A versatile microfluidics platform for enhanced multi-target super-resolution microscopy

Tsukanov, R.; Basak, S.; Chi-Vu, K.; Mougios, N.; Oleksiievets, N.; Pollack, Y.; Brandenburg, S.; Opazo, F.; Lehnart, S. E.; Enderlein, J.

doi:10.1101/2025.10.07.680900

bioRxiv · 10.1101/2025.10.07.680900

A versatile microfluidics platform for enhanced multi-target super-resolution microscopy

Tsukanov, R.·Basak, S.·Chi-Vu, K.·Mougios, N.·Oleksiievets, N.·Pollack, Y.·Brandenburg, S.·Opazo, F.·Lehnart, S. E.·Enderlein, J.

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Abstract

DNA-based Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) is a powerful variant of single-molecule localization microscopy (SMLM) that overcomes the limitations of photobleaching, offers flexible fluorophore selection, and enables fine control of imaging parameters through tunable on- and off-binding kinetics. Its most distinctive feature is the capacity for multiplexing, which is achieved through a process known as Exchange-PAINT. This technique involves assigning orthogonal DNA strands to different targets within a sample and then sequentially adding and removing complementary imager strands that are specific to only one target at a time. However, manual Exchange-PAINT workflows are often inefficient, prone to drift and variability, and lack reproducibility. Here, we introduce a custom compressed-air-driven microfluidics system specifically designed for multiplexed SMLM. Featuring a stackable and modular design that is, in principle, not limited by the number of channels, the system ensures robust, reproducible, and material-efficient buffer exchange with minimal dead volume. It operates in both manual and automated modes and can be readily adapted to a wide range of commercial and custom microscopes, including wide-field, confocal, STED, and MINFLUX platforms. We demonstrate robust 5-plex Exchange-PAINT imaging in cancerous U2OS cells and, importantly, we establish multiplexed nanoscale imaging in fragile primary cardiomyocytes. These applications highlight the unique power of our platform to extend super-resolution multiplexing into physiologically relevant systems, thereby opening new avenues for biomedical research.

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Tsukanov, R., Basak, S., Chi-Vu, K., Mougios, N., Oleksiievets, N., Pollack, Y., Brandenburg, S., Opazo, F., Lehnart, S. E., Enderlein, J.. 2025-10-07. A versatile microfluidics platform for enhanced multi-target super-resolution microscopy. https://doi.org/10.1101/2025.10.07.680900

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