NanoSIMS

Khimheng Lau

D.Phil. Student 2006 - 2011

Khim used the NanoSIMS to localize the distribution of target molecules and tracers in cultured cells and tissue.

Combined NanoSIMS and Fluorescent Microscopy of BrdU in HeLa Cells:

The aim of this project was to develop a combined methodology of high resolution NanoSIMS and fluorescence microscopy to provide sub-cellular information on the distribution of bromine from the well known BrdU molecule in HeLa cells. Samples were prepared by simple chemical fixation and desiccation on a numbered grid to allow easy co-registration of the same cell in the fluorescence microscope and the NanoSIMS with the NanoSIMS able to provide significantly higher resolution images. This project was in collaboration with the Gray Institute for Radiation Oncology and Biology.

EF5 in Tissue section:

The NanoSIMS was used to study the localization of immunofluorescence EF5 molecules in a chemically fixed tissue section. The treated tissue sample was co-registered with an inhibitor that reduces hypoxia in tumor and compared to an untreated sample to show the different degree of EF5 binding by mapping for fluorine. This project was in collaboration with the Gray Institute for Radiation Oncology and Biology.

Localisation of Non-Fluorescent molecules, ATN-224 and Cu[ATSM]:

The ability to show the precise location of BrdU and EF5 molecules using the NanoSIMS, and to confirm the same distribution in the fluorescence microscope for antibody-tagged BrdU and EF5, gave us confidence that the NanoSIMS could be reliably used for the localisation of non-fluorescent molecules. In order to demonstrate this, the NanoSIMS was used to determine the distribution of a non-fluorescent drug, ATN-224, in endothelial cells culture and halogen-tagged Cu[ATSM], in EMT-6 and HT1080 tumour cell lines. The sequestration and mechanism of actions for both non-fluorescent compounds at the sub-cellular level is not yet fully understood but high resolution NanoSIMS analysis was able to provide more detailed information on the distribution of these compounds. This project was in collaboration with the The Weatherall Institute of Molecular Medicine (ATN-224 in HUVEC) and the Department of Chemistry, University of Oxford (Halogen tagged-Cu[ATSM]).

Publications:

K.H. Lau, M. Christlieb, M. Schröder, H. Sheldon, A.L. Harris, C.R.M. Grovenor, Journal of Microscopy 240(1) (2010) 21-31