Elemental Bioimaging of Thulium in Mouse Tissues by Laser Ablation

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Elemental Bioimaging of Thulium in Mouse Tissues by Laser Ablation-ICPMS as a Complementary Method to Heteronuclear Proton Magnetic Resonance Imaging for Cell Tracking Experiments Olga Reifschneider,† Kristina S. Wentker,† Klaus Strobel,‡,∥ Rebecca Schmidt,‡ Max Masthoff,‡ Michael Sperling,†,§ Cornelius Faber,‡ and Uwe Karst*,† †

Westfälische Wilhelms-Universität Münster, Institute of Inorganic and Analytical Chemistry, Corrensstr. 30, 48149 Münster, Germany ‡ Westfälische-Wilhelms-Universität Münster, University Hospital, Department of Clinical Radiology, Albert-Schweitzer-Campus 1, 48149 Münster, Germany § European Virtual Institute for Speciation Analysis (EVISA), Mendelstr. 11, 48149 Münster, Germany ABSTRACT: Due to the fact that cellular therapies are increasingly finding application in clinical trials and promise success by treatment of fatal diseases, monitoring strategies to investigate the delivery of the therapeutic cells to the target organs are getting more and more into the focus of modern in vivo imaging methods. In order to monitor the distribution of the respective cells, they can be labeled with lanthanide complexes such as thulium-1,4,7,10-tetraazacyclodoecaneα,α,α,α-tetramethyl-1,4,7,10-tetraacetic acid (Tm(DOTMA)). In this study, experiments on a mouse model with two different cell types, namely, tumor cells and macrophages labeled with Tm(DOTMA), were performed. The systemic distribution of Tm(DOTMA) of both cell types was investigated by means of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICPMS). Using the high resolution of 25 μm, distribution maps of Tm in different tissues such as tumor, liver, lung, and spleen as well as in explanted gel pellets were generated and the behavior of the labeled cells inside the tissue was investigated. Additionally, quantitative data were obtained using homemade matrix-matched standards based on egg yolk. Using this approach, limits of detection and quantification of 2.2 and 7.4 ng·g−1, respectively, and an excellent linearity over the concentration range from 0.01 to 46 μg·g−1 was achieved. The highest concentration of the label agent, 32.4 μg·g−1, in tumor tissue was observed in the area of the injection of the labeled tumor cells. Regarding the second experiment with macrophages for cell tracking, Tm was detected in the explanted biogell pellet with relatively low concentrations below 60 ng·g−1 and in the liver with a relatively high concentration of 10 μg·g−1. Besides thulium, aluminum was detected with equal distribution behavior in the tumor section due to a contamination resulting from the labeling procedure, which includes the usage of an Al electrode.

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relaxation time T2, are affected.6 Nowadays, MRI is not limited to merely morphological imaging but is frequently used in unconventional applications such as noninvasive MR thermometry or cell tracking.7−11 For such experiments, thulium-1,4,7,10tetraazacyclodoecane-α,α,α,α-tetramethyl-1,4,7,10-tetraacetic acid (Tm(DOTMA)), which acts as T1 and chemical exchange saturation transfer (CEST) agent, can be used routinely. It shows a strong temperature dependence of its chemical shift and a narrow line width.12,13 Furthermore, the signal from methyl 1H of DOTMA can be detected without the interference from the background water signal. This possibility of directly imaging cells labeled with Tm(DOTMA) by means of the methyl protons opens the door to in vivo cell tacking experiments by MRI. Recently, in vivo studies were performed

are earth metals are favorable as labels for quantitative analysis of peptides and proteins by means of ICPMS due to low ionization energies, absence of isobaric interferences, and very low background resulting in excellent limits of detection down to the pg·L−1 level.1,2 Labeling is performed by means of chelate ligands that can be linked to reactive groups of proteins by means of a specially designed linker (e. g., isothiocyanate (SCN) or maleimido functionalities).3,4 Among all multidentate lanthanide chelate complexes, 1,4,7,10-tetraazacyclodoecane-1,4,7,10-tetraacetic acid (DOTA) tags show the highest stability.5 Ln chelate complexes are widely used in magnetic resonance imaging (MRI) as contrast agents, mainly based on gadolinium (Gd). MRI is routinely applied in medicine for diagnostic purposes. The application of Gd-based contrast agents provides an excellent soft tissue contrast due to the high paramagnetic moment of the element. This effect is based on reducing the relaxation times of water protons in the surrounding tissue, whereby both, longitudinal relaxation time T1 and transversal © 2015 American Chemical Society

Received: November 21, 2014 Accepted: March 20, 2015 Published: March 20, 2015 4225

DOI: 10.1021/ac504363q Anal. Chem. 2015, 87, 4225−4230

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Analytical Chemistry

demonstrated. Quantitative, spatially resolved data of different tissue sections of mouse organs were obtained by means of LAICPMS after cell tracking experiments by MRI of tumor cells and macrophages labeled with Tm(DOTMA). Moreover, a novel method for matrix-matched standards for external calibration based on egg yolk was developed. An excellent homogeneity and the possibility to prepare thin tissue sections analogous to organ tissue were achieved by the prepared egg yolk standards. The quantitative distribution of Tm was determined in tumor, liver, and gel pellet sections with high spatial resolution down to 25 μm laser spot size. The obtained distribution maps of Tm were in good agreement with the in vivo MRI data of parallel tissue sections published before.15

to monitor Tm(DOTMA) tagged tumor cells and macrophages in mouse models.14,15 Obtained results revealed the distribution of tagged cells in the living organism, using a novel approach termed highly shifted proton MRI or heteronuclear proton MRI. The detection specificity of this method was validated by using LA-ICPMS. However, the issue of quantification has previously not been addressed but is a prerequisite for future applications of heteronuclear proton MRI. Laser ablation in combination with inductively coupled plasma-mass spectrometry (LA-ICPMS) represents such a wellestablished method for the (quantitative) investigation of spatial elemental distribution in tissue sections.16 Remarkable advantages of elemental imaging by LA-ICPMS include a high spatial resolution depending on the respective ablation cell, laser unit, and lens system, as well as low limits of detection in the lower ppb range, provided by the ICPMS detection system. Recent work by the research group of Günther for example demonstrated a spatial resolution of 1 μm by means of a femtosecond (fs) laser and a novel ablation cell technology for investigation of human epidermal growth factor receptor 2-enriched breast cancer tissue.17 Investigations of single cells were performed with 5 μm resolution with respect to silver nanoparticle distribution inside the eukaryotic cells after spiking experiments.18 Furthermore, several strategies for quantitative analysis of the elemental distribution were presented in the literature. Some approaches include the internal standardization by covering the tissue section with a suitable standard in order to correct matrix effects. Furthermore, external calibration by means of so-called matrix-matched standards was performed in the past.19,20 Quantitative imaging experiments include the analysis of teeth, brain, kidney, and tumor sections, revealing precious chemical and biomedical information.21−24 Hare et al. introduced a novel approach to generate three-dimensional images, containing the quantitative information on the distribution of Fe, Cu, and Zn in the mouse brain, which once more demonstrates the enormous applicability of this technique.25,26 First attempts for the complementary use of LA-ICPMS imaging and MRI were performed by Kamaly et al. and Pugh et al., where the distribution of Gd in post-mortem samples from a mouse tumor and pig brain was qualitatively investigated.27,28 Quantitative studies of Gd-based contrast agents by means of LA-ICPMS were performed in articular cartilage using different quantification strategies such as standard addition, one-point calibration, and isotope dilution analysis. However, the spatial resolution was partially lost, as the sample preparation included the powder formation of the respective specimen.29 Single cell tracking experiments of Gd labeled cells by means of LAICPMS were reported for the first time by Managh et al. in 2013.30 In this experiment, single cells were incubated with two frequently used Gd-based contrast agents, Omniscan and Dotarem. Using a laser spot size of 25 μm, the samples were investigated either in vitro directly after the incubation or after in vivo experiments in the peritoneal lavage of mice. In order to obtain quantitative information on the total content of Gd inside the labeled cells, they were digested and analyzed using a solution-based ICPMS method. However, quantitative elemental bioimaging by means of LA-ICPMS of tissue samples after injection of labeled cells was not carried out prior to the current study. In this study, the great advantages of LA-ICPMS as a complementary technique to MRI in cell tracking experiments based on labeling with Tm-based contrast agents were

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EXPERIMENTAL SECTION Animal Experiments and Sample Preparation. All experiments were approved by the local Ethics Committee for Animal Experiments (ID 8.87-50.10.36.08.191). For the applied animal model, 8−10 week old nude mice (CD1 nu/nu, Charles River, Sulzfeld, Germany) were used. Labeling of the HT-1080 human fibrosarcoma cells (4 × 106) with Tm(DOTMA) (15 μmol/L, Macrocyclics, Texas, USA) was performed after resuspending cells in 500 μL of serumfree medium (Dulbecco’s Modified Eagle Medium, Gibco, Invitrogen) by electroporation (BioRad Gene Pulser XCellTM, CA, USA) using a voltage of 220 V. After this procedure, cells were allowed for reclosing membrane leakage at 4 °C for 30 min and were cultured under standard conditions overnight before implantation in mice. Labeling of 5 day old bone marrow derived murine macrophages (BMDMs) was performed through incubation with Tm(DOTMA) solution (5−15 μmol/3 × 106 cells/5 mL incubation medium) for 24 h. The mouse model included a local inflammation that was induced by subcutaneous injection of 150 μL of polyacrylamide gel (PAG) pellets in both flanks of 10 nude mice (CD1 NU/NU, Charles River, Sulzfeld, Germany). To strengthen the inflammation, lipopolysaccharide (100 μg/150 μL PAG) was added to one PAG pellet per mouse. 3−5 × 106 Tm(DOTMA) labeled macrophages were injected intravenously either 24 h before or immediately after pellet implantation. After 8 days, animals were sacrificed and PAG pellets as well as liver were explanted. Cryosections of 5 μm thickness were prepared for post-mortem imaging. Preparation of Matrix-Matched Standards. For quantification, matrix-matched standards based on egg yolk were prepared. In the first step, the egg white was separated from the egg yolk. The homogeneous interior of the egg yolk was carefully collected by a syringe. For each standard, 800 mg of the mixture of three different egg yolks was spiked with 200 μL of the respective thulium standard solution, previously prepared from thulium(III)chloride hexahydrate (99.99%, Sigma-Aldrich, Steinheim, Germany) by dissolving in doubly deionized water. After spiking, the mixture was homogenized and shaken for 1 h carefully to avoid the formation of bubbles, which may negatively influence the homogeneity of the prepared standards. After homogenization, the viscous homogenate was heated to a temperature of 90 °C for 10 min and was allowed to cool down in order to generate a solid structure similar to tissue. During the heating, vials were opened to allow for the evaporation of water. After cooling down, the solid blocks were removed from the vial and embedded in NEG 50 before sectionizing using a microtome cryostat (CryoStar NX70, Thermo Scientific, Walldorf, Germany). The resulting egg yolk sections were 5 μm 4226

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thick, equal to the investigated liver and tumor sections. The matrix-matched standards were analyzed by LA-ICPMS immediately prior to ablation of the samples. ICPMS Analysis of Matrix-Matched Standards. For the validation of the concentrations of the matrix-matched standards, a bulk analysis by means of ICPMS (iCAP Qc, Thermo Scientific, Bremen, Germany) was performed after microwave digestion (Mars Xpress, CEM GmbH, KampLinfort, Germany). For this purpose, 200 mg of each solid egg yolk standard was transferred to a 50 mL microwave vessel and 2 mL of HNO3 (65%, Suprapur, Merck KGaA, Darmstadt, Germany) was added. The obtained clear solutions were analyzed by means of ICPMS (iCAP Qc, Thermo Fisher Scientific, Bremen, Germany). For external calibration, standard solutions of Tm, Lu (SCP Science, Champlain, NY, USA), and Ho (Merck KGaA, Darmstadt, Germany) were used. 165Ho and 175 Lu (1 μg L−1) were used as internal standards, added before and after the digestion, respectively. For sample introduction, a PFA μFlow-ST nebulizer (Elemental Scientific, Omaha, NE, USA) within a quartz cyclone spray chamber and a quartz injector pipe with an inner diameter of 1 mm were used. Due to the absence of interferences, the analyses were performed in standard mode of the ICPMS instrument. For the plasma interface, a nickel sampler and skimmer with a 2.8 mm insert were used. Additional ICPMS conditions were as follows: RF power 1550 W; auxiliary gas flow rate 0.8 L min−1; nebulizer flow rate 0.88 L min−1. 169Tm, 165Ho, and 175Lu were monitored with a dwell time of 0.01 s each. LA-ICPMS Analysis. This imaging study was carried out by coupling a commercial laser ablation system (LSX 213, CETAC Technologies, Omaha, NE, USA) controlled by DigiLaz III software (CETAC Technologies) to a quadrupole-based inductively coupled plasma-mass spectrometer, model iCAP Qc (Thermo Fisher Scientific). The laser ablation parameters such as laser energy, spot size, scan rate, and carrier gas flow were chosen in order to obtain good spatial resolution and quantitative ablation of the whole section, avoiding the ablation of the glass slide and interferences by fractionation effects. All samples and standards were ablated quantitatively using a line by line scan method with no distance between lines. The laser energy was adjusted to 0.4 mJ per single shot at a repetition rate of 20 Hz, and a spot size of 25 μm (tumor and organs) or 50 μm (for PEG pellets) with a scan rate of 50 μm s−1 was chosen. A mixture of helium (0.7 L min−1) and argon (0.4 L min−1) was utilized as a carrier gas to transport the ablated material into the plasma. For tuning the instrument, a fully automated adjusting approach was performed daily using the respective functionality of the Qtegra software. For this purpose, parameters like position of the torch, extraction voltage, and the additional carrier gas flow of argon as well as the most relevant ion lenses in front of the mass analyzer were optimized for maximum intensity as well as low levels of oxides and doubly charged ions. Additionally, an indium solution (1 μg L−1) was introduced continuously into the ICPMS system via a PFA nebulizer and a cyclonic spray chamber in order to monitor the performance of the ICPMS system during the whole acquisition time. The isotopes 27Al, 79Br, 115In, and 169Tm were monitored with a dwell time of 0.125 s each. Data processing was carried out using Origin8.5G (Originlab Corporations, Northampton, MA, USA) and ImageJ 1.47n (National Institute of Health, Bethesda, MD, USA) for image generation.

Article

RESULTS AND DISCUSSION

Calibration by Means of Egg Yolk Standards. Since the response in LA-ICPMS is strongly influenced by the matrix dependent ablation behavior, aerosol formation, and transport, reliable quantification strategies based on internal standardization or external calibration with matrix-matched standards were investigated in the past. The use of carbon (13C) as internal standard was shown to be unsuitable because of the phase partitioning of carbon-containing gaseous species particles.31 Therefore, external calibration is nowadays the most widely accepted approach for quantitative elemental bioimaging by means of LA-ICPMS. A serious disadvantage of this approach is the lack of certified reference materials for varying matrices used in the investigations. Thus, homemade matrix-matched standards are well established, and good results for various applications have been reported. For matrixmatching, usually tissue homogenates were spiked with defined amounts of standard solutions of the respective elements. In this process, a homogeneous distribution of the spiking agent within the matrix was one of the main concerns. Furthermore, the degree of similarity between samples and standards regarding their ablation behavior was essential. In order to obtain homogeneous distribution at the μm scale, the homogenates were required to contain no large particulate material, which imposed a major challenge especially when starting with native tissue as matrix. Using egg yolk as a matrix, reliable calibration results were achieved for thulium in this study. For the formation of a solid structure comparable to the structure of the investigated soft tissues, the spiked and homogenized egg yolk mixture was heated to 90 °C. The optimized procedure for the standard preparation resulted in a very homogeneous structure without bubbles or coarse particles. Using these standards, a calibration curve with a correlation coefficient of R2 = 0.999 was obtained. Limits of detection and quantification were determined using the 3 σ-criterion with 2.2 and 7.4 ng g−1, respectively. The calibration procedure was performed directly before ablating the respective tissue sections. Signal stability of the ICPMS instrument was monitored during the analysis using a standard solution of indium (1 μg L−1) introduced by nebulization and mixed with the aerosol from the ablation chamber directly before entering the plasma. Verification of the analyte concentration within the matrix-matched standards was accomplished by conventional solution nebulization ICPMS. For this purpose, weighed amounts of the standards were digested by a microwave. The recovery rates ranged between 92% and 110% for all seven LA calibration standards with concentrations between 0.01 and 46 μg g−1. As the standards and the tissue sections were ablated quantitatively using the same ablation parameters, a high degree of matrix-matching could be concluded with respect to the ablation behavior. Analysis of Tumor Tissue. MRI allows an in vivo detection of distinct cell populations if a suitable contrast agent is used within the experiment. In the first part of the study, tumor cells were labeled with Tm(DOTMA). This particular contrast agent is beneficial for cellular MRI as the direct detection of methyl 1 H provides the possibility of an unambiguous detection of the cells. However, the spatial resolution achieved with this MRI technique is approximately 1 mm, which is not sufficient to investigate the distribution of labeled cells inside the tumor. For this purpose, a complementary technique such as LA-ICPMS providing a higher resolution is required. 4227

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detected with similar signal intensities as before in the stained section. With respect to the generated distribution maps of Tm and Al, again, a high similarity was observed. These results indicate that the presence of Al results from contamination in the implanted cells and, thus, a very similar distribution pattern to Tm was observed by LA-ICPMS. In order to achieve information on the origin of the Al contamination of the cells, their preparation steps were investigated. During the labeling procedure, prior to implantation, the tumor cells were electroporated using an aluminum electrode, which leads to dissolution and transfer of Al(III) ions into the cell compartment together with the Tm. To prove the origin of Tm and Al from the labeling experiment, a native tumor section grown on native implanted cells was investigated using the same LA-ICPMS parameters. When ablating the unlabeled material, neither Tm nor Al signals were detectable throughout the whole tumor section (data not shown). These results confirmed the efficiency of the labeling procedure with Tm and the origin of the contamination with Al during the labeling process by electroporation. For quantification purposes, cryosections of the tumor were investigated in order to avoid alteration of the analyte distribution or concentration by washing procedures, which are required for fixation and paraffin embedding. The prepared matrix-matched standards based on egg yolk were ablated directly prior to the tumor sections using the same parameters. In addition to Tm, the Al signal was recorded simultaneously. The observed qualitative distribution of Al and the quantitative distribution of Tm are presented in Figure 2. As expected, a

For the analysis of tumor tissue sections, a qualitative study by means of LA-ICPMS was performed in order to validate the data from the MRI experiments. For this purpose, paraffin embedded sections from the tumor were stained with hematoxylin and eosin (H&E). This way, differentiation between tumor, surrounding skin, and fatty tissue was achieved. The stained section was ablated using a spot size of 25 μm, and besides thulium, also aluminum and bromine as constituents of the H&E staining solutions were monitored. Figure 1a. shows

Figure 1. (a) Bright field micrograph of the investigated tumor section, paraffin embedded, dewaxed, and stained with H&E. (b) The corresponding distribution of 79Br from the staining agent eosin. The structure of the tissue section is completely visualized. (c) The distribution of 27Al from the staining agent hematoxylin. (d) The corresponding distribution map of 169Tm from Tm(DOTMA). (e) Bright field micrograph of the investigated parallel unstained tumor section. (f) The corresponding distribution map of 169Tm from Tm(DOTMA). (g) The corresponding distribution of 27Al as contamination due to electroporation for cell labeling.

the microscopic picture of the investigated H&E-stained tumor section, with tumor tissue appearing in the typical purple color. By means of the corresponding distribution maps of 79Br (Figure 1b) and 27Al (Figure 1c), the structure of the tissue section was easily visualized. Comparing the 169Tm distribution with the optical micrograph and the H&E distribution images, Tm was located exclusively in the tumor tissue. This indicates that no diffusion of Tm(DOTMA) from the implanted tumor cells into the surrounding tissue and subsequent uptake by other cells took place. The contrast agent remained inside the tumor cells even during the cell division process. The implanted cell area was determined by the highest signal intensity of 169 Tm indicated with an arrow in Figure 1d. Interestingly, this area also showed the highest aluminum content compared to other regions of the tissue (Figure 1c). To identify the origin of this high aluminum concentration, an unstained parallel section of the same tumor was investigated using the same LA-ICPMS parameters. The microscopic image of the unstained section is shown in Figure 1e. 169Tm (Figure 1f) as well as 27Al (Figure 1g) were detected in the unstained tumor section by means of LA-ICPMS. As expected, no bromine was found in the unstained tumor section (data not shown). However, Al was

Figure 2. (a) Bright field micrograph of the investigated tumor section, the area of interest is marked by the yellow oval. (b) The corresponding 27Al distribution resulting from the electroporation procedure of the tumor cells. (c) The corresponding distribution map of 169Tm from Tm(DOTMA) shows the maximum concentration of 32.4 μg·g−1 at the position marked by an arrow. (d) The distribution map of 169Tm in a selected concentration range from 0 to 1 μg·g−1.

good correlation between the distribution patterns was observed and confirmed the previous results. The area with the highest Al signal intensity also showed the highest Tm concentration, namely, 32.4 μg·g−1. Analysis of Labeled Macrophages in Explanted Gel Pellets and Liver. In the second part of this study, macrophages were labeled with Tm(DOTMA), injected into mice bearing subcutaneous LPS-containing PAG pellets, and 4228

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Analytical Chemistry tracked with MRI. Labeled macrophages were expected to migrate to the site of LPS-induced inflammation and infiltrate inflamed tissue and the pellets. Such cell migration was observed in the MRI experiments, showing the expected strong signal in the region around the pellets.15 By means of LA-ICPMS, a qualitative assessment was performed on pellets with and without LPS, as well as in the organs: liver, lung, and spleen. A strong Tm signal was observed in the investigated organs, with all samples showing comparable signal intensities when a spot size of 25 μm was applied (data not shown). In contrast to these observations, Tm signals in the pellets were much lower and only detectable using a larger spot size of 50 μm. As macrophages are expected to be present predominantly in the liver, both pellet and liver sections were investigated quantitatively by means of LA-ICPMS. Quantitative analysis was performed using the previously introduced homemade egg yolk standards. Figures 3 and 4 illustrate the quantitative data obtained from investigations of gel pellet and liver sections, respectively.

micrograph of the investigated gel section is presented. The skin tissue marked in yellow is the part influenced by injection of the pellets. The observed distribution was in good accordance with results from the in vivo study published earlier by Schmidt et al.15 The MRI experiments in their study suggested that the labeled macrophages have entered the gel pellet mostly near the tissue−pellet interface, which was validated by immunostaining of the macrophages. This distribution behavior of the labeled macrophages was confirmed by means of LA-ICPMS analysis, showing the maximum Tm concentrations in the outer region of the pellet (Figure 3b). In Figure 4, the results for the investigated liver section are shown. The micrographs, obtained by means of bright field microscopy (Figure 3a) and autofluorescence microscopy (Figure 3b), clearly show a very homogeneous structure of the section. The macrophages remain inside the liver until they are activated by an immune response. For this reason, relatively high Tm concentrations of up to 10.6 μg·g−1 were found in several areas of this liver section. The average concentration throughout the whole section was 0.36 μg·g−1, which is demonstrated in Figure 4d.

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CONCLUSIONS In this work, a method for cell tracking based on elemental bioimaging by LA-ICPMS was presented. Within the performed study, tumor cells and macrophages were labeled with Tm(DOTMA), which is well suited for ICPMS analysis due to the low background in tissue based on the absence of isobaric interferences for 169Tm. Therefore, LA-ICPMS was demonstrated to be a complementary method for in vivo experiments with MRI showing a much better resolution and the possibility to obtain quantitative data. Using LA-ICPMS for elemental bioimaging, an excellent resolution of 25 μm was achieved, which is approximately 40 times better than the resolution obtained by MRI for this particular marker. Hence, the exact location of initially implanted tumor cells and the systemic distribution of macrophages labeled with Tm(DOTMA) in mouse organs and in a bio gel pellet were specified. Additionally, contamination with aluminum was revealed within the tumor cells. Detailed investigations demonstrated that the Al contamination originated from the electroporation process, for which aluminum electrodes were used. Quantitative data were generated using a simple calibration approach based on an egg yolk matrix. The standards showed an excellent homogeneity and also a good linearity with low limits of detection and quantification, namely, 2.2 and 7.4 ng g−1. With the approach of external calibration, Tm concentrations of up to 32.4 μg·g−1 could be detected in tumor tissue and up to 10.6 μg·g−1 in liver sections.

Figure 3. (a) Bright field micrograph of the investigated gel pellet section. (b) The corresponding distribution map of 169Tm with a maximum concentration of 0.06 μg·g−1.

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Figure 4. (a) Bright field micrograph and (b) autofluorescence micrograph of the investigated liver section of a mouse treated with tagged macrophages. (c) The corresponding distribution map of 169 Tm with the maximum concentration of 10.6 μg·g−1. (d) The distribution map of 169Tm with a concentration scale of 0−1 μg·g−1.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +49 251/83-33141. Fax: +49 251/83-36013. Present Address ∥

K.S.: Bruker BioSpin MRI GmbH, Rudolf-Plank-Str. 23, 76275 Ettlingen, Germany.

A spot size of 50 μm was used for explanted gel pellets, as no Tm could be detected with a spot size of 25 μm according to the low Tm concentration. In Figure 3a, the bright field

Notes

The authors declare no competing financial interest. 4229

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ACKNOWLEDGMENTS This study was supported by the Cells in Motion Cluster of Excellence (CiM - EXC 1003), Münster, Germany (project FF2013-17).

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DOI: 10.1021/ac504363q Anal. Chem. 2015, 87, 4225−4230