I’m writing here because I know there are good connoisseurs of the Nikon systems around here and hope to get some pointers from you!
We are encountering an issue with Z-drift during long-term (>24 hours) time-lapse imaging across multiple positions using a 100X, 1.49 oil objective on our Nikon N-SIM system. For focus stabilization, we rely on the Perfect Focus System (PFS), which has performed reliably for shorter experiments and real-time sample exploration.
However, during extended multi-position acquisitions using the ND acquisition panel in NIS-Elements, we observe a gradual Z-shift of up to a couple of microns. Interestingly, the PFS does not indicate any error and appears to be functioning normally. This issue seems specific to multi-position imaging, as long time-lapses at a single position do not exhibit the same drift. We use an ample amount of oil on an 18 mm coverslip, making oil-related problems less likely.
We are seeking your expertise to understand the potential cause of this Z-drift and identify possible remedies. Could this be related to specific acquisition parameter settings, or is there a particular consideration or technique we might be overlooking for multi-position long-term imaging with the N-SIM and PFS?
HI Hazen, we’re cycling through the positions. There is no fast movement needed as we are imaging about 10 positions every 10 minutes. Actually I’d be happy with 1 position every minute (could help with avoiding oil inertia and such) but NIS Elements by default will image all the positions as fast as possible every 10 minutes.
Is your system incubated? If so, have you warmed things up in advanced, including the oil, to get them equilibrated; thermal drift is often the culprit? Troubleshoot with a couple of close points and see if you encounter the same issue. You might think there is sufficient oil but often that can be an issue when traveling across your slide or dish. Also, if your sample isn’t positioned properly, oil can spread unevenly. Depending on the nature of your sample, you could see if that is an issue when looking at the PFS offset for the different positions, does it differ by a great deal. The other thing you can try is to recreate the experiment with a dry objective. You might not see a change because of the lower power mag, but it is worth a try.
We have been instructed that only the Nikon Type F oil is officially PFS compatible. The caveat is that Nikon only guarantees PFS with oil immersion for several hours only and not overnight/weekend imaging sessions. In our experience the Type F oil hardens out and we presume this is why Nikon does not guarantee PFS stability on longer imaging sessions. We sporadically remove build-up of yellow hardened Nikon Type F oil with methanol in a fume hood using lens paper and toothpicks. Pitty the Nikon Type F2 oil was discontinued, non-hardening and less odour.
Two alternatives you could try: Ibidi immersion oil does not solidify in our hands but the Abbe number (dispersion) is not fully matching Nikon optics (40.8) and we see axial colour shifts on beads on confocal. I see there is an Ibidi immersion oil 2 oil now with improved abbe number (43.8), but I have not tested for chromatic aberration yet.
Second option: In other labs I see Olympus/Evident Type F oil being used on Nikon systems. The abbe number of this oil is I think identical to Nikon and this oil can be used at 37 degrees.
Please also check the blog.everydayscientist post from years back.