Advancing Multi-Color EM via Direct Detector-enabled 4D-STEM

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PROJECT SUMMARY This focused technology development project will advance into first practice a transformative new imaging technology for multicolor electron microscopy (ColorEM), enabling new capabilities for high resolution and 3 dimensional (3D) localization and differentiation of different molecular complexes in cells and tissues. With this technology, we will substantively improve upon presently practiced, multiple labeling strategies involving antibody labelling approaches detected using colloidal gold, quantum dots, etc.. We will further provide a more accessible, higher throughput, and dose efficient alternative to spectroscopic methods, like energy dispersive x-ray (EDX) and electron energy loss spectroscopy (EELS) / energy filtered transmission electron microscopy (EFTEM). At the heart of this effort, we will exploit the performance characteristics of a next- generation, ultra-high speed direct detection device (DDD), now being optimized and extended (by us with an industry partner) for use with scanning transmission electron microscopy (STEM), a broadly deployed capability on modern EM columns. The optimized application of this new pixelated STEM detector technology, in combination with an extended palette of elemental probes and advanced software analysis, will allow for unprecedented new capabilities for performing atomic number (Z) contrast imaging – a process we call ColorSTEM. To bring this new integrated methodology into first practice, we will build on recent feasibility studies and surmount remaining technical hurdles to 1) make multi-labelled EM specimens with optimized elemental probe combinations; 2) troubleshoot the first use of a potentially transformative new sensor technology and work out the methodology for employing it for Z contrast imaging; and 3) devise the associated analytical/computational methodology for processing the very large resultant data sets to enable unambiguous differentiation of molecular complexes selectively marked with elemental probes.