Hi
I am new to 3D culture, live cell imaging and confocal microscopy. Is it possible to visualize epithelial and endothelial cells by live cell imaging for a period of time using confocal? In addition, I would like to visualize the bacterial cells and the biofilm produced on the 3D culture over a course of time?
I do have the information on the antibodies to be used for labelling to visualize epithelial & endothelial cells, and biofilm. However, the protocol needs the cells to be fixed. Also, I could get information on the nucleic acid stains which can be used on dead cells to visualize bacteria and eukaryotic cells.
Any thoughts on this?
Thanks in advance
Warm regards
Bindu
The big problem with that experiment would be photobleaching of your stains. You would need a microscope able to give pulses of the excitation wavelength which coincide with taking your image. A lot of the stains are also quite toxic to cells. Consider using GFP/RFP constructs under the promoters for your cell type markers to tell cell types apart. Also you may need some kind of microfluidics device to keep normal nutrient supplies and gas exchange.
It is possible. You will need a special chamber that allows the cells to live (temperature, humidity, and carbon dioxide concentration), and you can take images periodically (i.e. every 30 min or 60 min).
Yuning Hou . Yes, we are setting up the same system. Thank you for your inputs.
Yes you need the preliminary conditions on temperature (may be 37 degree), CO2 (mostly 5%) and humidity. A live cell setup has this function and make sure you have the correct inserts for the microscope to place your sample. Adding autofocus will be good when dealt with 2D for long time depended experiments.
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