S2 cells have microtubules immunostained with Alexa Fluor 647. a, STORM images of six adjacent planes in the x'y plane. The advantage of light-sheet illumination in minimizing out-of-focus bleaching is evident from the similar image qualities from earlier- to later-acquired images (all using identical acquisition parameters). b, The projection of the images in a onto the xy plane. c,d, Diffraction-limited (c) and super-resolved (d) images of the area in the dashed rectangle in a. e, The cross-sectional profile along the dashed line in d. The super-resolved images clearly show the ultra-structure of the microtubules with a resolution (FWHM) down to 49.1 nm. f, The statistical distribution of the photon number of single molecules (data from the cell shown in a (z' = 0 µm)). The average photon number per photoswitching cycle is 2,568 ± 109 (average of 5 different datasets; the error is the s.d.). As a comparison, we imaged the same sample using another home-built STORM microscope. Under near-total internal reflection (TIR) conditions using a 60×/1.40-NA objective, the average photon number is 9,184 ± 287 (average of 5 different datasets, mean ± s.d.), 3.57 times of that collected with eSPIM. This difference was mostly the result of the higher collection efficiency of an oil-immersion objective, and the performance of an oil-immersion objective degrades quickly when imaging away from the coverslip surface because of refractive index mismatch between glass and water. On the other hand, using the same 1.27-NA water-immersion objective under high-inclined illumination conditions (TIR is impossible in this case) did not produce single-molecule data of sufficient quality to be reliably analyzed owing to a high background. Therefore, the oil-immersion objective with near-TIR illumination is better for flat samples near the surface, whereas the eSPIM system is more advantageous for thicker samples away from the coverslip surface. Five experiments were repeated independently, with similar results.