I am very new to the live cell imaging of actin. I am trying to visualise actin filament dynamics by transfecting Lifeact GFP by spinning microscope. I need to visualise the actin filament dynamics in response to some compounds for 2 hours. But the problem is the actin filaments that I am trying to image are losing the focus continuously. Does any one have any suggestions to cope up with this issue.
Hi, Ravindra.
You are not losing the focus, but actin are just showing dynamics of rapid polymerization and depolymerization in responses to the environmental cues. The cues may include temperature, gases, pH, nutrients and wastes produced by cells in medium. Of course, tungsten and UV light are one of major causes. The depolymerrization/depolymerization cycle occurs within a fraction of second when the environmental cues change and reach threshold of the cue magnitude.
You need first to establish control dynamics of actin dynamics whether you have changes or not. Then, record the drug effects preferably with time lapse exposure at appropriate intervals. Comparison of the two groups may tell the effects on the actin dynamics.
Thus, set up a similar condition to that of working CO2 incubator on the stage of the microscope including temp, gas, humidity and/or perfusion medium system with gas. (see the link.) http://www.the-scientist.com/?articles.view/articleNo/34173/title/A-Room-with-a-View/
If that is not all possible, then observe actins under lower magnification (20X objective instead 40X or 63X) in more number of cells. This will allow you to record overall dynamics of actin in larger No of cells, and also deeper focal planes without losing the focus, but dynamics of depolymerrization/depolymerization itself.
This is the limitation of dynamic study after tagging with GFP under fluorescence microscope. Good luck to your try.
Hi Ravindra,
Make sure you are imaging on good system with "perfect focus". This feature check small perturbations. Its a small setting on microscope.
Another issue to use correct concentration of lifeACT. Make sure its not limiting & not bleaching due to high laser power.
For 2hr - make a loop which will image every 5min. for 2hr. Try low laser power. Videos can later be enhanced in ImageJ.
Also keep cells healthy by adding 1mM-10mM HEPES, 37degree.
Good luck.
Anand
Dear Ravinda,
why don't you come to the mayor lab. we do a lot of imaging of actin dynamics and we can discuss things much easier here rather than using research gate.
see you soon!
Thank you all. Sure Darius. That's great idea. See you soon.
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