Hi everybody,
recently, I encounter a problem of sample drifting under confocal microscope. As I want to record mitosis and cytokinesis which take 1 hour in total in arabidopsis roots, but drifting is severe so that the tracking becomes difficult. Is there anybody having good suggestions to cope with this.
Actually, I have checked one paper published in Nature where they used agar pad to stabilize seedlings. Here is their protocol, i.e., Seedlings were mounted between a 24 × 60 mm cover glass and a 1-mm thick 0.8% agar pad affixed to a 22 × 22 mm cover glass. The agar cushion stabilized the specimen for sustained time-lapse imaging, minimized compression and mechanical damage, and allowed greater reproducibility in mounting conditions. I have some questions about this.
1. just put cover glass which has been affixed by agar pad on the other cover glass. Is it necessary to seal these two cover glasses?
2. are samples needed to be inverted under confocal microsope with regard to this protocol.
I am attaching the paper and the method is highlighted in yellow. Could anybody help me have a look at it?
I am really looking forward to any response!
Dear Feng,
you may try the technique by Jordi Chan (see attachment) with microscope chambers. For details, communicate with him, I think he will be eager to explain everything to you.
Manolis
If you are able to use an inverted confocal microscope then growing/using glass bottom dishes ( http://glass-bottom-dishes.com/pages/) might solve your problem. Arabidopsis will grown directly in an agar medium using those dishes.
However, common agar has the tedency for autofluoresence, noble agar however is a safe choice. I hope this will help.
regards.
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