I am using tapping mode AFM in MilliQ to image a plasmid PGEM3Z which is ~1um long circular DNA. I prepare the sample in the following way.
I take 15mm x 15mm mica sheet and cleave it using scotch tape. Then I incubate mica sheet in a solution of 10ml Methanol + 0.5ml acetic acid + 0.3ml APTES for 30min. This protocol is followed in Prof. Chirlmin Joo's lab. Then clean it with methanol twice and dry it using nitrogen purge. Then 0.3ng/ul PGEM3Z which is suspended in MilliQ water is added on the APTES coated mica and incubated for 10min. I can see the DNA but quality of image is not satisfactory.
I am not sure about the process of tuning the cantilever to a particular frequency, the amplitude setpoint and the gains required to get better image.
- Is there any order to be followed in which these parameters have to be set depending on their importance?
How do we choose the driving frequency and amplitude setpoint?
Please see the attached images:
In Image1, the DNA height is only about 0.7nm but it is supposed to be 2nm.
In Image2, the DNA is seen in phase image clearly but not in topography.
Why does this happen?
For details
http://www.ntmdt-si.com/spm-notes/view/preparation-of-samples-of-dna-molecules-for-afm-measurements
For details
http://www.ntmdt-si.com/spm-notes/view/preparation-of-samples-of-dna-molecules-for-afm-measurements
The first set of images isn't perfect but it isn't terrible either. You can clearly see the plasmid. Height in an AFM image can depend a lot on the specific conditions, so be very careful in drawing conclusions from height or comparing experiment to experiment.
On setting up the frequency and amplitude:
* Set the drive frequency to the resonant frequency of the cantilever.
* Adjust the drive amplitude for the desired free amplitude. 1V is maybe a good starting point. Bigger will exert more force on the sample, smaller less.
* As you lower the setpoint, the tip starts interacting with the surface, first with net attractive forces, where the phase increases, then with net repulsive, where it decreases. You need to decide which regime you want to operate in. You can try both and see what works best. You don't want it to be jumping between them. 80% of the free amplitude is a good starting point and in the repulsive regime on my system with 1V free amplitude and a typical cantilever.
* After that I'd adjust the gain. Typically for a feedback system you want the highest gain before the system begins to oscillate. But if it doesn't need to be perfectly tuned, I like to watch the lines in the two directions as they are acquired, and increase the gain until they match up.
Hope that helps!
This was asked a while ago, but... What exactly are you trying to improve about the images? Apart from the tip tuning parameters, the surface activation protocol may also affect the image quality. I used to bind my DNA to freshly cleaved mica in a buffer containing 10 mM MgCl2 and 10 mM Tris HCl2 pH 7.8, for 5 min, and rinse the mica with water. No activation etc. If you believe that you must use the protocol you outlined, I would take images at every step (prior to adding DNA), and check at which step the problem(s) arise.
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