Manual stage and focus drift

Hardware
1

Dear all
I am going to build an epi fluorescence (TIRF) microscope for single molecule super res imaging. I prefer to use manual stage, but I am worried about focus drift. Can anyone let me know the best thing that I should consider to properly decrease that (except thermal stability) ? Can a manual stage really work for such a microscope? What is the best manual stage and slide holder?

Thanks

2

I’m assuming that you are asking about a manual z stage. In this case a manual stage should work fine, at least for 2D super-resolution imaging. You will probably have to baby-sit it however to keep it in focus, especially if you are taking really long SMLM movies. Also even with a manual z stage the feedback from a focus lock can be useful as they are typically more sensitive than your eye at evaluating if the focus has drifted and in which direction. The focus mechanisms that come standard on an inverted microscope from Nikon, Olympus, etc. are pretty stable.

2 Likes
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Thanks for your helpful reply.

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The DAISY paper claims

“… most commercially available locking systems typically stabilize the focus position at +/- 30nm at best”

and references Supplementary Fig 4 for axial drift tracking over the span of six hours for Nikon’s Perfect Focus System.

I’ve had a tough time finding quantitative data for other systems.

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Is this a question about the stability of commercial focus locks? I’ve done 2D SMLM without a focus lock so I’m quite confident this is possible, if not that convenient.

I don’t think that the statement about Nikon PFS is unreasonable, but in my experience this is not the easiest thing to measure. I think it depends a lot on variables that are not easy to control from experiment to experiment let alone between labs, such as room temperature fluctuations. I’ve only done 10-20 minute measurements myself, and sometime the drift will be less than 5 nm, and other times it will be greater than 10nm. However the drift is also pretty slow, so it can usually be corrected for by using the same approaches that you’d use for XY drift correction.