Hi All,
Do you have tips to mount coverslips (Vecatshield + nail vernish) and make them horizontal ?
The issue I often have is that when take tile scans only the centre in in focus…
Thanks
This could be a result of the objective lens or the stage. What objective lens are you using? And what type (make, model) of stage?
I use an LSM Zeiss880 Airyscan with the objectives below and a standard slide holder.
So my bet is that when I put nail varnish on the coverslip if there is more on one side then the coverslip is tilted…
Do you put spacers between the slide an coverslip to prevent this from happening ?
Such as : (not available any more aparently)
https://www.sigmaaldrich.com/catalog/product/sigma/s7810?lang=en®ion=US
Thanks
I’ve had a lot of success using GraceBio imaging spacers (link). They are adhesive and remove the need for nail varnish and are always flat.
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From your description I thought you may have objectives that are not corrected to have a flat field of view, but these are (“Plan”).
It’s possible it’s coming from the sample, but quite often this happens because the stage or stage plate (the part of the stage that holds the sample and that can be swapped out to hold different types of samples) is tilted relative to the optical path. It’s a quick test to determine if the tilt in the image is coming from the sample or stage: Focus on a sample and note the direction of change of focus (ie, move the focus in one direction and note which side comes into focus first). Then turn the sample around 180 degrees and focus in the same direction. If the direction changes it’s the sample, if it doesn’t it’s the microscope - and most likely the stage.
If it is the sample, a spacer won’t make a difference if the specimen is attached to the coverslip glass (which it should be for optimal image quality). If you find it is coming from the sample and not the stage, I’m not sure what I would recommend. Hopefully others have ideas!
If you find it’s the stage, look for small set screws along the edge of the stage plate that can be moved up and down to adjust the height - on our Prior and Nikon stages you need a hex key to adjust them. There are usually 3 or 4 such screws (in addition to the (usually larger) screws that hold the stage plate in place). You can also use these screws to adjust the tilt of the stage to match any tilt in your sample. But if the tilt is coming from the sample, you would need to do that for each sample, which is a bit of a pain if you have a lot of samples.
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Thank you, I’ll check if it comes from the stage.
Forgot to mention that this happens with cells grown on coverslips…rather than tissue sections.
Even more likely that it’s coming from the stage then!
I have seen people use a coverslip snapped into quarters to act as “legs” to another full coverslip with the sample on it (for the little square coverslips), but be careful with the glass…
I have also noticed people using an excess of mounting media having more problems with sloped samples as well. It can be very noticeable over large tiles, while users that aren’t tiling have no problems at all regardless of their sample setup.
On the upside, we have had a lot of users with 24 well plates these days, which has bypassed the mounting problems entirely.
I have mounted many, many slips, and montaged large areas. Do you need to use Vectashield and nail polish? I do not receive any compensation of any kind from Thermo, but I’m a big fan of the Prolong reagents. After mounting the coverslip, I work at the edge of my bench to work parallel to the slide, and I use a Pasteur pipet and go around the perimeter of the slip to aspirate out all excess mounting agent, this acts to ‘pull’ the slip down flat, with a very thin layer of mounting agent between the slip and the slide (while removing air bubbles), gentle tapping across the top of the coverslip with the pipet can also sometimes help. Be sure there is no Kimwipe fiber, eyelash, dust, etc. under your coverslip, this will result on flatness issues also. All the points Jen W points out are true, confirm stage/sample holder flatness, also confirm field intensity uniformity, your sample may be flat, but it the field isn’t flat…its an issue. If this is cells grown on glass, it shouldn’t be too bad, as your likely to have massive signal to noise and no autofluorescent background, which will really reveal ‘issues’, if you have it, with sample and system. What magnification are you using and are you taking overlapping images in a montage?