Abstract
Imaging the dynamics and interactions of biomolecules at the single-molecule level in live cells and reconstituted systems has generated unprecedented knowledge about the biomolecular processes underlying many cellular functions. To achieve the speed and sensitivity needed to detect and follow individual molecules, these experiments typically require custom-built microscopes or custom modifications of commercial systems. The costs of such single-molecule microscopes, their technical complexity and the lack of open-source documentation on how to build custom setups therefore limit the accessibility of single-molecule imaging techniques. To advance the adaptation of dynamic single-molecule imaging by a wider community, we present the “K2”: an open-source, simultaneous triple-color total internal reflection fluorescence (TIRF) microscope specifically designed for live-cell and single-molecule imaging. We explain our design considerations and provide step-by-step building instructions, parts list and full CAD models. The flexible design of this TIRF microscope allows users to customize it to their scientific and financial needs, or to re-use parts of our design to improve the capabilities of their existing setups without necessarily having to build a full copy of the K2 microscope.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Data availability: Design files and data are available at 10.5281/zenodo.7380380. A ready-to-view model is available at https://autode.sk/3vdsOu0. Data analysis code is available at github.com/GanzingerLab. Up-to-date information about the setup is available at the K2 TIRF Github page.
Funding: This publication is part of project number VI.Vidi.203.037 of the Talent Programme which is financed by the Dutch Research Council NWO (K.A. Ganzinger). C. Niederauer and K.A. Ganzinger acknowledge support by the WISE program of NWO.
Competing interests: The author declare no competing interests.
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