Hi everyone,
A colleague and I are brainstorming ways to improve his acquisition of a 4-color fixed cell image using AlexaFluor405 (a membrane protein label, not DAPI), AlexaFluor488, AlexaFluor546, and AlexaFluor647 labeling. We were previously using a Zeiss LSM980 to successfully collect images, but he has now transitioned to a Nikon A1 for further collection. On the Zeiss LSM980, we had set up the acquisition in Zen Blue to have each fluorescent channel as its own track with its corresponding 1 A.U. pinhole size. On the Nikon A1, we are not as familiar with the Nikon Elements software for laser-scanner confocals and thus not sure how to set up analogous settings for different pinhole sizes per fluorescent channel. The Nikon A1ās SOP for this specific microscope has 1 pinhole size for all fluorescent channels.
A1ās SOP:
Question: Is it possible to set up a 4 color acquisition, where each fluorescent channel has its own 1 A.U. pinhole size, on the Nikon A1 in Nikon Elements? If yes, how?
Thanks so much in advance!
Nikon has only one pinhole for all the channels.
Maybe you can try the lambda tab in the ND acquisition pannel and set up every channel independently with 1 AU. It will take longer for sure (if itās possible than Iām not sure as nobody requested that when I worked with Nikon microscopes).
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Hi Eva,
The 980 also has only one pinhole for all detectors just like the A1. Even though the software allows you to change this aperture size between tracks, the question is - do you really need to do this?
The short answer is probably not. The axial resolution limit of your 20x/0.8 objective is ~1.5 microns. The calculated difference in optical section thickness when setting the pinhole to 1AU for AF647 vs. AF 488 is roughly 200nm. This difference is well below the theoretical resolution limit (even if you are sampling at Nyquist) - so I doubt you can get a measurable difference between running acquisitions by setting the pinhole to 1AU for just the longest wavelength track vs. having each track separately set to 1AU.
The side effect of doing this on the 980 (likely the A1 too) is that your track switching has to be done frame by frame, rather than line-by-line. There are programmed delays (~750ms between tracks for Zeiss) set to allow for hardware changes in the frame mode that dramatically increase your acquisition time compared to the line mode.
All in all - it is more useful to set the pinhole diameter based on the longest wavelength track and have it remain the same diameter for all the remaining, lower wavelength tracks.
A little history here:
Zeiss is funny about this - because depending on which vendor rep taught you how to use the 980 - you may have been trained to set the pinholes for each track to match optical section thickness. The emphasis on this method is a carryover from the 5-series (LSM 510) where on this system each detector had its own pinhole - so you HAD to adjust each independently. This was more due to the fact that the optical design of the 510 had such a long light path for Ch1 compared to Ch2, Ch3 that they could not use a single pinhole. Subsequently this multiple pinhole concept was sold as a āfeatureā and folks were trained to use them this way. The 510 was so popular that this technique crops up even decades after the 5-series was retired.
Bottom line is that in my experience on all modern confocal microscopes, the wavelength defined differences in resolution caused by a single pinhole are minute enough to reasonably ignore. If this was really that important youād find more commercial confocals designed with multiple pinholes.
Hope this helps!
-Jason
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Hi @virsicas, thank you so much for your response! Apologies, I should have been more clear - I did not mean multiple physical pinholes 
just multiple pinhole size settings for the 1 pinhole during a scan.
Ah ok, it sounds like there is an additional software tab that he is not playing with yet then! Thank you! Iāve only used Nikon Elements for a spinning disk confocal so I did not think to interact with the lambda tab for pinhole settings!
Hi @jasonkirk, thank you so much for your response! Apologies, I should have been more clear - I did not mean multiple physical pinholes for both the 980 and the A1 just multiple pinhole size settings for the 1 pinhole during a scan.
Thanks for sharing such a thoughtful response! What makes this imaging problem extra fun are a few additional details that explain why the pinhole size between tracks matters. My colleague is optimizing for resolution and SNR in every single fluorescence channel of the 4, so from AF405 to AF647 (including the 405!). He is also using a 63x/1.4 oil immersion objective to get the resolution down to ~ 100s nm order of magnitude. The AF405 is the one suffering here with a fixed pinhole size between tracks, as setting it to the longest wavelength track is letting in AF405 out of focus fluorescence from different z planes. It is enough to result in even a visible difference in resolution and SNR, as different AF405 labeled features are coming in and out of focus at different z planes depending on what the pinhole size is. Normally, the AF405 is DAPI and high resolution images of cell nuclei are not worth the trouble in a 4 color image, but here we are equally interested in all 4 channels having high resolution images of different interacting membrane proteins on a cell, so the range in pinhole sizes from 405 to 647 can play a bigger role than usual.
Yes, you point out a great difference in the acquisition type - on the 980 it must be frame by frame instead of line-by-line, which does take a lot longer to acquire and involves more moving parts. Since the priority is resolution and SNR, my colleague is more willing to make sacrifices when it comes to acquisition time and other consequences of more moving parts. I would also imagine the same from the A1 as well, but we are way less versed in how to communicate these instructions the Nikon Elements software to the A1 . It sounds like this type of analogous acquisition is indeed possible on the A1 too though from both your and Virginiaās responses. So he will be checking out that lambda tab during the next imaging session!
I also just want to say I REALLY enjoyed this little history note about Zeiss and pinholes! Thank you so much for sharing! Made my morning, because you are spot on in that a significant amount of the Zeiss LSM confocal knowledge Iāve learned has been from big LSM510/580 fans who did indeed emphasize pinhole size. I had no idea the 5-series had multiple pinholes too, that is really cool to hear how far the light path optimization has come! And of course Zeiss sold the multiple pinhole concept as a feature 
some things donāt change over time.
Thanks again!
You may need to create an Optical Configuration per channel, and then add them to the Lambda tab. I wish I have a Nikon around to see if that would allow you to set up the pinhole, but Iām pretty sure you can. Then the scan will go from all the OC, one by one, similar to what happens in the SDC.
(The Optical Configuration panel is not the one you sekect, thatās the A1 GUI). Right click in an empty space of the screen, select Acquisition Settings > Optical Configuration panel. then you can create New OCs for each channel, and hopefully with 1AU per channel.
Just a quick note about setting different pinholes for your different channels with regards to axial sectioning. Donāt forget that as you change the pinhole size, the optimal step size changes as well, meaning that if you want to truly collect each channel at the 1AU setting, youāll need to basically collect different stacks and will have trouble merging them due to the different number of slices. Of course, you could always collect fewer than needed for the blue-shifted channel or more than needed for the red-shifted channels, but thatās basically what setting a common pinhole size does - it sets the optical section thickness to the same value - albeit less than perfectly ā1ā. Best of luck! Iād be interested to see any use cases where this process makes the difference in the data, truly!
