(3eh) Molecular Engineering Chemical Imaging Probes for Super Resolution Fluorescence Microscopy

My research area spans from protein engineering to single molecule fluorescence microscopy extending application to protein formulation and synthetic biology. Through my PhD and post-doc training, I gained experience in the interdisciplinary environment based on chemical engineering education. My current post-doc projects will be presented.

1) New dye was developed for super resolution microscopy, stochastic optical reconstruction microscopy (STORM). Recent advances in imaging and single molecule fluorescence microscopy (SMFM) have enabled direct observation of biological processes at the molecular level. Super-resolution imaging techniques such as photoactivated localization microscopy (PALM)and STORM have been developed using photoswitchable fluorescent proteins and organic dyes. However, there is a strong need for development of bright and photostable nanoscale probes for biological imaging as pointed out from inventors of STORM and PALM. In this work, we suggest dye-conjugated dendrimers as nanoscale imaging probes for high-resolution fluorescence microscopy, which overcome the diffraction limited optical resolution. Collective effects in multichromophoric systems generated controlled blinking and enhanced localization precision compared to single organic dyes such as Cy5 or Alexa flour 657. With this new generation of fluorescent dye, we were able to achieve a 10 nm spatial resolution for reconstructed images from dSTORM, which surpasses the spatial resolution of single organic probes (25 nm). 

2) We demonstrate labeling of transcription factor proteins by incorporation of non-natural amino acids containing bioorthogonal chemical functionalites (azide or alkyne) followed by copper-free click chemistry. The cyclooctyne promotion [3 + 2] dipolar cycloaddition with azides, “copper-free click chemistry” provides rapid and site-specific labeling of transcription factors (TFs). Following expression, purification and labeling, transcription factor proteins retain their sequence-specific DNA-binding activity under native conditions, whereas the activity of TFs labeled by copper-mediated click chemistry was ablated. Copper-free click chemistry exhibited high reaction yields, even at low temperatures or in the presence of high salt concentrations, which are generally unfavorable reaction conditions for common methods involving hydrolysis-mediated protein modification. Residue-specific replacement of methionine by azidohomoalanine was used to achieve mono- or multi-functionalization of engineered or “synthetic” transcription factor proteins.