Filtern
Erscheinungsjahr
- 2016 (3) (entfernen)
Dokumenttyp
- Vortrag (2)
- Zeitschriftenartikel (1)
Sprache
- Englisch (3)
Schlagworte
- Antibody labeling (1)
- Bioimaging (1)
- LA-ICP-MS based immunoassays (1)
- Metal staining procedures (1)
- Single cell analysis (1)
- bioimaging (1)
- single cell analysis (1)
Eingeladener Vortrag
- nein (2)
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. 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.
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).
Immuno imaging by the use of Laser Ablation Inductively Coupled Mass Spectrometry (LA-ICP-MS) is a growing research field in life sciences such as biology and biomedicine. Various element labeling strategies for antibodies have been developed for the application of multiplex immunoassays analyzed by the use of LA-ICP-MS. High multiplexing capabilities, a wide linear dynamic range and the possibility of absolute quantification are the main advantages of ICP-MS. But in the context of immuno imaging by the use of LA-ICP-MS, quantification of analytes is limited due to non-controllable antibody labeling chemistry. In the presented proof-of-principle a novel antibody labeling technique has been investigated which results in a controlled labeling degree. A small affinity protein based on the C2 domain of protein G was modified with conventional metal coded tags (MeCAT) after introducing a cysteine into the C-terminus of the protein. The modified C2 domain photo-crosslinks to the Fc or Fab region of the IgG and allows specific and covalent labeling of antibodies for multiplex immunoassay analysis by the use of LA-ICP-MS. In combination with a house-made calibration membrane the amount of labeled antibody–antigen complexes in a multiplex western blot immunoassay was determined by LA-ICP-MS.