The Nanoscale World

Multimode AFM and cell

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ashkan posted on Wed, Aug 3 2011 5:41 PM

Hi everybody;

I want to find young modulus of OB6 cells by using AFM. I was wondering which tip is the best tip for me?

I am going to do the experiment on Fluid cell.

previously, I did this test in air by using SNL-10 tips (D and C) and on separation- force curve I got large negative deatach force. I do not know what was the reason.

Another question is I am using SPIP software to analize my data and it automatically gives sensitivity, I do not konw it is reliable or not. if some one has a idea please let me know about that.

 

Thanks for helping.

 

Ashkan  

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Hi, Ashkan,

It depends on how locally or globally you wanna measure the modulus. Usually a soft cantilever is commonly used but the tip shape can vary a lot (it also depends on what contact mechanics model you wanna apply to analyze and extract modulus from your data). Generally speaking, to measure the whole cell property, a sphere bead (diameter range from a few um to tens of um) attached at the end of a tipless cantilever would be a good choice and you can simply apply classic Hertz model for you analysis as many publication did. 

The large adhesion you saw in air is simply because of so called 'capillary force' or 'meniscus force'. This is due to the thin layer of water and other contamination (at normal huminity level) formed on the surface of the sample and the tip, upon contact they will form a 'viscous bridge' between the end of the tip and sample surface, which can pull the tip dramatically when you retract the tip from surface, thus you see this big adhesion. To reduce huminity or perform the test in liquid both can improve this issue.

As I remember (sorry it was long ago, now the software may have been updated), the sensitivity value in SPIP is different from what you have calibrated in Nanoscope, even in a reversed unit! So the algorism that SPIP used seems different from the other softwares, you probably have to stick to its own calculation of deflection sensitivity so as to get the final results even though you don't know whether it's reliable or not (since you don't have other means to replace or correct it). So you have to make sure you have a proper calibration curve collected on a hard surface that has been saved and analyzed before you work on your data curves.  

LA

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ashkan replied on Thu, Aug 4 2011 11:15 AM

thanks for your help;

actually, today I used SNL-10 tip to find the mechanical properties in Tapping mode, but the problem I had was because I used glass as a substrate for my cell, ( I plated cells on glass for 24 hours) when I wanted to take image in liquid, cells were not attached to the surface firmly.

Do you know what should I do?

 

Thanks a lot

Ang Li:

Hi, Ashkan,

It depends on how locally or globally you wanna measure the modulus. Usually a soft cantilever is commonly used but the tip shape can vary a lot (it also depends on what contact mechanics model you wanna apply to analyze and extract modulus from your data). Generally speaking, to measure the whole cell property, a sphere bead (diameter range from a few um to tens of um) attached at the end of a tipless cantilever would be a good choice and you can simply apply classic Hertz model for you analysis as many publication did. 

The large adhesion you saw in air is simply because of so called 'capillary force' or 'meniscus force'. This is due to the thin layer of water and other contamination (at normal huminity level) formed on the surface of the sample and the tip, upon contact they will form a 'viscous bridge' between the end of the tip and sample surface, which can pull the tip dramatically when you retract the tip from surface, thus you see this big adhesion. To reduce huminity or perform the test in liquid both can improve this issue.

As I remember (sorry it was long ago, now the software may have been updated), the sensitivity value in SPIP is different from what you have calibrated in Nanoscope, even in a reversed unit! So the algorism that SPIP used seems different from the other softwares, you probably have to stick to its own calculation of deflection sensitivity so as to get the final results even though you don't know whether it's reliable or not (since you don't have other means to replace or correct it). So you have to make sure you have a proper calibration curve collected on a hard surface that has been saved and analyzed before you work on your data curves.  

LA

 

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Hi, Ashkan,

I guess you mean you wanna image the cells in tapping mode but would measure mechanical properties under force mode (force curve or force volume or QNM if you have the option).

Could you first specify what cell type are you working on? If you are working on adherent type cells, growing them on glass (pretreat the glass with collagen or fibronectin may enhance cell adhesion in your case) for 24H should not be a problem. I guess the problem would more likely be the height of your cells (esp when your tip is short, cantilever will move your cells rather than tip scanning over). A good startpoint would be practising on imaging the edge of flat cells while optimising your imaging parameters, especially  tapping mode is a bit more tricky. You probably can also try contact mode using very soft cantilevers (such as MLCT) for imaging living cells. You may find this nicely presented app note helpful. http://www.bruker-axs.com/uploads/tx_linkselectorforpdfpool/Characterizing_the_Effect_of_Anticytoskeletal_Drugs_on_Living_Cells_Using_MIRO_and_BioScope_Catalyst_AFM_AN125.pdf

LA

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Answered (Not Verified) replied on Fri, Aug 5 2011 6:52 AM

Hi Ashkan,

My colleague Ang already gave you very good suggestions. If you have a MM8 with the PFQNM option, I also strongly recommend this mode to determine the Young's modulus. Moreover, OB cells are among the stiffest and the most robust existing cells so you can use ScanAsyst Fluid probes. Regarding the calibration of the Young's modulus, I recommend gelatin. Most of commercial gelatins have a Young's modulus of about 100 kPa which is perfect to image living OB cells.

A last remark regarding your last post: whatever the technique you use, if you are planning to image in liquid, which is the case when you work on living cells, you should also do all the preliminary calibration steps in liquid. And I would not use SNL probes for any type of mode on living cells...

Best,

Alex.

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Thanks Alex and Ang;

I am sorry but I do not know what PFQNM means. I addition, Can I use ScanAsyst Fluid probes to take picture in Tapping mode?

@ Ang, Actually , I do not think the problem is because of the cell's height, because as I mentioned before, I have done this experiment in air and the approximate height was 3-4 micro meter.

I do not know if I do the experiemnt in fluid, cell height would increase.

Thanks again.

Ashkan

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Hi, Ashkan,

if your cells measured 3-4um high in air, they could be higher in liquid and that's pretty challenging for normal setup. Also tapping mode doesn't help to reduce lateral force in such case since the side wall of the tip or even cantilever will actually hit the cells and move them away. ScanAsyst fluid tips are not optimized for fluid tapping, and they are pretty sharp and practically blunt tips would produce better images of living cells. You may wanna refer to Brukerprobe website for your selection of proper tip for your application following the link here: http://www.brukerafmprobes.com/t-faq.aspx

Peakforce QNM is a powerful quantitative mechanical property mapping technique introduced by Bruker recently. There are quite a few discussions in this forum on this technique and some of them particularly focus on biological applications. You can start by reading some of the app notes listed here:

http://nanoscaleworld.bruker-axs.com/nanoscaleworld/media/p/1548.aspx

http://nanoscaleworld.bruker-axs.com/nanoscaleworld/media/p/418.aspx

also please find here a nice summary from Adam on the unique beauties of the technique:

http://www.linkedin.com/groups/Anyone-used-PeakForce-Tapping-mode-2815697.S.48366474?qid=b4780d5d-4273-44eb-a5af-15338ab48d67&trk=group_most_popular-0-b-ttl&goback=%2Egmp_2815697

LA

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Answered (Not Verified) replied on Fri, Aug 5 2011 9:18 AM

Ashkan,

 

It looks like my last post didn’t go through so I will type it again:

PFQNM means PeakForce Quantitative NanoMechanical characterization. It’s based on the PeakForce technology. If you want to know more, you can have a look at this App Note:

 

http://www.bruker-axs.com/uploads/tx_linkselectorforpdfpool/Quantitative_Mechanical_Property_Mapping_at_the_Nanoscale_with_PeakForce-QNM_AFM_AN128.pdf

 

To make it simple, it’s a new investigation technique to probe mechanical properties of a very wide range of sample, from which you can extract many information like: topography, Young’s modulus, adhesion, deformation, dissipation,… in a quantitative manner.

 

It’s been tested successfully on a wide range of sample like DNA, bio or non-bio polymers and starts working pretty well on living samples. An application note will be released within the next weeks and a webinar is scheduled for August 17.

 

Please send me some representative images you took so far (if possible the raw AFM files). I would like to look at them.

 

Thanks,

 

Alex.

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Hi, Ashkan,

It seems my post also didn't go through and have to try again :(

if your cells measured 3-4um high in air, they could be higher in liquid and that's pretty challenging for normal setup. Also tapping mode doesn't help to reduce lateral force in such case since the side wall of the tip or even cantilever will actually hit the cells and move them away. ScanAsyst fluid tips are not optimized for fluid tapping, and they are pretty sharp and practically unsharpened tips would produce better images of living cells. You may wanna refer to Brukerprobe website for your selection of proper tip for your application following the link here: http://www.brukerafmprobes.com/t-faq.aspx

Since Alex has already explained to you in great details of PFQNM, I just would like to quote another link where Adam summarised nicely the unique beauties of the technique: http://www.linkedin.com/groups/Anyone-used-PeakForce-Tapping-mode-2815697.S.48366474?qid=b4780d5d-4273-44eb-a5af-15338ab48d67&trk=group_most_popular-0-b-ttl&goback=%2Egmp_2815697

LA

 

 

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replied on Fri, Aug 5 2011 12:44 PM

Hi Ang,

Scanning cells is actually not that challenging for a "normal setup" if you use the correct setup to start with. The Catalyst AFM has a z-range of 20 micrometer out of the box and is thus well suited for cell work amoung other things.

Stefan

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Thanks everybody;

Your comments are very useful,

@Alex: can I have your email please to send those images?

 

Thanks

 

Ashkan

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Hi, Stefan, Prob I considered catalyst as special AFM for bio applications but existing users may not have it :) Nevertheless, Z range is one of the necessary specifications out of others like tip height and geometry and scanning mode for this application, and for living cells exceeding 4-5um high, I often find difficulty to get good images since the tip often side contact the cell rather than contact only at the tip apex. By principle, scan asyst and peak forcce tapping should help to resolve the problem but unstable baseline detection due to hydrodynamics limit the peakforce amplitude and still need to be improved I guess.

LA

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@Ashkan

You can find Alex's email in this post:

http://nanoscaleworld.bruker-axs.com/nanoscaleworld/forums/t/563.aspx

Can also send me a copy to: ang.li@bruker-nano.com

LA

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replied on Sat, Aug 6 2011 3:17 AM

There you go:

alexandre.berquand@bruker-nano.com

Thanks,

Alex.

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ashkan replied on Fri, Aug 26 2011 10:30 AM

Hi everybody;

 

I have another question. can I use SNL-10 tips to get image in Contact mode or it is specified for Mapping mode? actually, I could not find anything in bruker's website related to that so I am going to take picture in contact mode from cell in fluid cell.

 

Thanks

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