Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a tilted light sheet illumination scheme and engineered point spread functions with axial ranges up to ten µm for lowbackground, 3D super-localization of single molecules. This system is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells.Several methods have been developed to extend the imaging capability of single-molecule super-resolution (SR) microscopy 1-3 to three dimensions (3D), including multiplane imaging, interferometric approaches, and point spread function (PSF) engineering (for a review, see 4 ). In the case of PSF engineering, the axial position of a molecule is encoded in the shape of the PSF, and strategies include astigmatism 5 and the bisected pupil PSF 6 with axial ranges of 1-2 µm, self-bending 7 and double-helix (DH) PSFs 8-11 with axial ranges of ~2-3 µm, and the recently developed Tetrapod PSFs 12, 13 which have a tunable range of up to 20 µm. PSF engineering typically only requires the addition of a small number of optical elements to a standard microscope, making the method relatively simple to implement while exhibiting high precision for 3D single-molecule localization.One method to improve the precision of single-molecule localization is to reduce the background coming from out-of-focus fluorophores by using light sheet illumination 14 , where the sample is excited by a thin sheet of light orthogonal to the detection axis. Numerous light sheet designs have been implemented for SR imaging [15][16][17][18][19][20] , but these designs have certain drawbacks such as being incompatible with imaging of fluorophores close to the coverslip using high numerical aperture (NA) imaging objectives 17, 18 or require complicated optical and electronic designs or many custom-made parts which are often expensive and difficult to build and operate, and thus may not be easily accessible to the general research community.Here, we present TILT3D, an imaging platform that combines a novel, tilted light sheet illumination strategy with long axial range PSFs. We alleviate many of the aforementioned difficulties in existing light sheet designs by tilting the illumination plane; for example, two perpendicular objectives in close proximity are not required. TILT3D: (a) yields high localization precision of single molecules in 3D over the entire axial range of a mammalian cell via scanning of the light sheet combined with imaging with engineered PSFs, (b) has the usual light sheet advantages of reduced photobleaching and photodamage of the sample, but most importantly (c) is easy and cost-efficient to implement and operate. We validated TILT3D for 3D SR imaging in mammalian cells by imaging microtubules, mitochondria, and the full nuclear lamina using both DH and Tetrapod PSFs.. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aTh...