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Open source microscope plans for a compact laser TIRF setup with automated x/y/z stage, adjustable TIRF angle, and 3d localization by astigmatism.


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    It seems highly promising. Tracking maps are really interesting. Can we follow one track like a video? It would be great to follow some proteins in signaling pathways upon induction.

    Another question: Is it possible to track 2 proteins simultaneously by fusing with different fluorophores? It would be nice to reveal some protein-protein interactions via this technique.

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      If the labels are sufficiently separated spectrally you can image 2 species quasi simultaneously by synchronizing laser outputs to camera frames. Switching at >1 kHz is possible; whether that’s fast enough depends on how fast things are moving and what resolution you need to answer your question. You would need multipass dichroic and emission filters.

      To do this in the same frame there’s a bunch of possible tricks that all would require modifying the microscope to some extent, but can be very compact as shown by the ONI nanoimager.

      If by “track like a video” you mean moving the microscope stage to follow an object that moves beyond the field of view, the hardware here is sufficient to do that but you’d need to add some software and it would only work for things moving fairly slowly that are do not photobleach quickly.