Olga Reifschneider

Quantitative bioimaging of platinum in polymer embedded mouse organs using laser ablation ICP-MS

A novel quantification approach for tissue imaging using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) based on tissue embedding in cold-curing resins (Technovit 7100) is presented. With respect to massive side effects on cisplatin, the platinum distribution at different time intervals after cisplatin treatment of mice was determined quantitatively in different tissues including cochlea, testis and kidney. For this purpose, cold-curing resin blocks spiked with different amounts of platinum acetyl acetonate prior to curing were ablated after sectioning at 5 μm thickness and were analysed using ICP-MS after microwave digestion. High spatial resolution and limits of detection in the low ppb range (8 μg kg(-1)) were achieved using a simple and efficient sample preparation. External calibration using the Technovit 7100 standards proved to yield precise and reproducible quantification results. The distribution and retention behaviour of cisplatin in the organs was investigated using the new calibration method.

Elemental bioimaging of haematoxylin and eosin-stained tissues by laser ablation ICP-MS

Haematoxylin and eosin are frequently used histological stains (“H & E-stains”) for the visualization of tissue structures, which is particularly important for the diagnosis of various diseases including cancer. These stains contain aluminum and bromine in their chemical structures, thus providing access for their visualization by means of ICP-MS imaging. Different tissues including appendix, lymph nodes, Fallopian tube and esophageal tumor were investigated. By means of this novel approach, orthogonal data in comparison to optical micrographs, commonly used by the pathologist, were obtained. Hence, different entities of the parallel or serial tissue sections can now be allocated to the manifold set of elemental information acquired by LA-ICP-MS. In surgically removed and H & E-stained human tissues, the images of aluminum and bromine with resolution down to 10 μm were generated. In addition, elemental distribution maps of carbon, aluminum, bromine and platinum in unstained and stained human esophageal tumor sections after Cisplatin therapy were generated, showing a further expanding the capabilities of this technique. As both stained and unstained samples showed a similar platinum distribution, integrity of the platinum distribution after the staining process could be verified. In addition, no significant background of aluminum or bromine in unstained samples was observed, showing the successful reduction of polyatomic, isobaric interferences by utilization of a collision/reaction cell.

Laser ablation based bioimaging with simultaneous elemental and molecular mass spectrometry: towards spatially resolved speciation analysis

RATIONALE Biological functions of metals are not only specified by the element itself, but also by its chemical form and by its organ, cell and subcellular location. The developed laser ablation based setup enables spatially resolved analysis with simultaneous elemental and molecular mass spectrometry (MS) and promises therefore localization, identification and quantification of metal or heteroelement-containing species in biological samples such as tissue sections. METHODS A UV laser ablation (LA) system is hyphenated in parallel both with an elemental and a molecular mass spectrometer via flow splitted transfer lines to simultaneously obtain data from both of the mass spectrometers. Elemental MS was performed using inductively coupled plasma (ICP)-MS, whereas atmospheric pressure chemical ionization (APCI)-MS with an orbitrap mass analyzer was utilized for molecular MS. RESULTS Simultaneous elemental and molecular MS imaging with high lateral resolution down to 25 µm was presented for the staining agents eosin Y and haematoxylin as well as for the chemotherapy drug cisplatin in thin tissue sections. For molecular MS, target compounds were identified by their exact masses and by characteristic fragment ions. CONCLUSIONS The first simultaneous elemental and molecular MS imaging approach based on laser ablation sampling was introduced for spatially resolved speciation analysis. The combination of the advantages of LA-ICP-MS such as low detection limits and high spatial resolution with information on the chemical identity promises not only localization of metals, but also identification of metal species in biological samples. Therefore, this novel technique opens up new possibilities to address complex challenges in life science research.

Elemental bioimaging of nanosilver-coated prostheses using X-ray fluorescence spectroscopy and laser ablation-inductively coupled plasma-mass spectrometry.

The distribution of different chemical elements from a nanosilver-coated bone implant was visualized, combining the benefits of two complementary methods for elemental bioimaging, the nondestructive micro X-ray fluorescence (μ-XRF), and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Challenges caused by the physically inhomogeneous materials including bone and soft tissues were addressed by polymer embedding. With the use of μ-XRF, fast sample mapping was achieved obtaining titanium and vanadium signals from the metal implant as well as phosphorus and calcium signals representing hard bone tissue and sulfur distribution representing soft tissues. Only by the use of LA-ICP-MS, the required high sensitivity and low detection limits for the determination of silver were obtained. Metal distribution within the part of cancellous bone was revealed for silver as well as for the implant constituents titanium, vanadium, and aluminum. Furthermore, the detection of coinciding high local zirconium and aluminum signals at the implant surface indicates remaining blasting abrasive from preoperative surface treatment of the nanosilver-coated device.

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.

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-tetraazacyclodoecane-α,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.

Ambient molecular imaging by laser ablation atmospheric pressure chemical ionization mass spectrometry

RATIONALE The functions and properties of compounds are not only specified by their chemical structures, but also by their location inside a sample. Mass spectrometry is a powerful tool for imaging, whereby the kind of sample and compound depend on the used sampling and ionization methods. The developed laser ablation mass spectrometry method delivers high resolution images of small molecules in native samples. METHODS A UV laser ablation (LA) system was combined with an atmospheric pressure chemical ionization (APCI) mass spectrometer. The spatially resolved sampling was performed by focusing the 213 nm laser beam onto a sample. The fine aerosol generated by the ns pulsed laser irradiation was then transported to the APCI mass spectrometer by a nitrogen stream. In the APCI source, post-ionization was accomplished by a corona discharge. The resulting ions were detected with an orbitrap mass spectrometer. RESULTS The properties of the novel LA-APCI-MS setup are demonstrated by spatially resolved analysis of several samples including tablets, TLC plates and dried droplets. The target compounds are detected with high spatial and mass resolution. For higher molecular weight compounds like thyroxine, fragmentation was observed, whereas small molecules like caffeine stayed intact. CONCLUSIONS LA-APCI-MS is introduced as an ambient molecular mass spectral imaging method for molecules with high resolution in space and mass. The combination of two independent instruments offers flexible ion source and mass analyzer exchange and therefore LA-APCI-MS opens up new possibilities for molecular imaging under ambient conditions.

Elemental bioimaging of manganese uptake in C. elegans

A new method for elemental bioimaging with laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was developed and applied to study the uptake of manganese (Mn) in Caenorhabditis elegans (C. elegans). C. elegans is a well-established model organism in neuroscience, genetics and genomics, which has been extensively studied to decipher mechanisms of heavy metal induced toxicity. Knowledge about the distribution of manganese (Mn) and other metals in this organism will be helpful in elucidating pathways and mechanisms of transport, distribution and excretion. The LA-ICP-MS method requires limited sample preparation and can be used rapidly and easily to visualize the Mn distribution in C. elegans. Due to thorough optimization of the analytical parameters, intense Mn signals in C. elegans wild-type (WT) and mutants were obtained at a spatial resolution as small as 4 μm, thus proving the suitability of LA-ICP-MS to study the uptake of metals in C. elegans.