Imaging DNA in liquid

posted on Tue, Apr 6 2010 10:11 AM

rated by 0 users

Hi,

I have recently purchased some SNL10s to image some short (100nm) DNA strands. I need to detect whether the DNA strands are bent or straight.

(sausage or banana in the AFM).

I am using a dimension 3100 at the department of chemistry in Cambridge University.

Initially I tried using tap150s in air but these do not seem to have high enough resolution to image the DNA. Is there a tap150 or tap 300 with a sharper tip?

Unfortunately, no. All the existing Tap150 probes (12100, 12120, 12200, 12220) have an average tip radius of 10nm. I fully agree that for DNA imaging, 5 nm would fit better. Could you send me some of your samples (if transportation is not an issue)? I would like to test them on our latest dimension tool.

I then tried imaging in fluid using the SNL10s, however I had difficulty increasing the free RMS amplitude above 0.15V. Has anyone else had this problem? Were they able to resolve it?  Once I had engaged the set point was about 0.04V and the tip was not still not engaged properly.

SNL10s are known to give reproducible results, so I don’t expect a loss of coating but such a low sum signal could be explained by a tilt of the cantilever (maybe it was completely flipped over). Did you have way to check in real time how it looked like?

Normally the sum signal for this type of CL should be at least 1.5V.

The support notes that arrived with the tips showed an excellent image of DNA in fluid with a driving amplitude of about 20mV or thereabouts and the typical RMS with the 'C' cantilever was quoted as 0.3 to 0.6V.

From my own experience, it should be much more.

However even with a driving amplitude of the 400mV the best stable RMS amplitude I got was only 0.15V.

Was this reproducible or did you have this only for one single probe?

Initially my hunch was that the tip isn't mounted properly on the fluid tip holder.

Good reflex. This is the first thing I would have checked.

However, once the spring is released the tip is being held very firmly. I checked to ensure that the tip of the tip was closer to the surface than the tip holder but there wasn't much in it! Then I thought the laser wasn't aligned on the tip but I am getting a sum signal of around 3.5 - 4 and I was able to see the laser on the tip in the reflection image.

Yes, this is the sum signal you can expect in AIR.

Am I missing something here??? Is there a secret increase amplitude in fluid mode button...?

I don’t think there is any secret recipe. Except maybe try to slightly move the knobs to see if the laser is still correctly aligned while switching from air to liquid.

What is the purpose of the z-modulation? The same example you gave with the tips had the z-modulation disabled, but during an earlier session a colleague managed to increase the amplitude of the SNL10s by enabling the z-modulation. However, when I enabled the z-modulation in a second session the amplitude of the tip stayed low.

z-modulation is a kind a fake “tapping mode” you can use when you are not able to get some tapping signal (typically when you operate in fluid environment and due to a mechanical or electrical issue, you are not able to excite the CL by using the fluid holder). But in your case, I think the only issue is that you don’t have any sum signal. You have to fix this problem first. Moreover, z-modulation usually don’t give as good results as tapping mode.

Do you have an FAQ/source of info/person I could speak to on phone who can help me with this imaging of DNA in fluid? Perhaps advise on appropriate tips and how to get the best out of the SNL10s. I am envious of your publicity images and wonder why I cannot achieve this quality of image!

Well I know a couple of excellent people who spend their last 25 years imaging DNA in liquid but they all work on MultiMode. I also used to image DNA with Multimode, Innova or Catalyst, never on a Dim3100. This is why I propose you to send me some of your samples (to the Mannheim office please; my address is in my signature above). Probe wise, I had quite good feedback with SNL and with DNPs.

I am based in the UK at Cambridge university and will be trying a session on the AFM this afternoon from about 3pm to 6pm (uk time).

OK, good luck.

I am also copying some of my colleagues from our Cambridge office. Maybe they can be more helpful.

Best,

Alex.

Thanks

Chris Forman