Elemental Mapping of Labeled Biological Specimens at Intermediate Energy Loss in an Energy-Filtered TEM acquired using a Direct Detection Device

ABSTRACT The multi-color or single-color EM that was developed previously, by the pseudo-colored overlay of the core-loss or high-loss EFTEM elemental map/s of the lanthanide onto the conventional image, the lanthanide chelates conjugated to diaminobenzidine being sequentially deposited as a result of selective oxidization by orthogonal photosensitizers / peroxidases. The synthesis of the new second generation lanthanide DABs, which contains 4 times more lanthanide per DAB, gives significant signal amplification and enabling collection of elemental maps at much lower energy-loss regions more favorable. Under the same experimental conditions, acquiring EFTEM elemental maps for the lanthanides at the lower energy-loss of N 4,5 edge instead of the core-loss M 4,5 edge, provides ~4x increase in signal-to-noise and ~2x increase in resolution. The higher signal at the N 4,5 edge, also allows for more sophisticated technique of EFTEM spectrum Image for the acquisition of elemental maps with very high signal fidelity.