Fig. 4: Applications to super-resolution microscopy of UnaG.

a Averaged photon numbers per frame from single holoUnaG as increasing the camera exposure time. b Distribution of photon counts per single switching event at 180 ms exposure time. c Comparison of the photon numbers per switching cycle for conventional super-resolution probes measured in an identical experimental setup, with 200 ms of camera exposure time. High illumination powers at 561 nm (mMaple3) or 488 nm (UnaG and Atto 488) were used so that most of the single fluorophores switched off in a single camera frame. As a result, UnaG gave more than 1200 photons, which stands between the mMaple3 and Atto 488. d Repetitive localization positions measured from surface-immobilized single UnaG molecules, projected in x axis and y axis in the lateral directions. Multiple localization distributions were aligned by their centroid positions obtained from Gaussian fits. The combined datasets were again fitted with Gaussian that yielded ~12 nm of localization precision and ~28 nm of full-width at half-maximum (FWHM) in lateral planes. The localization distributions were normalized by their own maximum counts. e Demonstration of SML imaging utilizing the photo-switchable nature of holoUnaG in various subcellular structures (left: ER, middle: vimentin filaments, right: clathrin-coated pits) in fixed Cos7 cells. f Close-up widefield and SML images from the yellow-boxed regions in e. Scale bars: 2 µm for e; 200 nm for f. Error bars: standard deviations (n = 5, each measurement contained more than 500 single-molecule information). CLC clathrin light chain.