Quantitative Imaging of Silver Nanoparticles and Essential Elements in

Article Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

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Quantitative Imaging of Silver Nanoparticles and Essential Elements in Thin Sections of Fibroblast Multicellular Spheroids by High Resolution Laser Ablation Inductively Coupled Plasma Time-ofFlight Mass Spectrometry Akihiro Arakawa,*,†,‡ Norbert Jakubowski,§ Gunda Koellensperger,∥ Sarah Theiner,∥ Andreas Schweikert,∥,¶ Sabine Flemig,† Daigo Iwahata,‡ Heike Traub,† and Takafumi Hirata⊥ Downloaded via GUILFORD COLG on July 24, 2019 at 06:50:11 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Bundesanstalt für Materialforschung und-prüfung (BAM), Richard Willstaetter-Strasse 11, 12489 Berlin, Germany Research Institute for Bioscience Products and Fine Chemicals, Ajinomoto Co., Inc., Suzuki-cho 1-1, Kawasaki-ku, Kawasaki-shi, Kanagawa 210-8681, Japan § Spetec GmbH, Berghamer Strasse 2, 85435 Erding, Germany ∥ Institute of Analytical Chemistry, University of Vienna, Waehringer-Strasse 38, 1090 Vienna, Austria ¶ Institute of Inorganic Chemistry, University of Vienna, Waehringer-Strasse 42, 1090 Vienna, Austria ⊥ Geochemical Research Center, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan ‡

S Supporting Information *

ABSTRACT: We applied high resolution laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) with cellular spatial resolution for bioimaging of nanoparticles uptaken by fibroblast multicellular spheroids (MCS). This was used to quantitatively investigate interactions of silver nanoparticles (Ag NPs) and the distributions of intrinsic minerals and biologically relevant elements within thin sections of a fibroblast MCS as a three-dimensional in vitro tissue model. We designed matrix-matched calibration standards for this purpose and printed them using a noncontact piezo-driven array spotter with a Ag NP suspension and multielement standards. The limits of detection for Ag, Mg, P, K, Mn, Fe, Co, Cu, and Zn were at the femtogram (10−15 g) level, which is sufficient to investigate intrinsic minerals in thin MCS sections (20 μm thick). After incubation for 48 h, Ag NPs were enriched in the outer rim of the MCS but not detected in the core. The localization of Ag NPs was inhomogeneous in the outer rim, and they were colocalized with a single-cell-like structure visualized by Fe distribution (pixel size of elemental images: 5 × 0.5 μm). The quantitative value for the total mass of Ag NPs in a thin section by the present method agreed with that obtained by ICP-sector field (SF)-MS with a liquid mode after acid digestion.

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Silver (Ag) nanoparticles (NPs) are currently applied in a variety of consumer products and are also attractive for medical applications.8 With their widespread use, the potential for human exposure to Ag NPseither intended or unintendedis increasing. Therefore, many studies have evaluated the toxicity and transport mechanism of NPs. For a quantitative study, inorganic metallic NPs can be determined by LA-ICP-MS7 if matrix-matched calibration standards are applied.9,10 However, in most cases, reference materials are not available for calibration of LA-ICP-MS for the analysis of soft materials, and matrix matching of laboratory-prepared standards is the most straightforward and common procedure. Becker et al. were the first to apply customized laboratoryprepared standards to quantitative imaging of biological

or bioimaging, many different analytical techniques using fluorescence, matrix-assisted laser desorption ionizationmass spectrometry, or optical microscopy are available and subject to continual improvements.1,2 However, none of these methods are quantitative.3 Recently, synchrotron X-ray fluorescence imaging4,5 and laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS)6,7 are shown as promising strategies for quantitative bioimaging. In this study, we chose LA-ICP-MS because of the following benefits: (1) little sample preparation, (2) multielement capabilities, (3) large dynamic range, (4) low limits of detection, and (5) calibration possible by matrix-matched standards. Additionally, the method is well-established for bioimaging, widely distributed, and already often applied for biological samples. For LA-ICP-MS, the sample introduction takes place by laser ablating a sample line-by-line. Detected multiple line scans of isotopes are converted into a two-dimensional intensity profile representing the local distribution of target elements. © XXXX American Chemical Society

Received: May 13, 2019 Accepted: July 2, 2019 Published: July 2, 2019 A

DOI: 10.1021/acs.analchem.9b02239 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry samples by LA-ICP-MS.6 Matrix matching by mixing liquid standards with homogenized biological materials is timeconsuming and challenging for investigations of thin tissue sections. Matrix-matched standards are usually made of tissue homogenate,11 gelatin,12 or synthetic polymers13,14 spiked with known amounts of a standard. In the work of Drescher et al., nitrocellulose membranes were applied as a matrix of carbonrich materials for single-cell analysis.15 In our previous work, we pipetted calibration standards by hand onto polymer substrate, and later, we used a pin-type array spotter. In that case,16 the droplet volume was reduced to 0.61 ± 0.14 nL and the droplet diameter to around 300 μm, which is much closer to a diameter relevant in tissue samples. To date, most NP uptake studies have been performed in two-dimensional models where adherent cells are grown on microscope slides. However, most of our organs and tissues have a three-dimensional cell distribution. Consequently, three-dimensional structures are becoming increasingly important in toxicological and medical studies. Among the three-dimensional structures, multicellular spheroids (MCSs) are the most prominent because of their spherical geometry. Some applications with tumor MCSs measured by LA-ICP-MS have already been published.17−20 Theiner et al. were the first to use LA-ICP-MS for imaging of platinum (Pt)-based drugs applied for cancer therapy in thin sections of tumor MCSs.17 A heterogeneous distribution of Pt was observed in thin sections of different tumor spheroids with the highest concentration in the centers and the outer cell layers of the tumor MCSs.18 In the past decade, new applications using mass cytometry have been reported.21 Mass cytometry,22 a single-cell analysis method based on ICP-time-of-flight (TOF)-MS, is a powerful tool to study single cells and allows (quasi)simultaneous detection of element-tagged antibodies across the mass range of 75 to 209 Da (with the new device “Helios”) on a single-cell level. This multidimensional high throughput analysis has been predominantly applied in the field of immunophenotyping since it was introduced in 2009.23 Then, its application has expanded to the absolute quantification of inorganic metal-NPs associated with single cells.24,25 However, even with the most advanced instrumentation, the present CyTOF technology is limited in the working mass range. Therefore, we applied an alternative ICP-TOF-MS instrument covering the whole mass range for investigation of essential biologically relevant elements (such as P, Fe, Cu, Zn) as well as NPs. Recently, to study NP transport through a biological cell barrier, we produced fibroblast MCSs, incubated them with NPs, and used a sector field (SF) ICP-MS instrument for detection of NPs in thin sections.26 Quantification was hampered by the settling time of the SF instrument, which caused severe data losses. Therefore, the scan speed of the device also limited the number of isotopes that could be investigated with high spatial resolution. LA-ICP-TOF-MS looks promising for quantification and improvement of the multielement coverage. Although ICPTOF-MS was pioneered by Hieftje’s group,27 a new instrument developed from the work of Günther’s group at ETH Zurich (Zurich, Switzerland) was launched commercially a few years ago.28 Initial applications showed the main advantages of ICPTOF-MS, and they later used this prototype instrument for laser ablation studies of biological samples.29 The aim of this study is to establish an analytical methodology using a LA-ICP-TOF-MS to quantitatively investigate the interactions between Ag NPs and fibroblast

MCSs as a three-dimensional in vitro model. Additionally, we investigated localization of Ag NPs in the model with cellular spatial resolution. For this purpose, we tried to apply a noncontact piezo-driven array spotter for the first time to print a series of multielement calibration solutions with picoliter volumes onto a polymer-coated plastic slide for multielement calibration of intrinsic elements in single cells and of NP suspensions. Finally, we compared the quantitative results from LA-ICP-TOF-MS with those measured by ICP-SF-MS after acid digestion.

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EXPERIMENTAL SECTION Preparation of Calibration Standards. Detailed information is described in the Supporting Information. Sample Preparation. Sample preparation for LA-ICP-MS analysis was carried out in the way shown in our previous study with minor modifications.26 A detailed description is provided in the Supporting Information. LA-ICP-TOF-MS Imaging. Detailed information regarding the instrumentation is described in the Supporting Information. Acid Digestion of a Thin Section. The experimental procedure for acid digestion of thin sections is described in the Supporting Information. Data Analysis. The method for data processing is described in detail in the Supporting Information. Each isotopic image was generated by iQuant2, which was developed by Suzuki et al.30 Integration of each signal intensity and line scans were carried out with ImageJ.31

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RESULTS AND DISCUSSION Multielement Calibration by Use of a Piezo-Driven Array Spotter. The calibration concept is based on an array spotter which is used for printing calibration spots on a polymer slide and was applied here for the first time for a multielement calibration approach. A prerequisite for calibration is knowledge of the volume of each droplet generated by the spotter. In the software, we set the droplet volume to 350 pL and then validated this by taking a photograph of a droplet and using it to measure the diameter and calculate the volume. Additionally, we validated the reliability of droplet generation by spotting (see the Supporting Information for a detailed discussion). From these measurements, we confirmed that the software settings were reliable and that picoliter-sized droplets could be generated with high accuracy and precision. Calibration spots on the polymer slide were arranged in a grid of 6 × 5 spots (30 spots in total) (Figure S-3). All eosin stained (red) spots were printed very reproducibly with a consistent diameter (130 μm). This diameter is smaller than the laser diameter used for calibration. A defined region (black dashed box, Figure S-3) was used for imaging of the calibration series. Only one spot containing PBS was visible in this region because of salt deposition. As an example, we ablated this region (black dashed box, Figure S-3) using the imaging mode for the element Ag to visualize the calibration spots (Figure 1). 109 Ag spots were visible in the concentration range from 10 to 1 μg mL−1. The line scan of the Ag image is provided as Figure S-2. As shown in the line profile, spike signals were observed. From that, we concluded that the NPs are not dispersed homogeneously, possibly forming aggregates locally during the drying step. Concerning the multielement solution, we observed for some elements a “coffee ring effect” with B


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Analytical Chemistry

the intensity values for both the solution and suspension standards were clearly above the blank value, and the error bars of five consecutive measurements overlapped. This means that there is no significant difference between Ag in ionic form or in suspension if we use single-shot mode. Additionally, we prepared calibration curves for those elements with intensities above the blank value (Figure S-5). The calibration curves for 26 Mg, 31P, 39K, 55Mn, 56Fe, 59Co, 63Cu, 66Zn, and 109Ag showed good linearity as a function of the total amount of the calibration standard. For 32S, the calibration curve was rather flat because this isotope was affected by spectral interference from oxygen (O2). However, the background signal was very stable, and it might be possible to apply calibration for those samples with higher sulfur contents. Sulfur is an important element for biological studies as it is a component of two amino acids in proteins. For Ca, the background signal was high and quite unstable even though 44Ca was chosen to avoid interferences originating from Ar. For all isotopes except 32S and 44Ca (Figure S-5), the limits of detection were estimated as three times the standard deviation (3σ) of the blank (Table 1). For the imaging mode, mean values and standard

Figure 1. 109Ag image of calibration spots. Columns 1−4 indicate each region for 50 Ag NPs (column 1) and 3500 (column 2), 350 (column 3), and 35 (column 4) fg Ag ionic forms, respectively.

enrichment at the outer rim or in the center. However, because we integrated the whole spot area, the integrated intensity is directly proportionally to the number of atoms (total amount) ablated, independent of their homogeneous distribution. In the imaging mode, calibration spots of the lowest concentrations were hardly visible because the average mass of Ag in each analytical pixel was only 1.2 ag if it was distributed homogeneously. For the lowest concentration applied (Figure S-4), the integrated intensity of 35 fg of Ag in the suspension was in the same range as the blank value, whereas the signals for the ionic Ag standard had much higher integrated intensities than the blank. This problem is obviously related to data acquisition and how intensity values are calculated by blank correction. The calibration spots measured in imaging mode can be used for calibration. In one of our recent publications, we used a different calibration approach by single-shot analysis.26 In this case, the laser diameter is larger than the calibration spot and is ablated by single shots. Therefore, we also investigated the single-shot mode for comparison with the imaging mode. The main advantage of single-shot for calibration is that it has a much shorter analysis time, which leads to higher sample throughput. The analysis time for a series of calibration spots measured in imaging mode (five spots at four concentrations) is about 2 h. We used single-shot laser ablation of the whole calibration spot as a faster alternative (less than 3 min for 20 spots). For this, we used a square laser ablation spot (side length of 150 μm) that fully covered the spherical calibration spot (ø 130 μm). We used 10 laser shots on the same calibration spot to completely ablate and wash out the aerosol in less than 1 s, although the dried droplet is already ablated in the first shot. This was done to guarantee that all material of the calibration spot is fully transported to the ICP-MS and not limited by the wash out time of the ablation cell. We plotted the 109Ag intensities measured in single-shot mode for five calibration spots of the calibration array for the ionic solution and the NP suspension (Figure 2). Figure 2b shows an enlargement for the lowest concentration range and the blank value. Compared with the result of imaging mode (Figure S-4),

Table 1. Limits of Detection of Elements Contained in the Ionic Solution for Calibration after Blank Correction Calculated for Imaging and Single Shot Laser Ablation Mode limit of detection imaging mode

slope of calibration curve (count s−1/ng g−1)

Mg (m/z 26) P (m/z 31) K (m/z 39) Mn (m/z 55) Fe (m/z 56) Co (m/z 59) Cu (m/z 63) Zn (m/z 66) Ag (m/z 109)

2.9 0.87 7.7 130 95 81 39 28 190

concentration (ng g−1)

amount (fg)

50 17 180 61 61 21 0.61 0.21 3.9 1.3 0.58 0.20 2.2 0.75 2.7 0.93 0.50 0.17 limit of detection

single-shot mode

slope of calibration curve (count s−1/ng g−1)

concentration (ng g−1)

amount (fg)

Mg (m/z 26) P (m/z 31) K (m/z 39) Mn (m/z 55) Fe (m/z 56) Co (m/z 59) Cu (m/z 63) Zn (m/z 66) Ag (m/z 109)

2.6 1.4 7.1 81 74 52 27 11 135

880 2300 600 39 500 19 13 150 18

307 808 210 14 175 6.5 4.6 52 6.4

deviations of all data points in the whole area of calibration spots were used. The sensitivities (slopes of calibration curves; see Table 1) in the two calibration modes were similar. This result indicates that the analytical calibration procedure is not limited by matrix effects even if we used a different size of laser diameters for both calibration modes. Concerning LODs, a high number of data points as it is the case in the imaging mode is an important factor for improving a standard deviation. Namely, we can use a few thousand data points to calculate a mean value of the intensity measured and thus we achieve a much better standard deviation to estimate the LOD

Figure 2. (a) 109Ag signal intensities of calibration spots and (b) an enlargement. Error bars show the standard deviations of five calibration spots. C


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Figure 3. Elemental images of a thin section of an MCS which was treated with Ag NP for 48 h. (a) An optical photo of the thin section. (b−m) Mg (m/z 26), P (m/z 31), S (m/z 32), Ca (m/z 44), Mn (m/z 55), Fe (m/z 56), Co (m/z 59), Cu (m/z 63), Zn (m/z 66), Br (m/z 79), Ag (m/z 109), and Au (m/z 197), respectively. The masses are color coded (and signal intensities for S, Br, and Au). A size scale is shown below each image. The amount or intensity (if calibration data were not available) are shown on the right-hand side of each figure with a color-coded scaling for the elements. The red dashed line in panel a shows the direction of a line scan, which is shown in Figure S-5.

compared with our previous work,15 where we pipetted droplets by hand to produce calibration spots with a diameter of about 2 mm. Compared with the pipet spots, the spots created by the spotter in the present study are smaller (ø 130 μm) so that they can be ablated by a single laser shot. However, the diameter of spots is still much larger than the cell diameter, which is approximately 10 μm for fibroblast cells in suspension. Thus, although the calibration approach using the array spotter is an improvement, it is still not appropriate for the single-cell dimensions. Multielement Distributions Measured by LA-ICPTOF-MS in a Thin Section of Fibroblast MCS. Elemental distribution maps of a thin section (20 μm thick) from the center of the spherical MCS after incubation with Ag NPs are shown in Figure 3. Only elements with intensities above background signals were selected. In case quantitative data were available, the scaling is shown color coded on the righthand side of each figure and is given in fg, while for other elements, intensities are given in counts per second (cps). Figure 3a shows a photo of the thin section of the MCS (around 350 μm in diameter) before ablation. It can be seen that an outer layer at the rim of the spheroid can be clearly distinguished from the center core region. Au was used in this study as an internal standard (Figure 3m) and was coated onto the polymer slide in a homogeneous layer by sputtering before the thin sections of MCS were loaded. The Au signal showed some morphological structures of the MCS tissue, and this effect has been observed for other tissue samples.33 It is not clear if this is caused by a matrix effect, but the constant Au signal inside the MCS confirmed that the biological material of the spheroid is completely ablated. The Au signal outside the MCS can be used to correct drift, which was not observed at all during all measurements. Bromine (Br) (Figure 3k) which is contained in the eosin molecule (C20H8Br2N2O9, molecular weight: 647) was examined to simulate the behavior of small molecules. As a result for Br, although the incubation time was

for the imaging mode (being proportional to the square root of the number of measurements taken assuming a Poisson distribution). Consequently, we could get better LODs from the imaging mode rather than from the single shot mode (Table 1). For 109Ag, the LOD in the imaging mode was achieved even below the total amount of Ag in a single Ag NP particle (ø 50 nm: 0.68 fg). This is proof that the instrument we used in this study has in principle the potential to detect single Ag NP. Of course, in a single laser shot, we will not achieve such low limits of detection due to the worsening of the results by the Poisson statistics so that it was not clear whether or not we can detect single NPs. Karla et al. reported that a transient signal coming from a single Ag NP (ø 50 nm) was less than 500 μs even though the sample is Ag NP suspension.32 In a previous study, we used liquid ICP-SF-MS as a detector for detection of NPs in single cells.26 In that case, we could also detect a single Ag NP of the same size; however, we had to sacrifice multielemental information due to the low scanning speed and data losses of the sector field instrument. From the results presented in this study, we concluded that TOF-MS provides a gain in multielement capability without a loss in the potential to detect a single Ag NP. For further quantification, we used calibration data from the single-shot mode for all elements investigated to improve the analytical throughput because of this good agreement for the sensitivity (slope of calibration graph). The reason for doing so is that in the single shot mode, we can use the mean value from five different calibration spots for each concentration, whereas in the imaging mode, we have a single calibration spot only. From this analytical point of view, it would be better to talk about LOD per voxel instead of pixel. However, because we present our data in a two-dimensional figure, we prefer to use pixel prospectively. Overall, the calibration approach using the piezo-driven array spotter already showed a significant improvement D


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limited by the spatial resolution of the laser system and by data losses caused by the stabilization time of the magnet of the sector field mass spectrometer. This means that we could not differentiate if the Ag NP was trapped in this collagen-rich structure or accumulated in single cells. However, in the present study, we achieved better spatial resolution at the cellular level for all elements with no data loss, which means that we had the opportunity to look for this correlation. Our findings are discussed in the last section of the text. We determined the total mass of each element within a thin section using integration with ImageJ (Table S-5). These masses ranged from 2.1 pg for P to 35 fg for Mn. Although these values were above the limit of detection, they were close to the limit of quantification in the imaging mode assuming compromising conditions for single pixels. This is not a contradiction if we keep in mind that we see some enriched areas with even higher element contents in the outer rim region (Figure 3). The total mass of Ag exceeded the masses for most essential and mineral elements, except for Fe and P, in the thin section by more than one order of magnitude (Table S-5). However, the Ag still did not affect the viability of the MCS. Comparison of the Quantified Values Determined by LA-ICP-TOF-MS and Nebulizing ICP-MS. To confirm the quantitative data obtained by LA-ICP-TOF-MS, total amounts of elements present in the thin section of the center section of parallel grown MCS were measured by nebulizing ICP-SF-MS after acid digestion (Table 2). In Table 2, only results for Ag

comparatively short (only for 1 min), Br signals were already diffused among the outer rim and even in the core of the fibroblast MCSs. In another study, a diffusion limit of small molecules and nutrients was reported as about 200 μm.34 Therefore, the eosin molecule might also be diffused freely into MCSs. We concluded that the eosin molecule can be used as a probe to simulate the behavior of small molecules which are presented in the cell culture medium such as nutrients. Phosphorus (P) is an element with important biological functions that is present as an energy transporter (e.g., ATP), RNA, DNA, and in phosphorylated proteins. We found P was mainly enriched in the outer rim but was also detected in the core (Figure 3c). Sulfur (S) is present in most proteins and can be resolved with the high mass resolution provided by this ICP-TOF-MS instrument. In the MCS, 32S is distributed like 31 P, which is mainly concentrated in the outer rim region (Figure 3d). The trace elements Fe, Cu, Zn, and Mn were also clearly visible. The Fe, Cu, and Zn signals confirmed that the isotope ratios were consistent with their natural abundances, which means that spectral interferences can be disregarded. All of these elements showed good correlations to the signals of P and S and were concentrated in the outer rim regions of the proliferating cells. Ag also accumulated in the outer rim of the MCS. In this case, we considered a possibility of Ag NP-based signal enhancement, which is caused by an improvement of transport efficiency through a composition of aerosol generated by a laser ablation process to a surface of nanoparticles.35 However, in the previous study, no enhancement effect was observed upon comparison of signals of both thin sections with and without Ag NP accumulation.26 The main difference between our experiments and the work of Holá et.al. is that they loaded a high particle number of NP particles on top of the sample, whereas we worked with quite low numbers of nanoparticles dispersed in our biological system. Therefore, we concluded that there is no significant enhancement effect from nanoparticles at least in our case in the thin section of the MCS. However, this surface and concentration effect needs to be investigated in more detail in a separate study. For better visualization of the Ag localization, data from ImageJ software were arranged along the red dashed line (Figure 3a) in Figure S-6 for P and Ag, and the latter was converted to a particle number (shown on the right-hand axis). P was used here to visualize the outer rim region of the MCS, which was rich in P symmetrically. Compared with P, the Ag NPs were distributed asymmetrically with a pronounced peak on the right-hand side of the line close to the top of the outer rim zone. Even after 48 h, no Ag NPs were visible in the core of the MCS. This means that all NPs are trapped in a thin layer with roughly the dimension of a single cell in the outer rim zone. This asymmetric distribution of Ag NPs is also seen in the image of the MCS thin section (Figure 3l), where Ag shows the highest intensity in the lower part of the image. This phenomenon was also seen in our previous study. In that case, we interpreted this enrichment zone using the orientation of the MCS during the incubation step of the NP. This part of the MCS was oriented toward the top of the culture flask where the NP concentration was highest.26 However, knowledge of the MCS orientation is lost during transfer of the MCS from the 96-well plate to the embedding block. In our previous study,26 we used trypan blue staining to identify a collagen-rich structure between single cells in the outer rim zone. However, our previous measurements were

Table 2. Quantification of the Total Amount of Ag in a Thin Section of MCS by LA-ICP-TOF-MS in Comparison to Liquid ICP-SF-MS Analysis after Acid Digestion Ag mass (ng)

LA-ICP-TOF-MS

nebulizing ICP-SF-MS (n = 5)

0.16

0.30 ± 0.42

are shown as the concentrations for all other trace and mineral elements measured after digestion have been below the limits of detection of liquid ICP-SF-MS analysis. This was because LODs were significantly lower for LA-ICP-MS due to the following facts: first, a dilution of sample by digestion is avoided in direct measurements by LA-ICP-MS; second, there is no contamination from solvent; and third, the transport efficiency of LA-ICP-MS is close to 100%, whereas much lower efficiencies of 5% at most have been reported for pneumatic nebulization.36 To determine other elements within the MCS, more than a few MCSs must be digested even in the case of phosphorus, which has the highest content of elements quantified. Consequently, the total integrated mass of Ag in a thin section quantified by LA-ICP-TOF-MS agreed with the masses measured by nebulizing ICP-SF-MS after an acid digestion for five individual samples of parallel grown MCSs. Considering the standard deviation calculated in Table S-4, we have to point out that this is not coming from the measurement uncertainty but is reflecting the biological variability. The total Ag mass was slightly higher in the results from the digestion method. As mentioned earlier, the detection of a single Ag NP in a single pixel is not possible by LA-ICPTOF-MS with the setup used in this study. Therefore, this could lead to underestimation of the number of particles in areas with low NP concentrations. Taking this into consideration, the accuracy we realized in this experiment is quite good and can be improved with a TOF instrument with a E


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Figure 4. (a) Photograph of a thin section with a red box showing an area of enlarged view of panels b and c. (b) The amounts of P and Fe are shown in blue and red, respectively. (c) The iron and Ag NP particle number are shown in red and green, respectively.

and the washout time. As discussed earlier in the text, the washout time of the chamber we used did not greatly limit the resolution. However, even with the used laser ablation system, our lateral resolution was limited by the laser spot shape. The shape of the area of ablated material (laser crater) of overlapping laser spots is critical for the spatial resolution. Signal tailing is caused by sample material remaining from a previous laser shot when a circular shape is used for overlapping. As shown in Figure S-1, we define a theoretical resolution of 2 μm in the x-direction because after 4 laser shots, most of the material (more than 50%) is coming from a new area. Therefore, we estimated that the spatial resolution of our setup was 5 μm in the y-direction and 2 μm in the xdirection. If a mathematical deconvolution strategy is used, this lateral resolution can be improved.40 Thus, we present the first image of single cells within an MCS detected by high resolution LA-ICP-TOF-MS analysis with cellular resolution (Figure 4c). In comparison to our previous work where single isotope measurements for single cells were shown, we now have extended our measuring capability to multielement conditions.26

higher sensitivity or with a new measurement strategy by using single particle (sp) LA-ICP-MS which was applied in the work of Metarapi et al.37 In that paper, the authors showed that NPs are ablated intact and can be detected time-resolved with integration times of 100 μs so that even the particle size can be calculated from the histograms achieved. This would be a nice tool to demonstrate if particles are dissolved in the biological system. So far, we only can confirm the stability of our Ag NP in the culture medium, as it was investigated in the work of Lopez et al.36 Of course, shorter integration times can be applied with a TOF-MS, but this was not used in this study. Single-Cell Analysis in MCS by LA-ICP-TOF-MS. The sensitivity of the ICP-TOF-MS instrument used is high enough to detect several elements with high resolution at a cellular level in fibroblast multicellular spheroids. We selected an area at the bottom of the MCS thin section shown in Figure 3l to identify single cells where it can be seen that cells are wellseparated from each other with low cell density. We produced an enlargement of the distributions of P (blue), Fe (red), and Ag NPs (green) in this area (Figure 4). The line structure in the y-direction of the concentration in this highly resolved image arises from the fact that our pixel size is 5 μm in this direction but 0.5 μm in the x-direction. The Fe (red) distribution showed a round geometry with a diameter of 10 μm. This is identical to the diameter of fibroblast cells after trypsinization,26 and we assumed that this element can be used for visualization of a single cell. P (blue) distributed comparably homogeneously at the area in the outer rim so that it was also contained in the extracellular matrix abundantly. In this figure, the measured Ag intensity was converted into the number of particles using the calibration graph. Each green pixel represents more than 700 Ag NPs. Areas of the high accumulation of Ag NPs were correlated to locally concentrated high amounts of Fe. The numbers of Ag NPs correlated to the Fe signals were 18 342 particles in cell A and 3223 particles in cell B (Figure 4c). The number of particles in cell B agrees with that measured in an earlier study by a different method (3138 ± 722). In our earlier measurement, we used a FIB/SEM technique for single-cell analysis for Ag NPs (ø 75 nm) using an incubation time of 24 h and the same concentration as the present study.38 In the case of FIB/SEM, it was clear that the particles were present inside the cell. To examine this issue with the presented methodology, a combination of the metal-tagged staining technique which we established recently would be helpful to visualize cell compartments.39 The pixels for single elements showed good resolution (Figure 4c), but our lateral resolution was not identical to the dimension of each pixel. The spatial resolution of laser ablation is affected by many factors such as the laser setup, area ablated by the overlapping beam shape,

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CONCLUSIONS We investigated a quantification strategy for Ag NPs, biologically relevant elements, and minerals in fibroblast MCSs by high resolution LA-ICP-TOF-MS. The defined spatial resolution of our setup was 5 × 2 μm, which is smaller than a single cell with a diameter of 10 μm. Therefore, cellular resolution can be achieved for all elements detected by ICPTOF-MS. As a proof of this claim, we obtained an image of the Fe distribution of single cells in a thin MCS section in correlation with the NP distribution. In this study, we established the quantitative methodology to investigate the interactions between Ag NPs and intrinsic minerals in fibroblast MCSs using a high-resolution LA-ICP-MS. We believe that the methodology presented in this study can be applied for many toxicological and medical applications to study transport of metallodrugs, nanodrug containers, or minerals in tissue and organs at cellular levels. It will be used in our future research to investigate the transport of NP in three-dimensional models at cellular levels as a function of incubation time, surface modifications, and NP diameter.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b02239. Sample preparation for LA-ICP-MS analysis, acid digestion of a thin section, instrumentation and data F


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processing for LA-ICP-TOF-MS, and validation of the spotter used for printing the calibration series (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] or qoo.nel.asb@ gmail.com. ORCID

Akihiro Arakawa: 0000-0001-7144-1928 Sarah Theiner: 0000-0001-5301-0139 Takafumi Hirata: 0000-0003-4683-9103 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Akvile Häckel (Charité Universitätsmedizin, Berlin, Germany) for providing access to and support with the use of a cryomicrotome, Sigrid Benemann (Bundesanstalt für Materialforschung und-prüfung) for support of Au sputtering, and Olga Borovinskaya and Martin Tanner for their help on how to optimize and run the icpTOF 2R ICP-MS instrument (TOFWERK AG). The authors acknowledge Teledyne Photon machines for technical support.

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REFERENCES

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