As conjugated laser-generated AuNPs are clearly reduced in their size range to about 5nm in total,5 these functionalized AuNPs are not accessible to light microscopic detection by their scattering cross sections. However, after clustering of a few single particles, for instance at a hybridization focus, the successful hybridization could be recorded without any washing of nonhybridized particles. This would provide one further important characteristics for applications in living biosystems.

Acknowledgments

The project was supported by the excellence cluster REBIRTH, Masterrind GmbH Verden, and the NBank Niedersachsen.

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©(2010) Society of Photo-Optical Instrumentation Engineers (SPIE)
Sabine Klein, Svea Petersen, Ulrike Taylor, Detlef Rath, and Stephan Barcikowski "Quantitative visualization of colloidal and intracellular gold nanoparticles by confocal microscopy," Journal of Biomedical Optics 15(3), 036015 (1 May 2010). https://doi.org/10.1117/1.3461170
Published: 1 May 2010
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