Fluorescence Nanoscopy by Polarization Modulation (SPoD) and Polarization Angle Narrowing (ExPAN)

Abstract

result in improved performance across multiple AFM modalities, including single molecule force spectroscopy. However, instrumental drift in AFM remains a critical issue that limits the precision and duration of experiments. Previously, we developed an active optical stabilization technique to improve tip-sample stability at ambient conditions. However, force drift also occurs via uncontrolled deflection of the zero-force position of the cantilever. We found that the primary source of force drift in liquid for a popular class of soft cantilevers is their gold coating, even though they are coated on both sides to minimize drift. While removing the gold led to~10-fold reduction in reflected light, we nonetheless achieved a 10-fold improvement in force stability of bioAFM, with a sub-pN force precision over a broad bandwidth (0.01-20 Hz) just 30 minutes after loading. We subsequently extended AFM's sub-pN bandwidth by a factor of~50 to span five decades of bandwidth (Df z 0.01-1,000 Hz by developing an efficient process to modify a short (L ¼ 40 mm) commercially available cantilever (BioLever Mini) with a focused ion beam (FIB). Measurements of mechanically stretching individual proteins showed improved force precision coupled with state-of-the-art force stability and no significant loss in temporal resolution compared to stiffer, unmodified cantilevers. Ongoing work in our lab extends this concept down to ultrashort cantilevers (L ¼10 mm) along with instrumental development to detect these cantilevers in a commercial AFM. Such cantilevers enable sensitive detection of protein unfolding and refolding with~1 ms time resolution. Importantly, these cantilevers were robust and were reused for SFMS over multiple days. Hence, we expect these responsive, yet stable, cantilevers to broadly benefit diverse AFM-based studies. Telomeres play important roles in maintaining the stability of linear chromosomes. A specialized protein complex, called shelterin or telosome, binds to and protects telomeres at chromosome ends. Telomere maintenance involves dynamic actions of multiple proteins interacting with long repetitive sequences and complex dynamic DNA structures, such as T-loops. Furthermore, it was shown recently that in contrast to cohesion along chromosome arms, sister telomere association is a specialized process requiring a tighter association provided by the cohesin subunit SA1 in conjunction with specific proteins from the shelterin complex. To better understand the telomere maintenance pathways, we established complementary single-molecule imaging platforms: a newly developed Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy (DREEM) technique capable of revealing DNA paths in protein-DNA complexes, fluorescence imaging of quantum dot-labeled proteins for tracking dynamics of proteins on DNA tightropes, and a nanochannel based imaging platform for studying protein-mediated DNA-DNA pairing/looping in real time. I will highlight our recent results on: 1) Revealing DNA paths inside TRF2 complexes during DNA co...