Fig. 3: Custom fiducials for 3D dSTORM imaging, unsupervised data acquisition and live-cell single-molecule tracking.

a SEM image of a spherical fiducial enclosed in a nano-fabricated protection box (n = 60 per array). b Transmitted diffraction profile of the fiducial inside the box. c 3D dSTORM images of the microtubule network in fixed COS-7 cells color-coded as indicated by the colored depth (n = 12). Cells were grown directly on the coverslide and acquired in an unsupervised manner. Inset shows the diffraction limited image. d Axial profile of a microtubule in (c) has a resolution of 50.2 nm. e–g Clusters of localization points for individual Alexa-Plus 647 molecules were selected and aligned by their centers of mass and fit to a Gaussian distribution. The standard deviation was 4.9 nm in the x-direction (e), 6.3 nm in the y-direction (f) and 13.4 nm in the z-direction (g). h–l Single-molecule tracking in living cells. Representative three-dimensional images images of CD47 molecules in live HeLa cells acquired using highly inclined and laminated optical sheet (HILO) microscopy. A time series was acquired at the surface (h), a depth of 1.5 µm (i), and 3 µm (j) (n = 4). k–l Details of a track. x and y displacement of a representative CD47 molecule (k) and 2D representation (l) using the color-coded time scale in (k). Scale bars = 10 µm in (a–c), 5 µm in (h) and 200 nm in (l).