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The technique of colour EM that was recently developed enabled localisation of specific macromolecules/proteins of interest by the targeted deposition of diaminobenzidine (DAB) conjugated to lanthanide chelates. By acquiring lanthanide elemental maps by energy‐filtered transmission electron microscopy (EFTEM) and overlaying them in pseudo‐colour over the conventional greyscale TEM image, a colour EM image is generated. This provides a powerful tool for visualising subcellular component/s, by the ability to clearly distinguish them from the general staining of the endogenous cellular material. Previously, the lanthanide elemental maps were acquired at the high‐loss M 4,5  edge (excitation of 3d electrons), where the characteristic signal is extremely low and required considerably long exposures. In this paper, we explore the possibility of acquiring the elemental maps of lanthanides at their N 4,5  edge (excitation of 4d electrons), which occurring at a much lower energy‐loss regime, thereby contains significantly greater total characteristic signal owing to the higher inelastic scattering cross‐sections at the N 4,5  edge. Acquiring EFTEM lanthanide elemental maps at the N 4,5  edge instead of the M 4,5  edge, provides ∼4× increase in signal‐to‐noise and ∼2× increase in resolution. However, the interpretation of the lanthanide maps acquired at the N 4,5  edge by the traditional 3‐window method, is complicated due to the broad shape of the edge profile and the lower signal‐above‐background ratio. Most of these problems can be circumvented by the acquisition of elemental maps with the more sophisticated technique of EFTEM Spectrum Imaging (EFTEM SI). Here, we also report the chemical synthesis of novel second‐generation DAB lanthanide metal chelate conjugates that contain 2 lanthanide ions per DAB molecule in comparison with 0.5 lanthanide ion per DAB in the first generation. Thereby, fourfold more Ln 3+  per oxidised DAB would be deposited providing significant amplification of signal. This paper applies the colour EM technique at the intermediate‐loss energy‐loss regime to three different cellular targets, namely using mitochondrial matrix‐directed APEX2, histone H2B‐Nucleosome and EdU‐DNA. All the examples shown in the paper are single colour EM images only.

Elemental mapping of labelled biological specimens at intermediate energy loss in an energy‐filtered TEM acquired using a direct detection device