The LEGO® brick road to open science and biotechnology

We have developed a high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be determined with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. We demonstrated an imaging resolution of 20 nm. This technique can, in principle, reach molecular-scale resolution.

μManager is an open-source, cross-platform desktop application, to control a wide variety of motorized microscopes, scientific cameras, stages, illuminators, and other microscope accessories. Since its inception in 2005, μManager has grown to support a wide range of microscopy hardware and is now used by thousands of researchers around the world. The application provides a mature graphical user interface and offers open programming interfaces to facilitate plugins and scripts. Here, we present a guide to using some of the recently added advanced μManager features, including hardware synchronization, simultaneous use of multiple cameras, projection of patterned light onto a specimen, live slide mapping, imaging with multi-well plates, particle localization and tracking, and high-speed imaging.

While super-resolution fluorescence microscopy is a powerful tool for biological research, obtaining multiplexed images for a large number of distinct target species remains challenging. Here we use the transient binding of short fluorescently labeled oligonucleotides (DNA-PAINT, point accumulation for imaging in nanoscale topography) for simple and easy-to-implement multiplexed 3D super-resolution imaging inside fixed cells and achieve sub-10 nm spatial resolution in vitro using synthetic DNA structures. We also report a novel approach for multiplexing (Exchange-PAINT) that allows sequential imaging of multiple targets using only a single dye and a single laser source. We experimentally demonstrate ten-“color” super-resolution imaging in vitro on synthetic DNA structures and four-“color” imaging of proteins in a fixed cell.