Hi,
I am a PhD student, and my project heavily depends on imaging. I’ve been using LSM880 for my confocal images; however, recently, my PI suggested I start using airyscan for some of my images. I am trying to visualize nuclear pore complexes, similarly as in this paper (Fig. 1h), so my images require using 63x objective and max zoom. I recently tried using airyscan on my samples, but my fluorescent signal photobleaches so fast that I can’t even find a field of focus before taking the picture (I used gain at 800 and laser power at 0.5; otherwise, I didn’t see any signal at all). In my staining, I used Hoechst stain, a secondary antibody conjugated with Alexa Fluor 568 (Invitrogen; # A10042) and ibidi µ-Slide 8 Well (#80826). I have successfully used this combination for confocal microscopy, but I started questioning this method since the airyscan imaging didn’t work. What kind of more stable fluorophores would you recommend for this technique? Could it be the fault of the ibidi slide, and should I switch to the glass slide/glass coverslip?
Thank you so much for your help!
Are you also using the oxygen scavenging system they used in the paper you referenced? Are the wells in your ibidi 8 well reasonably well shielded from the air? Or they are open?
Are you trying to do airyscan imaging of the A568 dye? The Hoechst stain? Both?
Hi, thank you so much for your response! No, I haven’t been using an oxygen scavenging system (and I am not even sure if our scope has those capabilities). My 8-well ibidi is opened, I haven’t tried imaging them with the cover on top… I am trying to get a nice crisp image of both the Hoechst stain and A568 because I want to be sure I am looking at the nucleus. But I am open to using different fluorophores or plates if this will bring better results!
Hi! There’s a number of different factors that can contribute to issues you’re having. Below are a few major points I’d recommend looking into or double checking.
For sample prep:
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The ibidi wells you’re using have a No 1.5 polymer coverslip, not a glass one. Certain immersion oils are known to dissolve that polymer coverslip. Additionally, it may not have as good of optical properties as glass, which is more impactful if you are trying to push the microscope limits for resolution and contrast. I’d really recommend switching to No 1.5 glass for imaging. If cell attachment is an issue, you can look into various coating strategies (e.g., collagen, fibronectin, etc.).
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Just to double check, when you say you’ve “successfully used this combination for confocal microscopy” you mean the fluorophore pairings for a different sample, right? Cells generally have lots of DNA, so it’s a little unexpected to not be able to detect hoechst. It could be acquisition related, but you might want to try to image that in regular confocal mode first on the LSM880 to make sure your fluorophores permeated into your sample. You may have to troubleshoot your AF568 staining as well - if you haven’t validated the antibodies yet, is it possible they are not working well?
For acquisition parameters:
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When you say “max zoom”, are you referring to the scan area? On the LSM880 there are three choices to specify the pixel size, which according to that paper you’re aiming to have as 40 nm. The first is objective, which the 63x/1.4 NA oil objective is a good choice. Second is the zoom, which refers to the specific scan area within the field of view. Third is the image size, which refers to the pixel dimensions. Zen Black has an “optimal” button for calculating the image size needed for a given zoom and objective to minimize pixel size. The pixel size calculation is displayed just above the zoom box info. You should double check your settings to make sure that you’re getting the desired pixel size. I would also recommend trying to get away with the lowest zoom you can. Smaller scan areas make drift increasingly more impactful on imaging.
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For setting up Airyscan, misalignment of the light path to the array detector can seriously mess up imaging. When you are setting up at the start of imaging, are you checking the alignment of the detector with either the hoechst or AF568 emission light? The alignment varies slightly by wavelength. Generally people pick the channel they care about the most when setting up alignment, and/or the brightest one so it’s easier to center the light onto the array. You should keep an eye on the alignment between scans. Not aligning the emission to the detector can result in getting not much signal in an image. For the hoechst, if you’re able to see signal in the eyepiece with widefield, then you should be able to image it with Airyscan. Before moving to the AF568 signal, it may be good to troubleshoot the hoechst. Since you image that with the 405 laser, which has a separate light path from the other lasers, there is a chance that you may have to align the 405 laser excitation light as well since it’s a separate path from the 561 laser. For alignment, I recommend setting it up at the start of your imaging with a sacrificial cell.
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Have you double checked your emission filter choices for Airyscan? They should be visible in the light path diagram after the ChA checkbox.
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One way you can try to minimize photobleaching is to focus on the cells with transmitted light first in the eyepiece. Then, you can use the LEDs to further refine focus on the hoechst signal and pick a sacrificial cell. From there move to set up Airyscan in the acquire tab.
Once you feel confident about your sample prep, the acquisition setup needs optimization. Just like laser scanning confocal, Airyscan requires lots of settings optimization, so it’s very common to take some time with optimization and bleaching cells in the process. It’s hard to give recommendations on things like laser power, because that input isn’t in power units so it’s only relevant to your setup. When I was first getting started with Airyscan to do high resolution imaging of weak/sparse fluorophores, I found it helpful to first optimize regular laser scanning confocal imaging for minimal acquisition time, max resolution, and max contrast. From there, I moved to Airyscan and further tweaked settings. That approach may be helpful in this case. Best of luck!!!
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