The covalent trimethoprim chemical tag facilitates single molecule imaging with organic fluorophores.

Chemical tags can be used to selectively label proteins with fluorophores that have high photon outputs. By permitting straightforward single molecule (SM) detection and imaging with organic fluorophores, chemical tags have the potential to advance SM imaging as a routine experimental tool for studying biological mechanism. However, there has been little characterization of the photophysical consequences of using chemical tags with organic fluorophores. Here, we examine the effect the covalent trimethoprim chemical tag (A-TMP-tag) has on the SM imaging performance of the fluorophores, Atto655 and Alexa647, by evaluating the photophysical properties of these fluorophores and their A-TMP-tag conjugates. We measure SM photon flux, survival lifetime, and total photon output under conditions that mimic the live cell environment and demonstrate that the A-TMP-tag complements the advantageous SM imaging properties of Atto655 and Alexa647. We also measure the ensemble properties of quantum yield and photostability lifetime, revealing a correlation between SM and ensemble properties. Taken together, these findings establish a systematic method for evaluating the impact chemical tags have on fluorophores for SM imaging and demonstrate that the A-TMP-tag with Atto655 and Alexa647 are promising reagents for biological imaging.