SMLM set up question

We have a Nikon microscope. When I focus through 100 nm tetraspeck beads with an astigmatism lens in place, the shape of the bead is oval in the y-axis below the focal plane but it is not oval in the x-axis above the focal plane. It’s stretched only ever so slightly in the x-axis above the focal plane. Do you know why this is and how to fix this? When we ran the calibration sequence, the Wx-Wy calibration curves are centered at 0 but the concaves of both curves are tilted down toward one side. Finally, my PSF is not symmetrical (I thought it was because my adherent bead sample is in water and we are using a 100x oil objective). How can I fix all these?

Tagging @Hazen_Babcock

Are you using the Nikon astigmatism system? Or is this something that you built yourself?

If you remove the astigmatism lens is the PSF now symmetric above and below the focal plane? Or do you see defocus rings on one side and not on the other?

Also, if you could post the curves you are seeing here that would be helpful.

Hi Hazen - thank you so much again for your help. I attached the curves. We were looking at the PSF without the lens - it was not symmetrical. I see airy rings below the focal plane but not above. I assumed it was because of water in the dish and oil on the lens, but I am also reading that this could be adjusted with the objective collar. But I can’t seem to. I have played with the collar for ours but I can’t make the PSF symmetrical

It’s Nikon’s astigmatism system.

You can also correct for this by moving the camera back and forth, assuming that your system has the flexibility for this. I’d expect the manufacturer to have the camera at the right distance though…

The immersion oil can also have an effect here. Are you using something that is close to what Nikon recommends (in terms of index of refraction)?

Also, it isn’t easy to get curves that are perfectly symmetric. However what you are seeing is worse than what I would expect.

Do you at all have a DIC prism under your objective?
If so try taking it out!
Just a thought - It’s not that I’m seeing the curve and thinking it’s that but it does affect the resolution a bit and can affect things at this level

Hi George, I checked - there is no DIC prism and there is no ND filter in the way. I’m using Nikon’s F oil.

Checking the oil was a good idea. I noticed when I mounted beads in Prolong Glass, things improved a bit, also pay attention to the correction collar of the objective (if it has one). Be sure the objective is very clean. Is this a laser scanning confocal or a spinning disk? If it’s a spinning disk, the suggestion about the camera position is interesting, and this can result in some distortion. I read an article once where beads were mounted in immersion oil, interesting idea. Good luck!

Hi, the curve should be more symmetrical than that. This is what I get on our N-STORM using 0.1 µm Tetraspeck beads in PBS on the 100X 1.49 SR objective. This is for the far red channel, but you should get similar results with the green one (although TS beads are not as bright in green, and I don’t think there is any good SMLM label using 488 nm).

Capture d’écran 2020-11-12 à 22.13.25

Regarding symmetry of the PSF without the astigmatic lens - it is normal that you see rings on one side but something more blurry on the other side. The PSF should be approximatively similar in terms of diameter when going ± the same distance from the best focus point.

Make sure to use #1.5 or better #1.5H (high precision ± 5 µm) coverslips, and set the correction collar to the corresponding thickness (0.17 mm). Also, check that your piezo stage is well calibrated in functioning by shifting the focus using the stand Z motor by 1 µm, and going back to the focus with the piezo by 10 steps of 100 nm on each side. We had problems to calibrate once that were due to a faulty piezo stage. Make sure your laser is properly aligned in the FoV, and use the same illumination modality for calibration as you’ll use for acquisition (eg HiLO, TIRF or vertical). Make sure to have 50-80 beads on the FoV for proper calibration (the N-STORM needs a denser bead field than most calibration procedures).

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Thank you. We will try all of these.

Hi Sophie,

The issue with poor astigmatism that you’re describing is usually due to spherical aberration. So to reiterate some of the comments above try adjusting the correction collar. This may need to be repeated as the system stabilises temperature - best done after it has been on for 3 hours or more. Also make sure the objective lens and the coverglass as clean as possible. I used to sometimes adjust the correction collar with the cylindrical lens in position and when you get equal astigmatism above and below focus you know you’ve got it right. If you still can’t get it to work it could be that the objective lens has some damage or that you have a bad batch of coverglass/chambers. I have also seen issues with old or incorrect oil. Check with Nikon what the latest recommendation is on that.

The graph that Christophe is showing of Wx vs Wy is definitely achievable with some fine tuning of what you’re doing!

One other tip with the N-STORM is to take the STORM lens out in the illuminator. This gives you more even illumination over the FOV and you don’t need the extra laser power density for a bead calibration.