A biosinpired genetically-expressible dual-modality peptide-based probe for super-resolution correlative light and electron microscopy - 1 abstract 2 the overall goal of this project is to…

A biosinpired genetically-expressible dual-modality peptide-based probe for super-resolution correlative light and electron microscopy - 1 abstract 2 the overall goal of this project is to rationally engineer a new genetically expressible lanthanide-based 3 photostable phosphorescent probe with time-integrated single molecule brightness and high electron density 4 for super resolution correlative light and electron microscopy (clem) to enable elucidation of cellular and 5 detailed subcellular structures at the pseudoatomic level. Imaging/monitoring single molecules with nanoscale 6 spatiometric resolution within cells is an essential step for investigating the details of cellular processes. Super- 7 resolution and electron microscopy techniques can achieve the resolution required for those purposes and to 8 those ends, fluorescent probes such as fluorescent small molecules, fluorescent proteins (FPS), and quantum 9 dots (QDS) have become essential tools for research and medical diagnostics. 10 despite the variety of probes that exist, there is an unmet need for a genetically encoded, dual-modality probe 11 suitable for super-resolution correlated light and electron microscopy (clem). Such a probe would have time- 12 integrated single-molecule brightness necessary for super-resolution light microscopy and be electron dense 13 enough to allow visualization/localization by electron microscopy, especially cryogenic electron tomography 14 (CET). To meet those needs, photon biosciences is engineering a novel and innovative enabling bioinspired 15 peptide-based probe that has the time-integrated single-molecule brightness and electron density necessary 16 for super-resolution clem. The objective of this phase i project is to rationally engineer a probe to have ≥25 17 LN3+-phosphorescent centers, which we hypothesize will have the phosphorescence and electron density 18 necessary to surpass current single-modality clem probes and then show the probe's versatility by using IT in 19 cryo-luminescence microscopy to guide focused ION beam (FIB) milling of pseudomonas aeruginosa PAO1 20 expressing a crispr-CAS genome-edited engineered construct of our innovative peptide probe fused to the 21 S15 ribosomal protein at native levels as a proof-of-concept for super-resolution clem. Aim 1 is to optimize 22 our initial peptide probe via rational engineering for high electron density and brightness. Aim 2 is to develop a 23 multimeric probe from its precursor by concatenation and increase its electron density and brightness further. 24 aim 3 is to demonstrate proof of concept by cryo-luminescence microscopy. Aim 4 is to demonstrate further 25 proof-of-concept by using the probe for probe-guided FIB milling and tomography. Successful completion of 26 this project will result in an electron dense, time-integrated single molecule brightness, genetically expressible 27 dual modality probe for super-resolution clem.