The Nanoscale World

How to use Point and Shoot Method to Calculate Modulus in Live Cells using the Catalyst

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warren.truong posted on Thu, Jun 7 2012 1:13 AM

Hi,

I am using the Bruker Catalyst AFM.

I'm trying to measure cell elasticity using the indentation method using point and shoot.

How do I analyse the curves I get from each indentation point using Nanoscope Analysis v 1.40?

I want to obtain the exact numbers; i.e. I want to get the deformation/reduced Youngs modulus values at those indentation points.

Regards, Warren and Iman from UNSW.

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Hi Alex,

I will give it a try and will let you know. Thank you.

Regards,

Iman

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Hi Alex,

Thank you for your response. The problem is when I go to indentation option, after baseline correction, to choose the proper model, when I choose sneddon fit it doesnot show me the modulus. However, when I choose Hertzian it shows me the Young's modulus. How can I fix this? Thank you.

Regards,

Iman

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Ang Li replied on Wed, Oct 17 2012 10:27 PM

Hi, Iman,

you need to update tip half angle under modify force parameters first before you can use sneddon model.

Ang Li

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Hi Guys,

Yeah, I figured it after I sent the email. But I am wondering what is the tip half angle for ScanAsyst fluid tip? Is it 15 based on the tip catalog?  Thank you for the response.

Regards,

Iman

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And, I also put 0.5 for Poisson's ratio.

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Hi

I am having problem again! This time is with batch processing! I have analyzed just one curve out of ~ 5000 curves. But I donot know how should I extend it to the rest of the curves? Which file should I add to run the history on? When I add the original file it doesnot work! It just gives me one Young's modulus info!

Also, if I need the specific part of the data (curves just on the nucleus), how should I separate that part from the rest of the force curves?

Thank you.

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Answered (Not Verified) replied on Thu, Oct 18 2012 8:53 AM
Suggested by Bede Pittenger

Hello,

Question 1. This is fairly easy: once you hav processed the curve, select Run History, select a destination folder and drag & drop all the curves into the main pannel. Then just press Run. But if you have 5000 force curves, it might take you a while... (several minutes).

Question 2. You have several options but the easiest is probably to use the very lastly released feature of 8.15: while you are imaging via peak force tapping, use the "capture force curve" feature. This new icon should show up on the top bar. You have to disable regular capture before. I guess that by looking at the PF error channel for instance, it's quite easy to distinguish the nucleus area from the rest. Then, export the curves as you are used to.

Best,

Alex.

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Hi Alex,

Thank you for your response. In regards to your second answer, I think there is another way of doing it using Nanoscope analysis. I did it like that:

I separated the caruves right above the nucleus using red dashed lines in QNM Hsdc-Forcecurve-Image and then exported multiple curves (for example: from 2389-2784). Then I analysed the first curve and then ran history for the rest of the saved curves. But there is a big problem!

The average I got from these curves (in excel) is 232.484 MPa!! It is way higher than what I was expecting from the elasticity of the live cells (in my case neuroblasts). I need them to be in KPa range as one would expect. How can I normalise the data? Thank you.

Best,

Iman

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Answered (Not Verified) replied on Fri, Oct 19 2012 3:21 AM
Suggested by Bede Pittenger

Hi Iman,

 

Yes, HSDC is another way to do it.

With respect to your weird numbers, there are 2 possibiities:

1) Your defl sens / k / tip radius were not properly calibrated.

2) For the vast majority of the curves, you were too close to the substrate. Please check your average indentation vs the cell thickness. If you are too close, no wonder you find very high values.

Best,

Alex.

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Hi Guys,

I would like to ask you that is it possible to use ScanAsist-Fluid cantilevers for indentation experiments and force curve measurements instead of DNP cantilevers? If it is possible, how is it going to affect my results? (Young's Modulus).

Thank you,

Iman

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Answered (Not Verified) Ang Li replied on Tue, Nov 27 2012 11:51 PM
Suggested by Bede Pittenger

Hi, Iman, you definitely can use scanasyst-fluid probe, just calibrate in the same way as other probes and half angle might be slightly different from DNP as well.

Ang Li 

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replied on Wed, Nov 28 2012 4:54 AM

Hi Iman,

Ang is right. I also recommend ScanAsyst Fluid (nominal k =0.7 N/m) and ScanAsyst Air (nominal k =0.4 N/m; they can also perfectly be used in liquid) probes, under the condition that you work on not too soft cells (they should be 50 kPa or stiffer). With SA Fluid probes I found a high discrepancy in terms of spring constant (the tip radius and deflection sensitivity should be rather consistant); it can sometimes go down to 0.1, which makes them the perfect probes to work on live cells.

Good luck,

Alex.

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Hi Guys,

Thank you for your response. I calibrate the tip (ScanAsyst-Fluid) accurately as per manual and also consider the actual tip angle for analysis. Thank you again.

Best,

Iman 

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Dear Mr. Berquand,

I was just reading your comments on how to gather modulus values out of HSDC data. First, I want to mention that it must be explained better how to extract single force curves out of the HSDC data. I managed to do so now, but it took me quite a while. I tried hard to obtain force-separation curves from the HSDC data directly before I understood that you must export and re-open the data before you can do so.

However, the reason for me to write you has to do with the export function itself. I tried to export two force curves from a HSDC data set: at pixel positions 666 and 727. But after I had clicked "Export Curves" and saved the FrcExport files their names were "FrcExport_312" and "FrcExport_623". Those are the pixel positions that were given as default. Additionally, the modulus I calculated from those curves are in no agreement with the values in the DMTModulus image. Now I don't know if these forces are the ones I want or if I got two other force curves. How can I check that?

Yours sincerely, Dietmar Haba

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Bruker Employee

Hi Dietmar,

  1. What version of Nanoscope Analysis are you using?  You can find this in the Help>About... menu.
  2. Did you enter the curve numbers or did you move the cursors to selelct the curve numbers?
  3. When you do the export, are you exporting as "2 time-evenly spaced curves in each file"?

I tried to repeat your results with the latest version and failed, so any other ideas about what might be going on would help a lot!

--Bede

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