Calibration Strategy to Size and Localize Multi-Shaped Nanoparticles in Tissue Sections Using LA-spICP-MS.

In the field of nanotoxicology, the detection and size characterization of nanoparticles (NPs) in biological tissues become increasingly important. To gain information on both particle size and particle distribution in histological sections, laser ablation and single particle inductively coupled plasma-mass spectrometry (LA-spICP-MS) was used in combination with a liquid calibration of dissolved metal standards via a pneumatic nebulizer. In the first step, the particle size distribution of Ag NPs embedded in matrix-matched gelatine standards introduced via LA was compared with that of Ag NPs in a suspension and nebulizer-based ICP-MS. The data show that the particles remained intact by the ablation process as confirmed by transmission electron microscopy. Moreover, the optimized method was applied to CeO2 NPs that are highly relevant for (eco-)toxicological research but, unlike Ag NPs, are multi-shaped and have a broad particle size distribution. Upon analyzing the particle size distribution of CeO2 NPs in cryosections of rat spleen, CeO2 NPs were found to remain unchanged in size over 3 h, 3 d, and 3 weeks post-intratracheal instillation, with the fraction of smaller particles reaching the spleen first. Overall, LA-spICP-MS combined with a calibration based on dissolved metal standards is a powerful tool to simultaneously localize and size NPs in histological sections in the absence of particle standards.

LA-ICP-MS and Immunohistochemical Staining with Lanthanide-Labeled Antibodies to Study the Uptake of CeO2 Nanoparticles by Macrophages in Tissue Sections.

Due to the increasing use and production of CeO2 nanoparticles (NPs), the likelihood of exposure especially via the air rapidly grows. However, the uptake of CeO2 NPs via the lung and the resulting distribution into various cell types of remote organs are not well understood because classical analytical methods provide limited spatial information. In this study, laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was combined with immunohistochemical (IHC) staining with lanthanide-labeled antibodies to investigate the distribution of intratracheally instilled CeO2 NPs from the rat lung to lymph nodes, spleen, and liver after 3 h, 3 days, and 21 days. We selected regions of interest after fast imaging using LA-ICP-MS in low-resolution mode and conducted high-resolution LA-ICP-MS in combination with IHC for cellular localization. The lanthanide labeling, which was largely congruent with conventional fluorescent labeling, allowed us to calculate the association rates of Ce to specific cell types. Major portions of Ce were found to be associated with phagocytic cells in the lung, lymph nodes, spleen, and liver. In the lung, almost 94% of the Ce was co-localized with CD68-positive alveolar macrophages after 21 days. Ce was also detected in the lymph nodes outside macrophages 3 h post instillation but shifted to macrophage-associated locations. In the liver, Ce accumulations associated with Kupffer cells (CD163-positive) were found. Ce-containing populations of metallophilic and marginal zone macrophages (both CD169-positive) as well as red pulp macrophages (CD68-positive) were identified as major targets in the spleen. Overall, high-resolution LA-ICP-MS analysis in combination with IHC staining with lanthanide-labeled antibodies is a suitable tool to quantify and localize Ce associated with specific cell types and to estimate their particle burden under in vivo conditions.

Quantitative dried blood spot analysis for metallodrugs by laser ablation-inductively coupled plasma-mass spectrometry.

A quantitative dried blood spot (DBS) method based on direct sampling by means of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) is presented. Gadolinium-based contrast agents were used as model metallodrugs with a significant relevance for pharmaceutical applications. Challenges regarding the ablation of the complex blood-filter matrix were characterized and successfully addressed by a thorough adaption of the laser ablation conditions. Especially the laser fluence was optimized with respect to the particle size distribution of the generated aerosol as monitored by an optical particle counter. Thus, generation of micrometer-sized particles could be minimized in favor of smaller particles increasing the transport efficiency of the DBS ablation aerosol to the plasma and the recorded signal stability. Inhomogeneous blood drying on the porous filter paper could be compensated by the addition of an internal standard prior to blood spotting. To preserve the advantages of DBS sampling, such as small blood volumes and minimal invasiveness, the combined use of DBS and a capillary blood sampling system is demonstrated. By placing the internal standard into the capillary prior to blood sampling, a simple workflow usable for clinical application was implemented. The applicability of the developed method, achieving limits of detection and quantification in the low µg L-1 range and covering a linear range of over four orders of magnitude, was demonstrated for blood samples containing different concentrations of the gadolinium contrast agents gadopentetate and gadoterate.

Complementary Molecular and Elemental Mass-Spectrometric Imaging of Human Brain Tumors Resected by Fluorescence-Guided Surgery.

Fluorescence-guided surgery (FGS) has been established as a powerful technique for glioblastoma resection. After oral application of the prodrug 5-aminolevulinic acid (5-ALA), protoporphyrin IX (PpIX) is formed as an intermediate of the heme-biosynthesis cascade and accumulates within the tumor. By intraoperative fluorescence microscopy, the specific PpIX fluorescence can be used to differentiate the tumor from healthy brain tissue. To investigate possible limitations of fluorescence diagnosis, the complementary use of molecular and elemental mass-spectrometry imaging (MSI) is presented. Matrix-assisted laser-desorption-ionization mass spectrometry (MALDI-MS) is used to examine the distribution of PpIX and heme b in human brain tumors. MALDI-MS/MS imaging is performed to validate MS data and improve the signal-to-noise ratio (S/N). Comparing the imaging results with histological evaluation, increased PpIX accumulation in areas of high tumor-cell density is observed. Heme b accumulation are only found in areas of blood vessels and hemorrhage, confirming the hampered transformation from PpIX to heme b in glioblastoma tissue. Investigation of non-neoplastic brain tissue and glioblastoma resected without external 5-ALA administration as control samples with true-negative fluorescence verified the absence of PpIX accumulation. Analysis of necrotic tumor tissue and gliosarcoma, one rare type of glioma appearing nonfluorescent during FGS, as case examples with false-negative-fluorescence diagnosis, revealed the absence of significant amounts of PpIX, indicating an impairment of PpIX formation. Molecular analysis is complemented by quantitative laser ablation-inductively coupled plasma (LA-ICP) MSI correlating heme b and Fe distribution. Mathematical pixel-by-pixel correlation of molecular and elemental data revealed a positive correlation with heteroscedasticity for the spatially resolved heme b signal intensities and Fe concentrations.

Visualizing protoporphyrin IX formation in the dura tail of meningiomas by mass spectrometry imaging.

BACKGROUND: The advantages of 5-aminolevulinacid (5-ALA)-induced fluorescence-guided surgery in meningiomas are increasingly discussed. In this context, despite detectable tumor tissue in histopathologial analyses, no fluorescence was shown at the dura tail using the standard operating microscope. Thus, 5-ALA metabolism in this surgically important site remains unknown but needs to be elucidated when further evaluating indications of fluorescence-guided surgery in meningiomas. METHOD: We here present the spatially resolved identification of protoporphyrin IX (PpIX) in sphenoid ridge meningioma cryosections from a patient who underwent fluorescence-guided microsurgery using molecular imaging analysis by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). RESULTS: Despite a strong fluorescence of the main tumor, no fluorescence could be detected at the dura tail using the standard operating microscope (blue-light, 405 nm). However, histopathological analyses clearly showed meningioma tissue. Remarkably, MALDI-MS/MS analysis revealed PpIX formation also at the non-fluorescing dura tail. However, no PpIX was detected in the tumor free dura mater. CONCLUSION: MALDI-MS/MS visualized a selective accumulation of PpIX within the tumor tissue including the dura tail. Thus, absence of fluorescence in the dura tail as visualized by the operating microscope is not caused by the lack of PpIX formation.

Quantitative bioimaging of platinum group elements in tumor spheroids.

Limited drug penetration into tumor tissue is a significant factor to the effectiveness of cancer therapy. Tumor spheroids, a 3D cell culture model system, can be used to study drug penetration for pharmaceutical development. In this study, a method for quantitative bioimaging of platinum group elements by laser ablation (LA) coupled to inductively coupled plasma mass spectrometry (ICP-MS) is presented. Different matrix-matched standards were used to develop a quantitative LA-ICP-MS method with high spatial resolution. To investigate drug penetration, tumor spheroids were incubated with platinum complexes (Pt(II)acetylacetonate, cisplatin) and the palladium tagged photosensitizer 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (mTHPP). Distribution and accumulation of the pharmaceuticals were determined with the developed method.

Quantitative Bioimaging to Investigate the Uptake of Mercury Species in Drosophila melanogaster.

The uptake of mercury species in the model organism Drosophila melanogaster was investigated by elemental bioimaging using laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS). The mercury distribution in Drosophila melanogaster was analyzed for the three species mercury(II) chloride, methylmercury chloride, and thimerosal after intoxication. A respective analytical method was developed and applied to the analysis of the entire Drosophila melanogaster first, before a particular focus was directed to the cerebral areas of larvae and adult flies. For quantification of mercury, matrix-matched standards based on gelatin were prepared. Challenges of spatially dissolved mercury determination, namely, strong evaporation issues of the analytes and an inhomogeneous distribution of mercury in the standards due to interactions with cysteine containing proteins of the gelatin were successfully addressed by complexation with meso-2,3-dimercaptosuccinic acid (DMSA). No mercury was detected in the cerebral region for mercury(II) chloride, whereas both organic species showed the ability to cross the blood-brain barrier. Quantitatively, the mercury level in the brain exceeded the fed concentration indicating mercury enrichment, which was approximately 3 times higher for methylmercury chloride than for thimerosal.

Evaluation of reversible interconversion in comprehensive two-dimensional gas chromatography using enantioselective columns in first and second dimensions.

The reversible molecular interconversion behaviour of a synthesised oxime (2-phenylpropanaldehyde oxime; (C6H5)CH(CH3)CHN(OH)) was investigated by both, single dimensional gas chromatography (1D GC) and comprehensive two-dimensional gas chromatography (GC×GC). Previous studies on small molecular weight oximes were extended to this larger aromatic oxime (molar mass 149.19gmol(-1)) with interest in the extent of interconversion, enantioselective resolution, and retention time. On a polyethylene glycol (PEG; wax-type) column, a characteristic interconversion zone between two antipodes of E and Z isomers was formed by molecules which have undergone isomerisation on the column (E⇌Z). The extent of interconversion was investigated by varying chromatographic conditions (oven temperature and carrier flow rate) to understand the nature of the behaviour observed. The extent of interconversion was negligible in both enantioselective and methyl-phenylpolysiloxane phase-columns, correlating with the low polarity of the stationary phase. In order to obtain isomerisation along with enantio-resolution, a wax-type and an enantioselective column were coupled in either enantioselective-wax or wax-enantioselective order. The most appropriate column arrangement was selected for study by using a GC×GC experiment with either a wax-phase or phenyl-methylpolysiloxane phase as (2)D column. In addition to evaluation of these fast elution columns, a long narrow-bore enantioselective column (10m) was introduced as (2)D, providing an enantioselective-PEG (coupled-column ensemble: (1)D1+(1)D2)×enantioselective ((2)D) column combination. In this instance, the (1)D1 enantioselective column provides enantiomeric separation of the corresponding enantiomers ((R) and (S)) of (E)- and (Z)-2-phenylpropanaldehyde oxime, followed by E/Z isomerisation in the coupled (1)D2 PEG (reactor) column. The resulting chromatographic interconversion region was modulated and separated into either E/Z isomers (achiral (2)D column) or into the respective (R) and (S) enantiomers of the E/Z isomers when using a (2)D enantioselective column. With this arrangement, the isomers underneath the broad interconversion plateau in 1D elution profiles, including the enantiomers, could be resolved, illuminating salient features and understanding of the molecular reversible process of the interconverting molecules during the chromatographic elution. The two-dimensional patterns (contour plots), resulting from the combination of interconversion process and chiral separation, are discussed phenomenologically.