NetNotes

Hello microscopists, one quite experienced user of our Zeiss 780 confocal noticed that, unexpectedly, the thickness of her samples (determined by z-stack) nearly doubled from 11 to 21–25 microns. This was observed while using a 20×/0.8 air objective imaging several colors. I checked and the thickness is the same with a 63×/1.4 oil. All signals are good and the confocal seems to be functioning normally. The user’s main concern is that the z-stack acquisition time doubled. Any insights or suggestions on how to fix the issue is highly appreciated. Arvydas Matiukas [email protected] I think you have a fish tank effect: https://svi.nl/FishtankEffect. Petro Khoroshyy [email protected] Are the samples (for instance) cultured cells that would be expected to have constant thicknesses from sample to sample? Or are they physical sections of brain tissue? I know that Bright cryostat microtomes have two sets of advance screws: one set that allows sections to be cut up to 30 microns thick, and a second that allows sections to be cut up to 60 microns thick. There is no way of knowing which was installed without someone telling you. This could result in physical sections half as thick (or twice as thick, as in this case) as you intended. Martin Wessendorf [email protected] That is a good possibility, however, the thickness observed is the same regardless of air or oil lenses, as stated in the post. I am not clear from the post if the issue is between samples or between imaging sessions with the same samples. With the information given, it is not clear if it is a sample or a microscope issue. Leoncio Bergara [email protected] Dear Dr. Wessendorf, we reimaged the same samples as before (slices of mouse retina), so the sectioning thickness did not change. I was thinking about possible refractive index mismatch effects as referred to by Petro (fish tank effect), however it is still not clear what caused the thickness change. I am going to clean the objectives and try to calibrate the z-dimension using objects of known thickness (for example, beads, coverslip, slide). I will report my findings. Arvydas Matiukas [email protected] In Zen software, there is a refractive index correction factor somewhere in the Z stack settings (try show all button to show all settings). Maybe it is set to the wrong value. Or someone messed up something in the expert mode settings (just a guess). Petro Khoroshyy [email protected] In theory, a dry objective should underestimate sample thickness by a factor of 1.5 relative to an oil-immersion objective. In this case, the factor is more like 2. Could it be that the oil-immersion objective pushed down on the sample? You can check this by drawing lines with a marker on two opposite sides of a coverslip and check the apparent distance ­between them with both objectives. Mike Model [email protected] If your microscope has a problem in the Z-dimension, it could register a different thickness depending on the direction of travel. Check to see if it takes smaller steps moving upwards and larger steps moving downwards. Tim Feinstein [email protected] Great idea. You could also detect the edge of the coverslip by its intensity. I like to use “Range Indicator” for this purpose. Caveat: a #1.5 coverslip can vary a lot in thickness, around 160 to 190µm. Zeiss cover glasses (or a #1.5H) will vary less but will still vary. Anyway, they will be around 170mm and not double that. Brian Armstrong [email protected] We are implementing the recently published vitreous ionic-liquid-­solvent-based volumetric inspection of trans-scale biostructure (VIVIT) ­approach (https://pubmed.ncbi.nlm.nih.gov/40795853/) for some of our light sheet studies. It certainly renders samples very clear. However, the RI of the imaging solution needs to be quite high, around 1.543. So, I am looking for potential liquids to use. We are trying various essential oils (wintergreen, clove, and anise) but they all have very strong odors, which when concentrated are unpleasant. Benzaldehyde is another option but it’s a solvent for lots of things. We can get RI-matched solutions from Cargille but they are quite expensive. So, does anyone have experience with the long-term stability of these solutions before we pull the trigger? I expect they will discolor over time in direct light (the spec sheets say so) but how quickly does this happen and how are they to work with? Simon C. Watkins [email protected] We are using a 2-component oil mix: phenylmethylsiloxane oligomer: pdm ri:1.556 (Product code: PDM-7040) and Thermo Scientific mineral oil:1.467 (product code: 15484929), with great results. ­Mohammad Goudarzi [email protected] I have used a mixture of ShinEtsu HIVAC-F4 silicone oil and mineral oil from Thermo Fisher to image organs cleared with the CUBIC L/R protocol. The HIVAC F4 oil has a RI of approximately 1.55 and can be mixed with mineral oil to reach the target RI. I filled the chamber of our light sheet system with the mixture and did not notice yellowing over the 2 years of my postdoc. It has no strong odor and is very hydrophobic so it can be recovered and reused after imaging samples cleared with water-based protocols. It may work for your application since it is not soluble in many polar organic solvents. We sourced a few liters from Inland Vacuum for a reasonable price; they are US-based but ship in the EU as well. Donato Sardella [email protected] We have encountered an issue with Z-drift during long-term (>24 hours) time-lapse imaging across multiple positions using a 100×, 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 18mm 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? Christophe Leterrier [email protected] The cause could be the distance between the stage positions. If the distance is too great between positions, the oil (between the objective and the sample) may not have the time to fully follow and that may cause the drift. I suggest, with oil objectives, using small distances between the stage positions so the oil will follow more easily. Louis Villeneuve [email protected] This might be a completely different issue, but what we sometimes experience with our Nikon spinning disk is that the oil is so viscous that it drags the glass/coverslip a little bit when moving the stage, especially in the z-direction when using the fast piezo stage. We therefore set the PFS focus position at the bottom of the stack (not in the center), which gives a bit more time for the coverslip to relax to the PFS a focus in the of the We imaging at a distance focus is a very stage to the oil between positions. Maybe you set it up like it might be a [email protected] I second what time the oil can more viscous and be the of the [email protected] We have these in the and have them using a of ND We also the bottom of the with oil to with the stage to and a after the stage before it an The way we set the is to the to the the and We also set up a to do an or the PFS up and down if the PFS I am to any we have and We also that we the for in the microscope before [email protected] In my experience I think that of imaging with oil will have it’s quite viscous and it will dry a not I have that is of time for it to on our system when some clean the system at the we our system. you using silicone which I think might not in quite the same We have also some imaging using our objective. 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