We are working on DMPC lipid bilayers. When imaged under liquid, we observe lot of defect features. As the bilayer is prepared using Langmuir Blodgett, we anticipate some defects.
But how do I understand whether this defects are inherent to the bilayer or formed due to tip force(breakthrough)??
Please find attached a typical image
Hi,
Probably the easiest way to check for tip effects, is as follows:
- measure a small area, say 2x2 micron, repeatedly: do you see more deterioration over time?
- zoom out to a larger area, say 5x5 micron. Does the 2x2 micron area that was scanned before contain substantially more defects then the surrounding area?
What Dr Willem wrote is good method. What I sometimes use to ensure what I see is change the speed of scanning and/or turn on another channel, eg for CM - LFM and for TM - PhaseImaging. On the other hand, changing the parametres of scanning (speed, scansize, force tip-sample) is the easiest method.
Hi Janusz, I usually take both topography and lateral force mode images simultaneously. Is it possible to sort out this problem using LFM images? Could you please clarify this.
Regarding change of speed,I usually don't go more than 1Hz,as imaging is done under liquid. I have worked with two speeds 0.70 and 0.80Hz. That did not show any effect in my images.
I will also get back soon with the images that Dr Willem suggested.
Thank you..
I was thinking about another mode/channel to see if any particles really exist. I usually get some samples and I do not know nothing of them (because if one would tell me the material is nano I would search nano only - not characterize real/all structure). Many times I obtain results with 'something' what shouldn't exist on sample. It may be fat from fingers, dust, etc. So, If I see something unusual on the topography image I turn on another channel mode (LFM of PI) what let me to ensure what I see on topography. In case of yours sample, LFM may illustrate if on the surface You got 'particle' or not . If so, LFM should be similar to topography.
Looking at image it hard to say - we can only shoot in the dark guessing what it may be. If it is on steel - maybe it is some corrosion? In case of polymers - places with not completely reacted components. Of course - I do not have idea if in the water there is no nanoparticles??? I do not have experience with DMPC.
Hi,
What measuring mode are you using to scan your samples? Since you record LFM I guess it is contact mode. Have you tried to scan them in Dynamic or "jumping" modes? With these modes you can be very gentle with the surface, applying forces of just tens of pN.
Hi Janusz, Definitely we have no confusion on the presence of our bilayers on the substrate. Our confusion is whether this 4nm depth defect is forming due to tip interaction with the bilayer.
Bilayer is completely filled with DI water(no nanoparticle presen, so that our bilyaers don't dewet.
Many thanks
Hi Pablo, We have done tapping mode in air imaging. But in liquid imaging, at present we haven't optimized for that. Thats the only reason we are doing it in contact mode. And the we are using is pyrex nitride triangualr tip( spring constant is 0.08N/m).
Thank you
Then I would reccomend you to try in any other mode (Dynamic or "jumping" ones) trying to minimize the force the tip is applying to the bilayers.
Good luck!
How much force are you exerting during imaging? DMPC is a typical gel phase and so would require a high force (~few nNs) to break through the bilayer. The image you attached looks fine to me, i.e. I don't see any streaking or visible signs that you are perturbing your bilayer so my guess is in your case, it is inherent to the bilayer. My suggestion is try to poke into your DMPC and see how much force is needed to break through it and then do your imaging in contact mode in the lowest possible force, much lower than the breakthrough force. These following links might be good comparison for your system:
1. http://www.ub.edu/bioelectronano/posters/poster1.pdf
2. Figure 6 of this reference, S. Garcia-Manyes, G. Oncins, F. Sanz, 2006Effect of pH and ionic strength on phospholipid nanomechanics and on deposition process onto hydrophilic surfaces measured by AFM, Electrochimica Acta, 512450295036
All the best!
The holes are not regular, and do not produce aberrations in the portion of the image after the hole, which suggests that nothing was picked up by the tip. I would say, they are likely real.
I also tried what Dr.Willem suggested, repeated scanning of 3x3um and zoomed out scan(9x9um) looks pretty fine.
please check this image
thank you
we have done some work in AFM characterization of LB films. And from your data the it does not looks as if the holes/discontinuities are due to tip (especially from the second set of images you uploaded).
For the films deposited by LB process you will have to pay attention to: How any layers have you deposited (try varying the number of layers deposited)? What was the substrate and its roughness? I would suggest you also check the depth of the holes and compare it with the height of the bi-layer?
Hello Loveleen,
we deposit two layers of lipid on RCA cleaned glass substrate. These holes are generally monolayer(1.5-2nm) and bilayer depth(3.5-4nm), matches with the expected thickness.
Thank you
Hi,
I think letting the vesicles on the substrate needs to happen above the lipid transition temp. and keeping the temp constant all the time and during imaging will give you flat bilayer in liquid phase. you can reduce the temp later under the transition temp to get it the gel phase but you will probably notice some defects again....
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