I am really interested in the technology, but I don't want to (read: can't) make the investment to build a new rig, or buy a water-immersion objective and modify the optical path of my current TIRF rig, which uses a standard TIRF oil immersion objective and illumination through the objective. I have some flow chambers that use 25mm or 40mm regular coverslips, so I was wondering if anyone has adapted a "standard" TIRF system to use this technology.
That should be fine, if the intensity variation in objective type TIRF is compensated by counting background signal on the individual fluorophore. I have used objective type TIRF (or standard TIRF) and prism type one for single molecule fluorescence FRET, and both work good and the quality of data is comparable. Because FRET is more sensitive to the intensity variation, simple fluorophore counting by SiMPull will be no issues in that sense.
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