Over the last quarter century, innovations in fluorescence microscopy have allowed scientists to take monumental strides towards a deeper understanding of the inner workings of the cell. The trimethoprim (TMP)–based tag takes advantage of the highly specific and high affinity binding of the TMP small molecule antibiotic to the E.coli dehydrofolate reductase (eDHFR) enzyme, allowing for super-resolution imaging with high signal-to-noise ratios. Through standard transfection methods, eDHFR is genetically encoded into the DNA adjacent to the gene for the protein of interest (POI) and thus fused to the POI when expressed. The covalent model of this TMP-tag involves a L28C mutation on the eDHFR enzyme reacting with an acrylamide group installed on the TMP-probe heterodimer. Because this reaction is induced by proximity, and the efficiency of the reaction is inversely correlated to linker length as shown by the progression from first to second generation covalent TMP-tags, my research in Professor Virginia Cornish's lab seeks to further shorten the linker on the TMP-probe to optimize reaction time and improve functionality. Additionally, we explore a few oxazine-based fluorophores to optimize fluorophore cell permeability.