I am working on imaging a fluorescent protein expressed in HEK293-T cells with 2-photon excitation for long time durations (1-2 hours). I notice movement and changes in morphology of the cells over the duration of the experiment, which is painful for analysis and post-processing. I wanted to know if there are any recommendations to limit movement/morphology changes for this cell line?
The cells are transfected using Mirus, and are adhered onto glass bottom imaging dishes coated with fibronectin. The dish is perfused with a buffer containing 125 mM NaCl, 2.5 Mm KCl, 15 mM HEPES, 30 mM glucose, 1mM MgCl2, 3 mM CaCl2. At the moment, we do not have a heated stage or inline heating element for perfusion, so the experiments are done at RT.
Jonas Wietek thanks for the feedback! We are using a home built perfusion system at the moment, but are upgrading it to be continuously heated at 37 deg with lower turbulence, which I hope will keep the cells happier. Using fixed cells is unfortunately not an option, as we need the cells to be alive for the experiment.
Hey Srividya Ganapathy :) ! I have observed the same phenomenon when imaging HEK293T cells confocally at RT for 1h or longer (makes drawing ROIs so much more enjoyable...) What might also have an influence on the movement of cells is how much you expose your cells to the laser(s). (On the other hand, you are already using 2P excitation, so it shouldn't really matter that much...) So unfortunately, the temperature of the bath is probably what will have the bigger effect. I'd appreciate some feedback if you've tried it! Happy imaging! :)
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