Automated 3D multi-color single-molecule localization microscopy

Since its inception, single molecule localization microscopy (SMLM) has enabled imaging scientists to visualize biological structures with unprecedented resolution. Particularly powerful implementations capable of 3D, multi-color and high-throughput imaging have yielded key biological insights although widespread access to such technologies has been limited. The purpose of this protocol is to provide a guide for interested researchers to establish high-end SMLM in their laboratories. We detail the initial configuration and subsequent assembly of the SMLM, including instructions for alignment of all optical pathways, software/hardware integration and operation of the instrument. We describe validation steps including the preparation and imaging of test- and biological samples with structures of well-defined geometry and assist the user in troubleshooting and benchmarking performance. Additionally, we provide a walkthrough of the reconstruction of a super-resolved dataset from acquired raw images using the Super-resolution Microscopy Analysis Platform (SMAP). Depending on the instrument configuration, the cost of components is in the range $80,000 - 160,000, a fraction of the cost of a commercial instrument. A builder with some experience of optical systems is expected to require 3 - 6 months from the start of system construction to attain high-quality 3D and multi-color biological images.