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. 2017 May 8;7:1542. doi: 10.1038/s41598-017-01606-6

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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The schematic diagram of the low-cost SMLM setup. ND filter is a step variable metallic neutral density filters (NDL-10S-2, Thorlabs). Lens 1 and Lens 2 are achromatic lenses with a focal length of 100 mm (AC254-100-A, Thorlabs). Rotating diffuser is an engineered diffuser (EDC-5-06217-A, RPC Photonics) driven by a small DC motor (RB-Ada-142, RobotShop) with a rotation speed of 9100 rpm. Lens 3 is an achromatic lens with focal length of 200 mm (AC254-200-A, Thorlabs). The objective lens is a 100x oil immersion infinity-corrected Plan Fluor objective lens with a NA of 1.3. Dichroic mirror (FF660-Di02, Semrock) and emission filter (ET700/75 m, Chroma) are designed for the commonly used fluorophore, Alexa-647. The tube lens is an achromatic lens with focal length of 100 mm (AC254-100-A, Thorlabs), corresponding to an optical magnification of 60X. The camera (Chameleon3 CM3-U3-31S4M, PointGrey) is an industry CMOS camera without cooling, the sensor model is IMX265 with a pixel size of 3.45 µm, QE of 71% and read noise of 2.89 electrons. In this setup, 2 × 2 physical pixels are binned as one image pixels, corresponding to 115 nm per pixel in the sample plane.