BRIEF COMMUNICATIONS NATURE METHODS | ADVANCE ONLINE PUBLICATION | 1 methods. In special cases, when proteins are confined in cellular regions, single-molecule-based nanoscopy of structures deeper inside biological samples can be achieved 4 . Combining SPIM illu- mination with single-molecule detection is a good alternative 6–12 , but it always requires a complicated and non-versatile two- objective-based experimental setup. Here we show how a single objective can be used to produce SPIM 3D optical sectioning with capabilities ranging from single- molecule detection at the cellular level to whole-embryo imaging. soSPIM relies on the reflection of an excitation beam onto a 45° mirror to create a light sheet perpendicular to the optical axis, with fluorescent light collected through the same objective used to generate the illumination (Fig. 1a,b). We created a multi- wavelength light sheet either by using a cylindrical lens placed in the excitation path or by means of linear scanning along the y-axis of the mirror. By steering the beam along the x-axis of the mirror, we controlled the axial position of the illumination plane along the z-direction. We matched the objective’s focal plane with the illumination plane (Supplementary Fig. 1) and positioned the thinnest part of the light sheet across the sample by defocusing the incoming laser using an electrically driven tunable divergent lens (Supplementary Fig. 2a). We were able to adjust the thickness and length of the light sheet with an exchangeable telescope that tuned the size of the Gaussian beam in the back focal plane of the objective. We achieved sizes ranging from 265 × 500 × 5.2 µm 3 (length × width × thickness) to 13 × 30 × 1.2 µm 3 with objectives ranging from 10×/0.3 numerical aperture (NA) to 100×/1.3 NA working at distances between 10 and 600 µm from the 45° mirror (Supplementary Fig. 2b–d). We used the same tunable lens to synchronously compensate for the translation of the beam waist along the x-axis upon axial displacement of the objective (Supplementary Fig. 3). The light-sheet thickness compared favorably with values obtained for single-cell SPIM 6–12 (Supplementary Table 1) using high-NA objectives. The 60-kHz scanning speed of the galvanometric mirrors and the 500-Hz response time of the tunable lens were fast enough that the acquisition frame rate was limited solely by the sample brightness and the camera’s maximum acquisition speed. In addition to the beam-steering unit, the fabrication of the mirrors is important to our technique. We molded disposable micromirror chips by imprinting a master mold in a UV-curable polymer (NOA 73 or MYpoly134). We adjusted the dimensions of the mirrors according to the size of the samples (Supplementary Fig. 4) and used two different protocols to fabricate cavities for cell (20–200 µm) and embryo (700 nm to 3 mm) imaging (Online 3D high- and super- resolution imaging using single-objective SPIM Remi Galland 1–3 , Gianluca Grenci 3 , Ajay Aravind 3 , Virgile Viasnoff 3–6 , Vincent Studer 1,2,6 & Jean-Baptiste Sibarita 1,2,6 Single-objective selective-plane illumination microscopy (soSPIM) is achieved with micromirrored cavities combined with a laser beam–steering unit installed on a standard inverted microscope. The illumination and detection are done through the same objective. soSPIM can be used with standard sample preparations and features high background rejection and efficient photon collection, allowing for 3D single- molecule-based super-resolution imaging of whole cells or cell aggregates. Using larger mirrors enabled us to broaden the capabilities of our system to image Drosophila embryos. Over the past decade, selective-plane illumination microscopy (SPIM) has demonstrated its superior capacity to image thick, live samples in 3D for a prolonged duration compared to wide-field, confocal or multiphoton-excitation microscopy. This technique has been widely used in developmental biology for 4D reconstruc- tions of live embryos and thick tissues. SPIM is usually achieved through the use of two objectives arranged perpendicular to each other—one for generating a planar illumination, and the other for collecting fluorescence. SPIM’s high sectioning capability, high contrast, reduced photo-bleaching and reduced phototox- icity relative to other approaches are appealing properties for 3D single-molecule-based nanoscopy. Single-molecule-based super- resolution microscopy is usually constrained to 2D observation at the surface of the glass coverslip using total internal reflection fluorescence illumination. By combining oblique illumination 1 and point-spread-function engineering methods 2,3 or biplane imaging 4 , one can obtain 3D images of material within the first micrometer above the coverslip, with an axial resolution less than 50 nm. Interferometric techniques such as interferometric pho- toactivated localization microscopy (PALM) use two objectives in a 4Pi illumination configuration and improve the axial resolu- tion below 10 nm (ref. 5), but the constraints on imaging depth are similar to those associated with the previously mentioned 1 Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France. 2 Centre National de la Recherche Scientifique, Bordeaux, France. 3 Mechanobiology Institute, National University of Singapore, Singapore. 4 Bio Mechanics of Cellular Contacts, Centre National de la Recherche Scientifique, Singapore. 5 Department of Biological Science, National University of Singapore, Singapore. 6 These authors contributed equally to this work. Correspondence should be addressed to V.V. (dbsvvnr@nus.edu.sg). RECEIVED 15 AUGUST 2014; ACCEPTED 12 APRIL 2015; PUBLISHED ONLINE 11 MAY 2015; DOI:10.1038/NMETH.3402