Exploring Detection Limits of Advanced SIMS Technology in Cellular and Sub-Cellular Biological Systems

Institution CRP Sante , Laboratoire National de Santé
Partenaire(s) : CRP Gabriel Lippmann
Du : 01/01/2003
Au : 31/12/2008
Budget : 1 040 000,00€
Contact(s) : Müller Claude P.

Summary

Fluorescence microscopy is the unchallenged technique for structural analysis of cells and subcellular organelles. Some of the shortcomings can be resolved by alternative methods such as microautoradiography and more recently SIMS microscopy. This technology which combines chemical elemental microanalysis with lateral resolution is based on the detection of secondary ions emitted upon the impact of an energetic primary ion beam. The present project investigates potential applications and limitations of SIMS technology in cell biology.

During the initial phase of the project, SIMS specific sample preparation protocols were developed using either chemical or cryofixation of specimens. Additionally, ways to improve the analytical conditions of NanoSIMS image acquisition of organic samples were investigated. They focused on the characterisation of primary ion implantation and its effect on secondary ion emission during analysis of cells and tissues. Several modifications to standardize acquisition protocols with respect to the duration of implantation increased the daily throughput of properly implanted samples. These optimized protocols improved the quality of NanoSIMS images.

Recent studies highlighted a relationship between sources of heavy metal pollutants, such as industrial Cr and Cd emissions, and modifications of lichen physiology and cellular ultrastructure. A study on the distribution of atmospheric pollutants in lichens revealed that NanoSIMS is a valuable tool in identifying subcellular compartments involved in the retention of trace elements and is of particular interest in biomonitoring studies. An approach combining quantitative measurements of elemental concentrations by ICP-MS with qualitative ion maps by NanoSIMS allowed the determination of concentrations of various elemental pollutants and their distribution at cellular level.

Furthering the aggregated results obtained on measles virus nucleoprotein in an earlier phase of the project, 15N-labelled antibodies against nucleoprotein were produced. The incorporation of 15N was characterized by MALDI-TOF analysis. Immunolabelling experiments on the NanoSIMS showed an 15N enrichment, corresponding to the specific binding of the antibody to the viral protein in nucleoprotein rich subcellular regions. These experiments demonstrated that immunolabelling studies by NanoSIMS were possible. The data obtained by SIMS were confirmed by confocal microscopy and by a SIMS pulse labelling experiment developed in the frame of this study (Figure 1 and 2). This is the first report of an immunolabelling study using a stably labelled isotope in SIMS analysis.

Refereed Scientific Publications:
  • A Billing, J Turner, CP Muller. Rapid non-genomic effects in rat thymocytes. In preparation.
  • E Prodhomme. Identification of seven new post translational modifications in Measles virus nucleocapsid protein. In preparation.
  • JP Lasserre. Effects of the endocrine disruptors atrazine and PCB 153 on the protein expression of MCF-7 human cells. In preparation.
  • AB Schote, T Bechet, P Pirrotte, JD Turner, CP Muller. Characterisation of a new DNA binding domain deficient Chicken Ovalbumin Upstream Promoter- Transcription Factor IIΔ isoform in the human brain. Submitted.
  • F Fack, J Kessler, P Pirrotte, J Kremer, D Revets, W Ammerlaan CP Muller. Detection of differentially modified pathogen proteins by Western Blot after 2-D gel electrophoresis and identification by Maldi-Tof-Tof. COST Booklet. In press.
Other Publications

  • Patrick Pirrotte wins the 3rd prize in the Image processing using ImageJ Video Tutorial Competition, Second ImageJ User & Developer Conference, 6-7.11.2008, Luxembourg.
  • Patrick Pirrotte successfully defended his doctoral thesis “Biological applications of the NanoSIMS 50”, University of Strasbourg, France, December 2007
Project Website:

Figure 1: opsonized 15N conjugated bacteria mixed in a 1:10 ratio with M1241, observed on SIMS. (a) 31P in red, 12C14N in green (b) 12C14N in green, 79Br in yellow (c) 12C15N in yellow, 31P in green (d) 79Br in yellow, 31P in green