Build and operation of a custom 3D, multicolor, single-molecule localization microscope

Single-molecule localization microscopy (SMLM) enables imaging scientists to visualize biological structures with unprecedented resolution. Particularly powerful implementations of SMLM are capable of three-dimensional, multicolor and high-throughput imaging and can yield key biological insights. However, widespread access to these technologies is limited, primarily by the cost of commercial options and complexity of de novo development of custom systems. Here we provide a comprehensive guide for interested researchers who wish to establish a high-end, custom-built SMLM setup in their laboratories. We detail the initial configuration and subsequent assembly of the SMLM, including the instructions for the alignment of all the optical pathways, the software and hardware integration, and the operation of the instrument. We describe the validation steps, including the preparation and imaging of test and biological samples with structures of well-defined geometries, and assist the user in troubleshooting and benchmarking the system’s 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. Depending on the instrument configuration, the cost of the components is in the range US$95,000–180,000, similar to other open-source advanced SMLMs, and substantially lower than the cost of a commercial instrument. A builder with some experience of optical systems is expected to require 4–8 months from the start of the system construction to attain high-quality three-dimensional and multicolor biological images. The buildup and operation of a custom single-molecule localization microscope with state-of-the-art performance and advanced features bridges the gap between entry-level open-source projects and costly commercial systems. Key points The authors describe the complete configuration and assembly of a custom-built single-molecule localization microscope, including optical alignment, software and hardware integration, validation steps and benchmarking of the system’s performance. The microscope is optimized for ultra-stable three-dimensional imaging performance and multichannel functionalities, while remaining substantially less expensive than similar commercial systems.