NetNotes


planapochromat lenses. Tis includes newer microscopes with wider fields of view and a spinning disk confocal with a Cairn twin camera setup. (Te cameras, however, required substantial alignment to attain this). We use standard and unusual stage inserts and a variety of samples and are able to focus across the entire field. Tis includes imaging in reflection mode, TIRF, Nomarski, epifluorescence, and fluorescent confocal. We have a new microscope, however, which does not have focus uniformity. I believe it is a dual camera alignment issue and we have been discussing this for almost a year as an installation issue, but recently the manufacturer has insisted that the only way to fix the problem is to use screws on the stage to adjust the tilt. I am concerned that this is going to be a major problem in a core facility with users who will play with anything. It is going to add substantial burden on staff. Also, it means that standard inserts will not work. And it makes me wonder what this will do to the PSF if the coverglass isn’t perpendicular to the light path. Have we been unusually lucky all these years to never have encountered this problem before? Are we unreasonable to expect the microscope to be installed with all planes parallel for routine use? Is this type of stage alignment standard acceptable procedure in other labs? Tank you. Michael Cammer michael.cammer@med.nyu.edu


As a former ZEISS affiliate service tech, back when I was young,


and this still applies today, we used a stage square to make sure that the flat surface of the nosepiece and the surface of the stage were parallel with each other. Te device was made on a lathe. Te base was about 24 mm diameter and screwed into the nosepiece and extended 45 mm to a flat disk about 75 mm round. I would carefully lower the 75 mm disk to the stage surface to make sure it touched evenly. Tis can be used on uprights or inverts. Tis enabled the user to check for parallelism issues caused by either the stage mount or the detent in the nosepiece. If the detent is worn it may cause a rotational misalignment which causes the focus to be nonuniform across the field. It is also best to have a crosshair objective to check alignment of the optical axis of the scope. Check with your scope manufacturer to see if they can or will provide these reference parts. Dan Focht dan@bioptechs.com


We have a dozen confocals and none of them have any appreciable


focus shiſt across a single field of view. In fact, on the inverted microscopes with a thin sample, if a user has a paper label close to one edge of the slide and no label on the other, this should cause an obvious tilt as the slide sits higher on one side than the other, but from a single field of view it isn’t very obvious unless you’re looking for it. Te effect of the label becomes obvious if you move the stage as the sample quickly goes out of focus as you move side-to-side. If there is a substantial tilt across the field of view, I would agree that the manufacturer should address this, and not with stage inserts: we usually remove the stage insert tilt screws as they tend to jiggle their way down with gravity and end up misaligning the stage tilt more than anything else. Maybe you want to create a shared cloud folder (Dropbox, OneDrive) and drop an image or 2 in there so that we can see how bad it is? If it would help, I would be willing to take some images across several of our confocals and upload them to the same folder. I’m thinking a short z-stack of a thin sample (Molecular Probes Prepared Slide #1, BPAE cells for example) would illustrate the problem nicely. And maybe a second test would be to focus the same slide in the middle of the field of view, then take a tiled image (say, 5 x 5). Choose an objective and we can try to match it. Anyone else interested? James Jonkman james.jonkman@uhnresearch.ca


As a follow-up to our previous conversation: Tis microscope (not


a confocal) comes standard with a unique levelling stage insert. It has X and Y adjustment screws in opposite corners and a pivot point in the other


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corner. Tis stage insert can correct for any tilt throughout the system, al- though it can take a long time to get it right. Since you mentioned TIRF, I assume you’re not using that insert, but a live-cell heated stage insert. I do not like the standard heated stage that ships with that system. In our facility it eats objectives. Terefore, we ordered the PeCon push and click system for live cell holders (and rely on the plastic incubation box plus time to maintain temperature). We had to sign a waiver acknowledging that this insert was not approved for use with the system and that we may experi- ence unwanted thermal driſt and uncorrectable tilt. It turns out we didn’t see any of this and now exclusively use the push and click system for live and fixed samples as it can also hold slides. Te adjustable stage now col- lects dust in a box and our images look great. No need for adjusting stage screws. To be fair, we don’t do TIRF, but we image 200 μm beads at every SIM session, and these are always in focus across the field. It was very diffi- cult to achieve similar uniformity with the adjustable stage without a good 30–45 mins of adjustments. Doug Richardson ds.richardson@gmail.com


For our ZEISS confocal with Airyscan we have a leveling stage


insert that came with a protocol for using it. If you want to spend a few minutes you can get a small field of view very flat. Some stage inserts are fairly uniform across vendors so perhaps this could be adapted to your system. Brian Armstrong barmstrong@coh.org


Does your microscope have eyepieces? If so, checking for a flat


field with the eyepieces should solve the issue whether the tilt comes from the camera alignment or from the stage. Beads on a glass surface or any other thin sample should be a good sample for this. Also, how does the tilt behave with different objectives, say 40x and 100x? You should get a different outcome depending on whether the problems arise from the sample/stage or behind the intermediate image. Steffen Dietzel lists@dietzellab.de


Or (seconding to Steffen), does the microscope stand have another


pot where you could connect a camera directly? As already mentioned, if your sample does not go too much out of focus when you move the stage a long distance (1 cm) then you can’t fix your issue with stage insert leveling screws. Te issue then must be somewhere between the objective lens and the camera (filters in the main turret are not suspect, as these are in transmission and in infinity space; thanks Dan for pointing out the nosepiece). Alternatively, the whole XY stage is not bolted correctly to the microscope frame. Zdenek Svindrych zdedenn@gmail.com


Beam Pulsing Issues Microscopy Listserver On our Philips CM12 TEM, the beam is pulsing in SA mode, the


technician has changed the filament and checked the HT voltage. Tis helped but did not eliminate the pulsing. We do not know why this issue is occurring and the technician has not been able to fix it properly. Has anyone encountered this problem and if so, what do we need to check or replace? Tank you in advance. Sara Rizzo sararizzo125@gmail.com


You do not mention if it’s a very regular (an electrical issue) or


irregular pulsing (charging, including vacuum leakage). Only in the SA mode? Tat sounds like a charging issue. Clean the SA aperture holder and apertures. It could also be a bad relay for switching to diffraction. Tis would focus/defocus which would result in beam pulsing. Richard Edelmann edelmare@miamioh.edu


Tere are several possibilities. Abrupt changes in visual screen


brightness might be caused by charging, arcing, too high emission current (not necessarily high setting), electronics issue, etc. Vitaly Feingold vitaly@sia-cam.com


www.microscopy-today.com • 2021 September


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