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Hi,
I am performing high-resolution imaging of biomolecules (antibodies) in fluid using tapping mode.
I am using Nanoscope IIIa and MTFML liquid cell.
I know that amplitude depends on the distance of the tip from the surface and that there exist two stable regions (attractive at higher separations and repulsive at lower separations). In the literature I noticed that for high resolution I have to work at higher working frequency (relative to the resonant frequency) to obtain two stable regions and to increase the probability to being in attractive regime. What is the reason for bistability behavior at higher working frequency and that for lower working frequency there is no bistability?
I know that to minimise tip-sample interaction, I have to keep the free amplitude as low as possible and then slowly increase to reach the repulsive regime. How can I know what is the lowest possible free amplitude when I am tuning cantilever before engagement? What is the best procedure to start with low free amplitude and then slowly increasing the drive amplitude? Now I start with setting drive amplitude at some small value when I tune the cantilever, then go to image mode and engage the tip. After engagement I go to force mode to precisely set the amplitude setpoint as high as possible, go back to image mode. But, when I am back to image mode I can't increase the drive amplitude anymore because the option for changing this setting is not shown anymore. Why is not possible to change drive amplitude during imaging after entering force mode?
I have also one question regarding estimation of cantilever free amplitude. What is the best way to estimate cantilever free amplitude in fluid? Is it right that at the beginning I should calibrate cantilever on substrate (mica) without adsorbed sample in fluid in contact mode to set the sensitivity or is enough to set the sensitivity looking the amplitude vs distance in tapping after engagement of the tip on the adsorbed sample on the substrate (this I am doing now)? When I obtained sensitivity I still don't know which value to use for calculating the amplitude. Is it right way to multiply sensitivity value to value of voltage from free amplitude? I am asking this because on the y-axis I have in units of nm/div and while using triggering is not possible to estimate how many divisions is the amplitude without damaging the tip.
I know that I have many questions, but I think that I need only small tricks to solve problems.
Thank you for your consideration of this matter,
Dejan
Hi Dejan,
You again !
At least I can help you on a few points. It's true that you generally have 2 regions that should be avoided.This was demonstrated by Spatz in 1995 and 1997. In those regions the tip brutally goes from a purely attractive to a purely repulsive regime. One of them is far below the frequency peak and the other one slightly above it. This is why we usually defined by default a 5% peak offset but to be very accurate, one should do the following experiment each time the sample is different and each time the tip is exchanged:
Engage the tip on a representative part of the sample you want to image and tune the cantilever with a 0 nm tip offset so that you are still on the surface. Then make a sweep from low to high frequencies and another one from high to low frequencies. Once you have the 2 images, use Paint Shop Pro or Photoshop to overlap them including the scale of the x axis. Then you should easily see the 2 regions to avoid! Not easy to explain but rather easy to do.
Regarding the calibration, the operation in fluid is the same as in air: in any case you have to calibrate on mica, glass, quartz or silicon (something not compliant) by recording a force curve and look at the linear portion after the contact points. This will give you a value in nm/V. Step 2: calculate the spring constant in N/m. Knowing those é values, you should be able to estimate the force directly in N.
Best,
Alex.
Hi Alex,
yes, me again I am dealing with this really intensively these days and it seems that you are only one who is willing to reply on my questions.
Now I am using approach which is written really extensively for imaging of antibodies in air by Neil H. Thompson and Santos (Cantilever dynamics in amplitude modulation AFM: continuous and discontinuous transitions and book chapter High resolution imaging of immunoglobulin G antibodies and other biomolecules using amplitude modulation atomic force microscopy in air). I start with calibration in fluid on mica to estimate the optical sensitivity and to later estimate the amplitude of cantilever. Here I have question. In this calibration step I want to preserve tip sharpness (I also have set of really sharp probes) and so I am using triggering to limit the range of the linear portion. What do you think how can I determine which triggering value is the smallest possible to obtain good enough force curve to estimate sensitivity and to preserve the tip?
Then I use adsorbed protein as sample, do the sweeping to get a peak and set one small value of drive amplitude for that peak and drive frequency slightly larger than resonance value. After that I engage the tip at 0nm scan size and after engagement go into Ramp mode to monitor amplitude-distance curve (phase-distance curve I have still to establish). Then I use steps described in the mentioned literature above until I obtain jump from attractive to repulsive part of amplitude-distance curve. In fluid is not so easy to monitor this jump because it is really small. For imaging I use the Drive amplitude value slightly larger than value when the jump is obtained and Amplitude setpoint slightly larger for the value of the jump. With this approach I obtained quite decent images. What is your opinion on this approach? I know that it takes a long time.
Thank you for your suggestion to obtain regions! I will try also that. Maybe I will obtain better images and it seems easily. How can I do a sweep after engagement because the button to go in sweeping mode I can't click on it (after engagement) and I never do sweeping after engagement? Or you just change the values of Drive frequency in the image mode by yourselves?
Thank you again for your patience with my questions !
Best regards,
I am not sure if your goal is to obtain great images of your samples using the method you describe or if your goal is simply to find a method to obtain great uimages of your sample. I can not really offer help with your lietrature method but suggest that you do not calibrate the optical sensitivity with each tip. After you have calibrated a few tips you should should have good enough statistics for that specific batch of levers, given that your laser alignment is similar, to allow for a good enough number. I wioyuld simoply be afraid to compromise the super sharp tip that you mention you are using each time I perform that procedure.
My approach to imaging is often rather simple but nevertheless works the majority of time. A start would be to simply use a rather small amplitude for which you should have settings right now and after going into feedback slowing perform a linescan. I then carefully adjust the cantilever drive amplitude until tracking is satisfactory. It is so simple that it might be worth a try in your case.
Stefan
Since you are using tapping mode, you might need to calibrate the amplitude sensitivity instead of deflection sensitivity. You can either use TM amplitude or TM deflection plot to do so and I guess you already have got amplitude-distance curve so you can just update the amplitude sensitivity by linear fitting of the repulsive part of the curve. Regarding the so-called noncontact mode imaging method you are following, I don't think it would work nicely in liquid, or as easy as in air. In air, a thin layer of adsorbed water on the sample surface usually gives rise to quite good signals of jump to attractive in the amplitude distance curve where you can easily define the working amplitude. Whereas in liquid, the attractive force reduces so much that I guess you would hardly see a clear jump to attractive part and it would be difficult to find a stablely workling amplitude to keep you working in attractive region.
If you have access to Bruker's new scanasyst mode, I recommand you give it a try and compare with your tapping mode images, we can easily get decent molecular resolution images by controlling the force well below 50pN.
La
thank you for all suggestions.
Stefan, I agree with you about using simple imaging procedure. But, at the beginning I want to estimate the range of setpoint amplitude which I could use and then is of course easier to use simplified procedure with known parameters.
Ang, unfortionally I don't have access to scanasyst mode yet. And I agree that that is hard to see the jump, but is possible because I got the jumps.
But I still don,t know what is the best setting for drive frequency. Is it better to use higher frequency than resonance one or smaller one?
Best regards
I would always retune when "on" the surface as already mentioned by Alex and then simply select the peak.
it looks like forum is time consuming for detailed discussion. Would you like to Skype me: FalconDot?
Cheers, Dmitry
Great idea Dmitry,
Maybe you can post a detailed receipe after your Skype discussion for the benefit of the community.
Hi Dmitry,
sorry for late reply. Yes, Skype is great idea. I will contact to to end of the week or next week (name: arzensekd)?
Cheers,