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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.
Hi all,
In relation to this question, what would be the advantages of a .hsdc line file captured over the nucleus of a cell in addition to the area surrounding the nucleus versus the point and shoot method (where I would draw a box over the nucleus and take 20 indentation points for example).
Which method would be the most accurate in determining the modulus of the cell? Regards, Iman.
Dear user,
The HSDC file will allow you capturing a very high number of force curves (up to 5400) on the same line. The line position is defined by the user. In general, people image entire cells so the nucleus portion of the line can very well be the center part, the rest being cytosol and organeles. With Point & Shoot, you can (just by capturing an OPTICAL image) select various locations where to make force measurements, using a single point, several customized points, a line, or a matrix (number of rows and columns defined by the user).
But whateevr the technique you chose, both types of curves can be exported and analysed in Nanoscope Analysis:
1) open one representative curve.
2) modify the tip parameters if needed.
3) correct the baseline.
4) select a model and extract the Young's modulus.
5) use the batch processing feature to apply those changes to all your curves.
You will end up with the average Young's modulus and the standard deviation.
Best,
Alex.
I forgot to add that both techniques have their advantages and drawbaks: in your specific case, if you want to get the YM for different organeles or cell parts (nucleus, mitochondria,...), I think that the P&S option is more accurate. I mean that with this one, it's probably easier to target the right place, either by making single shoots or by drawing a box.
Hi Alex,
Thank you for your useful advice. Could you please check if the procedure we are using is correct for opening a representative curve? We open the .hsdc file and select the deflection error channel (not the height channel) and load the associated image. From this, we select the curve we want and right click and select export XZ data. We then paste the data from this file into Excel and plot the graph. When you say correct the baseline, do you mean if the Force (nN) starts at say -10 nN, we add 10 to all the data points in the y-axis? Or have I got it totally wrong? We are lost in the method you have provided. We tried to look it up in Nanoscope Analysis' help file for "QNM Force Curve procedure" but there was a problem with accessing this help file. I presume from your suggestion that we can correct the baseline in Nanoscope Analysis and select a model to extract the Young's modulus.
Thank you for your help.
Hi Iman,
Exactly. The procedure you described herup is correct but I think you just don't have the latest version of Nanoscope Analysis. This one allows you, among others, to correct the base line (coming back to you remark: yes, I mean having a totally flat base line), modify probe info (tip radius,...) or other parameters like the Poisson's ratio, chose a fit model (like Sneddon), batch process,...
You should download it. It will make your life much easier!
Thank you for your response. Could you please tell me what is the difference between DMT modulus and Young's modulus?! The Nanoscope analysis gives us the cell stiffness (from DMT channel) in DMT modulus whenever we measure the cell stiffness. However, when we do the HSDC we can extract the Youn'g modulus from the obtained curves. Am I right?
Is there going to be any difference between the numbers we get from these two methods? I mean in biological samples.
Thank you.
Regards,
Iman
You are right, Iman. DMT stands for Derjaguin Mueller and Toporov. The DMT theory is one of the possible contact theories to define how the AFM probe interacts with the surface. I can give you more details if you need but to make it short, it fits well when samples are not too compliant (like high density polymers) but is not reallu well adapted to biological samples. DMT is the default fit in Nanoscope probably because originally, Peak Force Tapping was not developped to work on biological samples. But now that we figured out that it also works quite well on bio samples, I agree that we need other possible fits. This will come very soon.
But coming back to your remark, yes, one of hte best way to extract force curves is to make HSDC files and then re-process the force curves by using Nanoscope Analysis (you can select Stiffness, Hertz, or Sneddon). You should not use DMT if you work on biological samples.
Simply speaking, DMT modulus stands for the elastic modulus calculated from DMT method (can also considered as young's modulus calculated from DMT method), and DMT method is the extended Hertzian method considering adhesion. So you can obtain DMT modulus from single force curve analysis by selecting 'including adhesion' and 'Hertzian model fitting method', then the offline calculated young's modulus should be the same as DMT modulus shown in real time.
And as Alex pointed out, the assumption of DMT model may not apply if a nonsphere probe is used, usually the indentation is pretty deep and contact geometry can't be assumed as sphere anymore, so snedden model is often more appropriated, though not perfect yet.
Ang Li
One quick comment: after making HSDC files and exporting the corresponding force curves, the files can be processed in Nanoscope Analysis: the SW will give you 3 possible options in terms of contact theories:
- Stiffness.
- Hertz model (colloidal probe).
- Sneddon model (cone of infinite length).
Also the batch processing feature makes the calculation much easier.
But something much better is coming up. Stay tuned!
Hi Guys,
Thank you for your useful responses. I have a problem opening my saved Hsdc. files. Every time I try to open a file, it opens up but when I click "QNM Hsdc Force Curve-Image" to open the file as it shown in help section, the software shows me this message:
'The QNMOffline needs either QNMOffline or HarmoniX or PeakForceQNM Key'.
Do you guys know where can I get the key from?! I really need to extract the Young's Modulus from my data ASAP.
Thank you for your support,
You can send me an email at Ang.li@bruker-nano.com, I will check it for you.
Dear Alex,
For extracting the Young's modulus from Hsdc. files, as you suggested I do the followings:
1) open one representative curve and export it.
2) then open the exported file and correct the baseline using the given option.
3) then select a model (Hertzian) and at this point I am not sure what is next!?
I drag the marker pairs over the curve and it shows me some numbers but they are not the Young's modulus I suppose! I do this using multiple line plot option.
Hi, the procedure sent to your email.
Yes, this is correct except that you should not use Hertz except if you are using colloidal probes. Sneddon is more appropriate. Then step 4) would be "batch processing".
Let me know if it works for you or if you have any problem.