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This protocol can be used as a routine for cells smaller than 5 um and stiffer than 40-50 kPa.
1) Preferentially use a probe having a tip height > 7um and a k between 0.05 and 0.3 N/m.
2) Make sure ScanAsyst AutoSetpoint is turned off. You can also turn AutoGain off.
3) Engage on the glass.
4) Go to ramp and capture a few force curves. Make sure the defl. sens. is more or less the same and update it. It should be < 25 nm/V. Otherwise try a new probe.
5) Withdraw and calculate the k. You will typically obtain the best results with a slightly dulled probe (30 nm), a defl. sens. of 10 nm/V or <, and a k around 0.1 N/m. Once you have such a probe meeting those requirements, keep it safe. By cleaning them after use, you can very well re-use them.
6) Image the tip check sample to estimate the tip radius . The other technique is more time consuming but can also be used.
7) Engage on the glass (if you can't avoid engaging on cells because they are too confluent, never engage on the nucleus but rather on the edges) not too far from the cell of interest. Click Auto-Config (never do this on the cell!).
8) Make sure the gain is minimized. Decrease the setpoint until you loose contact. Then increase it until you get a decent tracking. Only now you can start optimizing the gain, which should always be kept as low as possible, especially when capturing HSDC files, which is also a very good way to see if the gain is not too high.
9) Don't hesitate to scan very slow (like in HMX), like 0.1 Hz in regular feedback or up to 0.3-0.35 Hz if Adaptive is on.
10) For rather flat cells, you can very well use the std amplitude (300 nm) and frequency (1 kHz) but you will get better results with the 0.5 kHz workspace. For higher and/or softer cells, you absolutely need the 1 um version and the 0.5 kHz workspace (or slower).
Alex.
Berquand Alex:8) Make sure the gain is minimized. Decrease the setpoint until you loose contact. Then increase it until you get a decent tracking. Only now you can start optimizing the gain, which should always be kept as low as possible, especially when capturing HSDC files, which is also a very good way to see if the gain is not too high.
Dear Alex,
Could you, please, explain a bit more about the gain optimization - why it should be kept as low as possible (most of the time people try to work at gains as high as possible) especially during HSDC? And how do you judge whether the gain is correct or not? You can have the gain as low as 0 but that won't do any good to your cells, I guess.
Kind regards,
Cvet
Like in any AFM mode (Tapping or Contact), the first parameter to adjust is always the setpoint. only when you get acceptable tracking conditions (trace and retrace more or less overlapping), you start working on the gains. It's exactly the same with Peak Force tapping. On cells I generally found that putting a gain higher than 1 (with the Bioscope Catalyst) will cause the noise level to increase. When capturing High Speed Data Capture files, it should even be lower if possible. To check if your noise level is not too high, you can capture a HSDC file on the glass, open it in Nanoscope Analysis, zoom in on the deflection channel and check the base line. You will immediately see whether it's noisy or flat. This will be a good indicator on how to set the gain in imaging mode.
Alex,
You can not get good tracking if your gains are completly off. Maybe you can elaborate with some example settings?
Stefan
I know this. I don't think I ever talked about turnning the gains completely off (if yes, please let me know so that I can correct it). I just said "minimized". Or maybe I said that on this type of sample, AutoGain can be turned off.
Example settings? Well, for instance with a probe having k ~0.1 N/m and a deflection sensitivity of ~20 nm/V, you can easily image live cells with a setpoint of 500 pN and a gain of 0.5-0.6 if you don't scan too fast (max 0.4 Hz with Adaptive on).
Thanks for sharing your experience, Alex, it's pretty helpful.