Filtern
Dokumenttyp
- Vortrag (6) (entfernen)
Referierte Publikation
- nein (6) (entfernen)
Schlagworte
- Bioimaging (6) (entfernen)
Organisationseinheit der BAM
Eingeladener Vortrag
- nein (6)
Biological systems exhibit a very high complexity, because they consist of various cell populations showing heterogenic characteristics. Therefore the individual analysis of single cells is important to understand cellular processes as well as their function in a cell system. Especially investigation in the question about what is biological variability and what is a substantial difference between two cells of the same type (which might lead to dysfunction or disease) is of high interest.
Today improved spatial and temporal resolution enable the use of laser ablation inductively coupled mass spectrometry (LA-ICP-MS) for element microscopy of single cells. Next to the characterization of natural trace elements the introduction of artificial metal labels into cells is of high interest. Element labeling of cell compartments and/or proteins allows its simultaneous analysis and localization within a cell via element microscopy. Two fast and simple metal staining procedures of adherent cells for identification of single cells via LA-ICP-MS at sub micrometer resolution are presented.
Two labeling reagents were selected which were already described in literature for applications around protein and cell analysis. An Iridium-intercalator is utilized to stain the cell nuclei whereas the whole cell is stained by maleimido-mono-amide-DOTA-complexes (mDOTA) loaded with lanthanide(III) isotope ions.
The metal staining procedures allow the visualization of single cells by element microscopy independent of a superposition of analyte’s 2D element intensity profile with a prior taken bright field image of the sample. The applicability was shown on an actual question in nano-toxicology (the cellular uptake of nanoparticles).
LA-ICP-MS finds widespread use in the analysis of biological samples and its importance as an analysitical method is still increasing. Often tissues or single cells are ablated in imaging mode on glass slides, but the analysis time increases by reducing the spot size of the laser. Additionally, with increased lateral resolution a loss in sensitivity is inherently connected and this holds true in particular for single cell analysis in imaging mode. However, if only the composition of elements in a single cell needs to be measured, complete ablation of cells by single shots looks promising. This approach was already discussed by Managh et al. and will be discussed in more detail in this presentation. The aim of this study is to develop a calibration strategy for single cell analysis based on the total consumption concept. It will be shown that this concept provides quantitative results at single cell level, if the target material is ablated. We compare two different laser spot sizes, 30 µm and 110 µm. The smaller spot size of 30 µm is used for imaging and the larger spot size of 110 µm is used for the targeted ablation of complete single cells. Cells were stained by two novel cell dyes, mDOTA-Tb (CheMatech, Dijon, France), which binds to free thiol groups of proteins, and Ir-DNA intercalator (Maxpar®, Fluidigm, San Francisco, USA), which stains DNA quantitatively. Finally, quantification of Ir and Tb will be performed by matrix matching calibration, following the approach of Drescher et al.[1], i.e. spotting of a dilution series of Ir and Tb standards on nitrocellulose membrane. Results obtained so far show a good correlation between imaging and single spot ablation, indicating that the total consumption concept may serve as an acceptable assumption.
The interaction of nanoparticles (NPs) with cells has become a major field of interest, ranging from therapeutic applications to nanotoxicology. The cellular uptake depends on the primary characteristics of the NPs (e.g. size, shape, surface modification) and on the cells interacting with the particles. Thereby the quantification of NPs in cells is of particular importance to obtain information under different experimental conditions. Presently, the number of NPs internalized is often determined by inductively coupled plasma (ICP) optical emission spectrometry (OES) or ICP mass spectrometry (MS) after acid digestion of a cell suspension or a cell pellet. The result is an average value and no information about the distribution among cells or within a cell is available. Therefore we developed a method based on laser ablation (LA) in combination with ICP-MS to localise and quantify metallic NPs in single cells.
LA-ICP-MS is a powerful analytical method which offers excellent sensitivity at high spatial resolution and multielement capability without time-consuming sample preparation steps. Recently, LA-ICP-MS was established for elemental mapping of biological samples like tissues.
In our experiments, fibroblast cells were incubated with gold or silver containing nanoparticles and grown on sterile coverslips under standard conditions. For LA analysis the cells were fixed with formaldehyde and dried. Subcellular resolution is achived by careful optimisation of laser energy, ablation frequency and scan speed. The elemental distribution was determined by continuous ablation line by line of cells incubated with NPs. Our results show that LA-ICP-MS is able to detect NP aggregates within cellular substructures. After 24 h of incubation the NPs were found in the cytosol, preferencially in the perinuclear region, but do not enter the nucleus. Additionally, a quantification strategy at single-cell level was developed. For this purpose nitrocellulose membrane was spiked with Ag or Au nanoparticle suspension at different concentration levels and analysed by LA-ICP-MS. Based on this calibration the number of NPs taken up by individual cells was determined and variations within the cell population become visible. The cells show a strong dependence of NP uptake on concentration and incubation time.
Our results demonstrate the potential of LA-ICP-MS providing insight into NP uptake and intracellular distribution dependent on experimental parameters.
Elemental imaging of biological samples using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) provides spatially resolved information on element distribution in thin sections. By rastering with a laser across the sample, a two-dimensional image of the elemental distribution can be reconstructed that shows the intensities of the respective elements. Using overlapping laser shots the area ablated from single cells or thin sections (thickness 5 to 10 µm) can be reduced significantly so that the pixel size of the intensity measurement is significantly reduced. Having in mind that a laser shot ablates thin biological samples completely, we can make use of a new concept for calibration in the laser ablation method: the concept of total consumption. This calibration strategy allows production of simple matrix matched standards and provides an internal standardisation by ink jet technology, where a metal containing inks is printed on as thin layer on top of a biological sample.
Different applications will be presented where our concepts have been applied. In the first example we used nanoparticle suspension of given particle numbers to quantify the uptake of metallic nanoparticles by biological cells. In the second example antibodies have been tagged by metals to measure protein expression in prostata cancer. In this approach application of house keeping proteins are investigated additionally to compensate variations in thickness and density of the biopsy samples. In the third application different nephrotoxic behaviour of Pt containing drugs have been investigated to study the local enrichement in kidney samples of mice treated with these three different compounds. Here the internal standard is required to allow intercomparisons between different individual mouse tissues.
At the end of the lecture future trends will be discussed for elemental microscopy.
In der klinischen Diagnostik werden für zytometrische Messverfahren bereits eine Reihe von Reagenzien eingesetzt zur Markierung von Antikörper eingesetzt, um die Detektion von Biomarkern mittels Fluoreszenz- oder Flugzeitmassenspektrometrie zu ermöglichen.
Seit kurzem ist auch eine Imaging Mass Cytometry Kombination direkt erhältlich, wodurch der Nachweis von Biomarkern in Gewebeschnitten erreicht werden kann. Dazu wird eine Kopplung von Laser Ablation und induktiv gekoppeltem Plasma Massenspektrometrie eingesetzt, wobei ähnlich der Massenzytometrie, zuvor Antikörper mit Metallen markiert, und im Anschluss mit dem Gewebeschnitt inkubiert werden. Durch die hohe Ortsauflösung können die Biomarker lokalisiert, und zukünftig vielleicht auch quantifiziert werden.
Insbesondere Lanthanide eignen sich als Markierungsmetalle, da sie einen niedrigen Untergrund und chemisch ähnliches Verhalten zueinander aufweisen. Allein durch diese Elemente können bereits etwa 15 Parameter unterschieden werden, was durch isotopenreine Standards weiter gesteigert werden kann.
Vom Markierungsgrad abhängig werden unterschiedlich viele Metalle am Antikörper gebunden, und beeinflussen so die Sichtbarkeit im ICP-MS. Nanopartikel könnten daher eine deutliche Steigerung der Sensitivität bewirken. GdVO4 Nanokristalle scheinen bisher sehr vielversprechend und bieten neben multiparametrischen Anwendungen auch Multimodalität.
Die Synthese der Nanokristalle zeigte hohe Homogenität und Reproduzierbarkeit in Partikelgröße in der Zusammensetzung. Ein erstes Experiment mit einer Zellkultur konnte bereits die effiziente Markierung der Zellen unter Beweis stellen, wobei durch hohe Signalstärke auch subzelluläre Auflösung in der LA-ICP-MS erreicht werden konnte.
Atherosklerotische Läsionen können durch Magnetresonanztomographie (MRT) unter Verwendung von sehr kleinen Eisenoxidnanopartikeln (VSOP) sichtbar gemacht werden. VSOP akkumulieren in atherosklerotischen Plaques und dienen somit als Atherosklerosesonde.
Das Ziel dieses Projektes war die Abbildung der Verteilung von Europium-dotierten VSOP (Eu-VSOP) durch Laserablation ICP-MS in histologischen Dünnschnitten der Aortenwurzelregion des ApoE-Knockout-Mausmodells, das reich an atherosklerotischen Plaques ist. Darüber hinaus wurde untersucht, ob die VSOP-Akkumulation in den Plaques mit anderen Biomarkern der Entzündung wie Makrophagen und verändertem Endothel korreliert. Somit wäre eine Beurteilung, ob es sich hierbei um instabile bzw. vulnerable Plaque-Regionen handelt möglich. Zu diesem Zweck wurden Antikörper mit verschiedenen Lanthaniden markiert und mit der Eu-VSOP-Verteilung unter Verwendung von LA-ICP-MS in einem Multiplex-Messmodus korreliert.
Eine mögliche Korrelation von reaktiven Stickstoffspezies (RNS) mit endogenem Eisen oder Eu-VSOP kann ebenfalls durch LA-ICP-MS nachgewiesen werden. Zu diesem Zweck wurden RNS-spezifische Antikörper auch mit Lanthaniden markiert.