***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****Hi Yevgeniy
Flip the dish to see if the slant is due to the insert or to the sample.
If it comes from the insert, check if your stage insert has small screws on its sides. If yes, make sure none of them is sticking out towards the stage. If they don’t, your insert might be warped. You can use the small screws to adjust it or get a new insert.
If it doesn’t come from the insert but from the sample (the most common case in my experience), you cannot use any device to fix the problem. You will need to adjust for every plate you need to image.
In that latter case, here are a few possibilities:
- when someone image coverslips mounted on a slide, they sometimes place the coverslip too close to the edge of the slide. This leads to the slide resting on one side on the stage insert and on the other on the coverslip. This is easily corrected by placing the coverslip a bit away from the slide edge.
- If you are imaging dishes or plates, it is unfortunately likely that your dish/plate is not flat. If I understood well from a previous conversation on the confocal server, it is because plates are moulded warm and they tend to warp when they cool down. We often have this problem with dishes that are made of rather thin plastic like the 35mm MatTek dishes but multiwell plates are clearly not flat either. We also experience this problem more with thin polymer bottom plates than with thin glass bottom plates.
- Check if the slant you experience is seen within 1 field of view or if you only see it from one field of view to another
- if you do not see it within one field of view, you can use a hardware autofocus or a z slope calculated by your software to deal with it.
- If you see it within 1 field of view (the toughest case), the solutions above will not work. You can try using the stage insert screws to correct the slant for every plate but it is likely that it will not be precise enough. You can start a painful hunt for plates that are more flat. We have sometimes experienced that it was a bad batch of plates, we complained to the company and they sent us another batch. This sometimes solves the problem and sometimes not.
When buying multiwell plates for a screen for example, always request a free sample and check if there is a bad (within 1 FOV) slant.
Med vänlig hälsning / Best regards
Sylvie
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: +46 (0) 73 733 5008
office: +46 (0) 08-524 811 72
From: Confocal Microscopy List [[hidden email]] On Behalf Of WAINWRIGHT James
Sent: den 31 oktober 2016 09:52
To: [hidden email]
Subject: Re: digital levels for microscope stages
***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/
wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****Hi Yevgeniy,
I’ve never found using a level works either. I’ve assumed this is partly the lack of sensitivity and partly because we don’t care (in this case) that the stage is level with the floor or “gravity” – only that it’s level with the *imaging plane*.
If I want / need the stage really flat, then I’ve used a method similar to Craig’s – i.e. imaging different positions on a known flat sample and adjusting the insert each time through a few iterations.
This is probably easiest with something like this slide from Thorlabs (no commercial interest):
https://www.thorlabs.com/
thorproduct.cfm?partnumber= R1L3S3P
Focus on one corner of the largest grid, then move the stage to another corner and adjust the stage insert levelling screws. Repeat this a few times with each corner.
You could also use a multi-well plate (probably cheaper than the above if you have them lying around) for even more accuracy (i.e. larger distance travelled by stage = more sensitive detection of focus shift). It’s easy enough to focus on the bottom of the wells by stopping down the condenser aperture to increase the contrast.
James
From: Confocal Microscopy List [[hidden email]] On Behalf Of Craig Brideau
Sent: 28 October 2016 20:26
To: [hidden email]
Subject: Re: digital levels for microscope stages
EXTERNAL EMAIL
ATTACHMENT ADVISORY
***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/
wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****I have used the interference pattern off a coverslip to level a sample. You can also use a thin layer of a fluorescent material or a thin grid to make sure your stage is aligned with your imaging plane.
Craig
On Fri, Oct 28, 2016 at 12:56 PM, <[hidden email]> wrote:
***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/
wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****Hello everybody
Does anybody have any experience with using levels to make sure the stage inserts on their systems are as flat as possible? We use a little bubble level sometimes to adjust our stage inserts as well as make sure that the AFM head on one of our systems is flat. I feel like it's not sensitive enough. Would anybody be able to recommend a good digital level with high sensitivity that can be used for the purposes that I described.
Thanks very much in advance,
Yevgeniy Romin==============================
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