Mackey, M. R. | Search bioRxiv

bioRxiv2023

Fe-TAMLs as a new class of small molecule peroxidase probes for correlated light and electron microscopy

We introduce Fe-TAML, a small molecule-based peroxidase as a versatile new member of the correlated fluorescence and electron microscopy toolkit. The utility of the probe is demonstrated by high resolution imaging of newly synthesized DNA (through biorthogonal labeling), genetically tagged proteins (using HaloTag), and untagged endogenous proteins (via immunostaining). EM visualization in these applications is facilitated by exploiting Fe-TAMLs catalytic activity for the deposition of localized osmiophilic precipitates based on polymerized 3,3-diaminobenzidine. Optimized conditions for synthesizing and implementing Fe-TAML based probes are also described. Overall, Fe-TAML is a new chemical biology tool that can be used to visualize diverse biomolecular species along nanometer and micron scales within cells.

cell biology↗

bioRxiv2020

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

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 N4,5 edge instead of the core-loss M4,5 edge, provides ~4x increase in signal-to-noise and ~2x increase in resolution. The higher signal at the N4,5 edge, also allows for more sophisticated technique of EFTEM spectrum Image for the acquisition of elemental maps with very high signal fidelity.

systems biology↗