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Good day, microscopists. I wonder if you can help me quantify how 
smoothly my stage is moving?

I've built a 3-axis stage around my 2-photon using breadboards and 
translation stages (e.g. Thor PT1). I am actuating the stages with 
micrometers. All three axes will be motorised by coupling the 
micrometers to stepper motors. At the moment I've only motorised Z, to 
check that everything works roughly as planned. My immediate concern is 
quantifying how smooth the motions are in all three axes. I've not done 
anything like this before, so I wanted to bounce ideas off you more 
experienced people.

The steppers won't have encoders, since I can't be certain these would 
reliably convey what's happening at the stage. Quantifying smoothness in 
X/Y should be simple: I'm thinking of translating the X/Y stage at a 
constant speed whilst imaging a target (e.g. pollen grains). Since I 
know how many microns per pixel there are, I can extract velocity and 
hence jerk in real units. I'd base measurements on the whole image, so 
imaging-related noise should have a negligible impact.

I'm less sure what to do about z motion, however, since targets will 
enter and leave the image plane. Perhaps fluorescent nano-beads are the 
way to go? Using these I could measure PSFs in Z. If I move at a 
constant speed in Z, intensity as a function of time should produce a 
Gaussian. Maybe I can use the known PSF of the objective to figure out 
velocity in real units? One problem might be that, since the target is 
small, imaging noise could contaminate the velocity measurements. Anyone 
have suggestions on this procedure or ideas for better targets?

Thanks!


-- 
Rob Campbell
turnerlab.cshl.edu