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Although it is the most commonly used technique for the chemical analysis of cells, mass spectrometry of cell lysate can only provide bulk analysis of chemicals present in cells. Energy dispersive x-ray spectrometry (EDS) is a well established technique that can provide the high resolution sub-cellular elemental mapping information. However EDS analysis is often performed only on a specific point or small region of a sample surface, providing a high resolution but limited localized chemical information. A manual 3D elemental mapping method for resin embedded biological samples was developed using the EDS and the Focused Ion Beam (FIB) SEM techniques. Although both the EDS and the FIB SEM are well established techniques, they are seldom used together because there is no automated way to perform these techniques in an interlaced manner.
Changes in cellular chemical signature often precede morphological changes in diseased cells. Also it is now widely accepted that the chemotaxis or chemical signaling is implicated in cancer metastasis. Therefore the ability to accurately detect and measure intra- and intercellular chemical and elemental distribution can lead to the identification of novel biomarkers for early detection of diseases and is crucial for the understanding and the proper interpretation of chemotaxis in biological systems.
One of the main difficulties of the EDS-FIB analysis is establishing a consistent mechanism to mill away proper amount of material at each step. Existing automated "Slice & View" technique cannot be used here because of the need to stop the process after each milling step for the EDS mapping. After exploring various ways to make consistent cuts, we settled on the use of FIB milled fiducial marks as the most reliable method to make consistent thickness cuts.
EDS-FIB analysis was performed on an osmium tetroxide fixed and resin embedded T. Pseudonana (diatom) sample using the FEI Nova NanoLab 600 FIB SEM instrument equipped with an Oxford Inca EDS system. SEM imaging and EDS analysis were performed using 5 KeV electron beam and milling was performed using 30 KeV ion beam. Once an area of interest with a high concentration of diatoms was identified, fiducial tick marks were FIB milled onto the sample surface near the area of interest at 250 nm interval. Initial milling was performed to line up the imaging surface (10 mm x 10 mm) with the first fiducial mark. After the high resolution imaging and the EDS mapping of the exposed surface, sample was milled until the next fiducial mark was reached. These steps were repeated until total of 7.5 mm thick layer was removed. Resulting SEM images and EDS data were aligned and processed using ImageJ, Matlab and Amira to produce 3D morphological and elemental maps of the diatoms as shown in Figure 1.
3D reconstruction of the silicon oxide shell of a diatom using the x-ray map and the SEM image stacks.
Start Date:June 8, 2007
Lead Organizational Unit:mml
FEI Nova NanoLab600 Focused Ion Beam
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