Abstract
DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20–25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time.
Highlights
Super-resolution light microscopy is developing rapidly, and a growing number of cell biologists are embracing this technology to study proteins of interest (POI) at the nanoscale
We optimized the transfection of mammalian cells (COS-7) with plasmids encoding for to EGFP
Golgi apparatus proteins present in different organelles fused to various fluorescent proteins
Summary
Super-resolution light microscopy is developing rapidly, and a growing number of cell biologists are embracing this technology to study proteins of interest (POI) at the nanoscale. Single molecule localization techniques like PALM [1], (d) STORM [2,3], and others [4] achieve resolutions that allows for distinguishing molecules that are separated by only a few nanometers. Among these localization techniques, DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) [5] has demonstrated to achieve a resolution below 5 nm on DNA origami structures [6,7] and offers the. In a DNA-PAINT experiment, the imager strands continuously bind to the docking strands and unbinds due to thermal fluctuations. The continuous transient binding of the imager strands results in sparse “blinking-like” fluorescence detection events
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