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Howdy all,
We have a spinning disc confocal, Yokogawa CSU-10, mated to a TiE base from Nikon, a three diode laser launch and two EMCCD 512 Cascade:II cameras on a DC2 image splitter. A second imaging path is present for widefield imaging as well. The entire machine resides on an table top isolation table. This machine is heavily used for live cell imaging and remains on for up to 120 hrs at a time and with time permitting multiple experiments in a week. The CSU-10 has been operational since circa 2002 first as an Ultraview LCI and now as a custom assembly. It has been very stable since it has come under my care (3 yrs), until very recently. Recently, the imaging intensities have fluctuated wildly; experiments which previously could be imaged with 16 bits of dynamic range have been cut to 14 bits. Importantly, this phenomena is observed across all wavelengths and the samples appear within normal range as confirmed by the widefield imaging path. Unfortunately, the phenomena isn't consistent, as the following day, imaging intensities have returned to normal (as verified by bead intensity calibration; molecular probes/invitrogen). The laser intensity has not appeared to vary (We don't have a power meter to measure precisely), and either EM:CCD, in either position on the DC2, capture similarly low intensities when the phenomena is present. The base, Ti-E, and filter wheels, are functioning as expected with analyzers staying out of the imaging path and proper imaging ports and filters being used. The only component left in the puzzle is the CSU-10. I've checked the dichroic for dirt and tightness, although the intermittent nature would suggest a non-static component. The only possibility remaining, is an issue with the disc itself. My question is whether this phenomena, intermittent lose of imaging intensity, has been observed with the CSU-10 or related spinning disc confocal heads (20, 22, 1x) by others or if any other insight can be offered into this frustrating problem. I'm leaning towards the disc because the the age of the CSU-10, the heavy use it has been under, and the elimination of other explanations. Thanks in advance for any insight or suggestions!
Cheers,
Seth
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