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Hi Niko,

This might seem like an obvious question, but have you checked that your laser power isn't oscillating over the same period you measured? A power meter measurement at the back of the objective should be able to rule this out first. Do other laser wavelengths on your system produce the same effect?

Have you ruled out that this is not the "scan line" artifact sometimes observed with spinning disk systems? See this paper for an explanation of that:

Chong, F.K., et al., Optimization of spinning disk confocal microscopy: Synchronization with the ultra-sensitive emccd, Cogswell, and T. Wilson, Editors. 2004, Spie-Int Soc Optical Engineering: Bellingham. p. 65-76.

It's possible that if you exposure time is not an integer number of disk sector turns, then you might get some kind of intensity beat frequency with you acquisition period of every 6 seconds, but the intensity change you're seeing here seems a bit too big for that. What is your exposure time of the images captured every 6 seconds?

Beyond that, there might be something off with the spinning of the pinhole disk (or pinhole and lenslet disk if this is a CSU) which is a little more difficult to determine. Can you hear the disk(s) spinning? Does it sound like it's turning at a constant speed? Do you know what speed it is spinning at?

Maybe there's a few ideas to troubleshoot there. Let us all know if you solve the problem.


John Oreopoulos
Staff Scientist
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca



On 2013-08-16, at 11:37 AM, Niko Ehrenfeuchter wrote:

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> *****
> 
> Dear all,
> 
> we're recently facing a very strange behavior on one of our spinning disk systems. One of our users noticed a repeated fluctuation in recorded timelapse experiments (this was a FRAP experiment, but we don't think that's related to FRAP).
> 
> The timelapse was recorded every 6 seconds, and after background subtraction a repeating drop of signal is visible in the intensity plot (please find the corresponding graph under [1], see below). The fluctuation is also visible by eye when scrolling through the timelapse.
> 
> We noticed a frequency of 66 seconds and therefore think we can exclude any external vibrations as source of this problem (plus the system is mounted on a Melles Griot optical table). On some experiments the oscillation almost follows a sine-like function, on others (like the mentioned one) it looks more rectangular with a very rapid drop and raise. Also, the frequency does *not* change if we double the acquisition frequency, it's still on 66 seconds when acquiring every 3 seconds.
> 
> One very weird thing is the stability of the background. The plots from above show the intensity in selected ROI's over cells. The same plot for an "empty" region in the sample shows a more or less constant signal (at least we could figure a pattern in it, even after normalization and using a big scaling factor to visualize the deltas).
> 
> Has anyone of you come across a similar behavior?
> 
> Any ideas about this would be highly appreciated!
> 
> Many Thanks,
> Niko
> 
> [1] https://www.biozentrum.unibas.ch/fileadmin/redaktion/Forschung/Research_Groups/IMCF/3i_intensity_fluctuation.png
> 
> -- 
> Niko Ehrenfeuchter
> Imaging Core Facility
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> Biozentrum, University of Basel
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> 
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