Improved Imaging Surface for Quantitative Single-Molecule Microscopy
Yu P. Zhang, Evgeniia Lobanova, Asher Dworkin, Martin Furlepa, Woo Suk Yang, Melanie Burke, Jonathan X. Meng, Natalie S. Potter, Renata L. Sala, Lakmini Kahanawita, Florence Layburn, Oren A. Scherman, Caroline H. Williams‐Gray, David Klenerman
Abstract
Preventing nonspecific binding is essential for sensitive surface-based quantitative single-molecule microscopy. Here we report a much-simplified RainX-F127 (RF-127) surface with improved passivation. This surface achieves up to 100-fold less nonspecific binding from protein aggregates compared to commonly used polyethylene glycol (PEG) surfaces. The method is compatible with common single-molecule techniques including single-molecule pull-down (SiMPull), super-resolution imaging, antibody-binding screening and single exosome visualization. This method is also able to specifically detect alpha-synuclein (α-syn) and tau aggregates from a wide range of biofluids including human serum, brain extracts, cerebrospinal fluid (CSF) and saliva. The simplicity of this method further allows the functionalization of microplates for robot-assisted high-throughput single-molecule experiments. Overall, this simple but improved surface offers a versatile platform for quantitative single-molecule microscopy without the need for specialized equipment or personnel.