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Hi all,
I'm relatively new to AFM. Recently I've been working on how to use our Dimension Icon AFM to image live cells. I tried to use SNL probes in contact mode (in fluild), but I haven't got any useful images yet. Would someone help me with these questions:
What kind of probes are recommended for live cell imaging?
What 's the appropriate set up for imaging live cells? especially, how should I choose the deflection set point value? I used 0.6V before, but the picture was still really bad.
Thanks a lot!
Hi, Djuenne
My experience with live cell imaging using contact mode
Probes: Soft & blunt probes. Generally silicon tip (~2 nm tip radius) are much sharper than silicon nitride tip (~ 10 to 20 nm tip radius). While SNL probes can be soft with silicon nitride cantilevers (down to k ~ 0.06 N/, the probe sharpness often still cause cells to destabilize. Recommend silocon nitride probes: MLCT C (k~ 0.01 N/m), MLCT D (k ~ 0.03 N/m), DNP-D (k~ 0.06 N/m), or ORC8-D (~ 0.06 N/m)
Setting: Set sample per line to 128x128, and scan rate to 0.25 Hz. Scan angle 90 deg. Intergral gain 1 or 2, and proportional gain 3 or 4. Engage with zero scan size at delta 2 V deflection between setpoint and deflection, then lower your set point until the piezo is retracted. Now increase your scan size to a desire value (~ usually 15 to 25 um) and start to increase your set point slowly until you start tracking the cell. May need to adjust the gains at this point. Once the setting is optimize. You can start to increase your sample per line and scan rate. But usually, I won't go more than 0.5 Hz at a large scan size.
Cells: I would start with easy cell type first (e.g. fibroblast) which are adhered well and are flat (< 2 ~ 3 um tall), then move to harder cell types (stem cells 7 to 10 um tall)
Substrate: I would treat my substrate with fibronectin, poly-L-lysine, collagen, laminin, entactin, CellTak™, or polyethylene glycol (PEG) derivatives to make the cells more adhered to the surface.
http://www.cma.fcen.uba.ar/files/prepmuestras.pdf
This is a good link for bioAFM prep protocol.
Optical: Icon's optics may not be optimized for visualizing transparent cells. You may be on a fishing expedition for your cells if they are not very confluent.
Wish you the best, and have fun!
Please let us know how it turns out!
-James
Hi, Djuenne,
SNL may not be a good start point for imaging live cells, it's a bit too sharp and stiff. You may try MLCT first, its type C cantilever (the softest one with spring constant of 0.01N/m) is classical for imaging live cells in contact mode. The setpoint depends on your system and may vary dynamically due to thermally induced deflection drift of your cantilever, basically, just try to minimize the setpoint (lower it until you loss contact and adjust back a little more) to achieve lowest contact force for imaging live cells. In ICON, I guess you also have ScanAsyst mode so you can try this instead as well.
Good luck with your experiment!
LA
Hi Djuenne,
While I have no experience with running the ICON, I hope however that it’s operation principles are not very different from conventional AFM.
Please find the permalinks on the topic at MIAWiki:
http://confocal-manawatu.pbworks.com/w/page/44087106/Living%20Cells%20Imaging%20AFM
Cheers,DmitryMIAWiki for Mass Collaboration
Thank you very much, Ang!
I'll order the MLCT, and try to image in ScanAsyst to see what I can get.
Thanks again！
Hi Dmitry,
The URL link is very helpful.
Thanks very much!
Djuenne
Hi James,
Thanks very much for sharing soooo much details. This information is really useful for my expriment. I'll definitely try your method and let you know the image results.
Thanks again and have a wonderful day!