The scheme of 3D STORM. (A) Three-dimensional localization of individual fluorophores. The simplified optical diagram illustrates the principle of determining the z-coordinate of a fluorescent object from the ellipticity of its image by introducing a cylindrical lens into the imaging path. The right panel shows the images of a fluorophore at various z positions. (B) The calibration curve of the image widths w_x and w_y as a function of z obtained from single Alexa 647 molecules. Each data point represents the average value obtained from 6 molecules. The data were fit to a defocusing function (red curve) as described in the Supporting Online Materials (27). (C) Three-dimensional localization distribution of single molecules. Each molecule gives a cluster of localizations due to repetitive activation of the same molecule. Localizations from 145 clusters were aligned by their center-of-mass to generate the overall 3D presentation of the localization distribution (left panel). Histograms of the distribution in x, y and z (right panels) were fit to a Gaussian function, yielding the standard deviation of 9 nm in x, 11 nm in y, and 22 nm in z.

Three-dimensional STORM imaging of microtubules in a cell. (A) Conventional indirect immunofluorescence image of microtubules in a large area of a BS-C-1 cell. (B) The 3D STORM image of the same area with the z-position information color-coded according to the colored scale bar. Each localization is depicted in the STORM image as a Gaussian peak, the width of which is determined by the number of photons detected (5). (C-E) The x-y, x-z and y-z cross-sections of a small region of the cell outlined by the white box in (B), showing 5 microtubule filaments. Movie S1 shows the 3D representation of this region, with the viewing angle rotated to show different perspectives (27). (F) The z profile of two microtubules crossing in the x-y projection but separated by 102 nm in z, from a region indicated by the arrow in (B). The histogram shows the distribution of z-coordinates of the localizations, fit to two Gaussians with identical widths (FWHM = 66 nm) and a separation of 102 nm (red curve). The apparent width of 66 nm agrees quantitatively with the convolution of our imaging resolution in z (represented by a Gaussian function with FWHM of 55 nm) and the previously measured width of antibody-coated microtubules (represented by a uniform distribution with a width of 56 nm) (5).

Three-dimensional STORM imaging of clathrin-coated pits in a cell. (A) Conventional direct immunofluorescence image of clathrin in a region of a BS-C-1 cell. (B) The 2D STORM image of the same area with all localizations at different z positions included. (C) A x-y cross-section (50 nm thick in z) of the same area showing the ring-like structure of the periphery of the CCPs at the plasma membrane. (D, E) Magnified view of two nearby CCPs in 2D STORM (D) and their 100 nm thick x-y cross-section in the 3D image (E). (F - H) Serial x-y cross-sections (each 50 nm thick in z) (F) and x-z cross-sections (each 50 nm thick in y) (G) of a CCP, and an x-y and x-z cross section presented in 3D perspective (H), showing the cage like structure of the pit.