Fig. 1: Imaging single proteins and mechanical oscillations.
From: Optical imaging of single-protein size, charge, mobility, and binding
a Proteins are tethered to an ITO surface with a flexible polymer tether, and driven into oscillation by an alternating electric potential applied to the surface with a three-electrode electrochemical configuration, where WE, RE, and CE are the working (the ITO surface), reference (Ag/AgCl wire) and counter electrodes (Pt coil), respectively. The oscillating protein molecules scatter the evanescent field generated by illuminating the ITO surface with light from a SLED, and the scattered light is collected to form an image captured with a CMOS camera. b The polymer tether is a 63 nm long polyethylene glycol (PEG), which links the protein (hydrodynamic diameter, DH) to the ITO surface via surface chemistry described in “Methods”. c Time sequence images of oscillating molecules (bovine serum albumin or BSA) recorded at 800 frames/s with potential modulation amplitude and frequency of 8 V and 80 Hz, respectively. d Fast Fourier Transform (FFT) filter is applied to the time sequence images shown in c to produce an FFT image, which resolves a single BSA molecule. e Spatial Fourier transform of the FFT image (k-space) in d showing two rings, indicating the FFT image pattern is due to the interference between a planar wave (evanescent) and circular (scattering by a molecule) waves. f FFT image contrast vs. potential amplitude, showing a linear regime at low electric fields, and a plateau regime associated with fully stretching of the PEG tether at high electric fields. The two regimes are indicated by the blue dash line. g Oscillation amplitude (Δz0) of a BSA molecule vs. potential amplitude (U0). Diameter and charge are determined from the amplitude of the plateau regime (black dash line) and the slope of the linear regime (blue line), respectively. Scale bars in c, d, f represent 3 µm.
