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AFM color scale conversion factor

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tadayyon posted on Mon, Jan 17 2011 4:20 PM

Can someone let me know how to find the conversion factor from the color scale (pixel intensity) to height? I capture an image of AFM topography and use it in different software. I have to convert the pixel intensity back to height again. I have a BioScope Catalyst machine and Nanoscope 8.0 software.

Thanks

Seyed Tadayyon

 

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Does your ROI have enough channels to cover all of the displayed layers?  The exported AFM image is created from the non- overlay images in the ROI.  The non-overlay images in the ROI are determined by the top N displayed layers on the canvas (where N is determined by the ROI configuration dialog and is <=8).

You can try this:

  1. Hide all of the layers in the canvas except one AFM image channel and one channel from the optical image.  If you have done the import correctly, you should be able to change the color table of the optical image by selecting the optical layer in the tree and then clicking on the color tab.
  2. Create a new ROI.  I think the default is to have two non-overlay images and the overlay image, so both of your layers should be showing.
  3. Do the Export as you described above.  It should create an image with two data channels which you can then read back into Nanoscope or Nanoscope Analysis

Good luck!

--Bede

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In Nanoscope Analysis, you can copy and paste cursors between different channels as long as they have the same analysis on them.  Try this:

  1. Load up your image and do a section on both of the channels that you want sections from.
  2. Draw a section cursor on one of the channels
  3. right-click on the channel tab on the left and select "Copy cursor"
  4. right-click on the other channel tab and select "Paste cursor"

The cursors should be in exactly the same position in both of the channels allowing you to see a section in exactly the same area.  If you need to plot them on the same plot, you will need to export the section data to a text file and plot it up in Excel or something.  The export function can be found by right-clicking on the plot.

--Bede

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The conversion depends on the color table, the data scale, the offset and contrast.  Most of the color tables are not linear, but color table 25 is an exception to this rule.  I would suggest starting out with an image of a calibration standard setting the color table to 25 and the data range to something like -200 to +200nm (assuming a 200nm deep standard).  The data should be linearly mapped so that the -200nm is black (R,G,B=0,0,0) and the +200 is white (R,G,B=255,255,255).  Right clicking on the image and selecting Export>Screen Display does this nicely.  Using the export from the browser causes the color table to be applied differently (there are no offset and contrast for example).

Hope that helps!

--Bede

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tadayyon replied on Tue, Jan 18 2011 12:38 PM

Your explanations helped me in terms of understanding how the color scale works and that it’s not as simple as I thought. However, it’s too much work for what I am going to do. I have to think of something else then.

 

What I want to do is to get a line profile of AFM topography and plot it with the line profile of an optical image I overlaid on using MIRO. I can get the ASCII file of the AFM profile but I need to know the pixel coordinates of the profile line (start and end) so that I can use those coordinates in a different software to draw the same line for the optical image. Therefore, the question is that “is there a way to know the pixel coordinates of the start and end of the profile line?”.

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Probably the best way to do this would be to create a Region Of Interest (ROI) on the MIRO canvas with both the AFM and optical channels displayed.  You should be able to export the ROI to a Nanoscope format AFM image with both AFM and optical channels.  You can then load up the exported image and use the usual Nanoscope Section line on both the AFM and optical channels (which will be precisely registered).  If you are using Nanoscope Analysis, check out the copy cursors functionality to make sure the section is at the same location.

--Bede

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The export function (top blue icon) exports only AFM channel but not the optical. I created the ROI and then "export image" --> "Single AFM File (First 8 Channels)" --> then clicked on "Export Layer Images". The saved file has only the AFM image but not the optical. Please let me know if I am not doing it right.

Seyed

 

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The trick is that the optical image must be imported using either the "Split to 3 channels" option or the "To gray" option in the import configuration dialog.  If you import as "Bitmap (1 channel)" the optical image cannot be saved to a Nanoscope image because there is no way to map it to 16 bits.  Other than that you are doing the export correctly.

--Bede

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tadayyon replied on Fri, Jan 21 2011 11:05 AM

I've tried both options you mentioned for import but the result is still the same. Is there any other trick that may seem obvious to you I am missing?

Seyed

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Does your ROI have enough channels to cover all of the displayed layers?  The exported AFM image is created from the non- overlay images in the ROI.  The non-overlay images in the ROI are determined by the top N displayed layers on the canvas (where N is determined by the ROI configuration dialog and is <=8).

You can try this:

  1. Hide all of the layers in the canvas except one AFM image channel and one channel from the optical image.  If you have done the import correctly, you should be able to change the color table of the optical image by selecting the optical layer in the tree and then clicking on the color tab.
  2. Create a new ROI.  I think the default is to have two non-overlay images and the overlay image, so both of your layers should be showing.
  3. Do the Export as you described above.  It should create an image with two data channels which you can then read back into Nanoscope or Nanoscope Analysis

Good luck!

--Bede

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Thanks for all your helps. It worked and now I can create AFM topography and optical images as a two channel AFM format. My purpose of doing this was to have the optical image with the AFM so that I could draw a section line in one image which could be synchronized on the other. However, it seems that the Nanoscope software is not capable of doing it. It can do the sectioning of the image one at a time. Please let me know if it’s otherwise.

  • | Post Points: 12
Top 10 Contributor
280 Posts
Points 6,219
Bruker Employee
Verified by Ben Ohler

In Nanoscope Analysis, you can copy and paste cursors between different channels as long as they have the same analysis on them.  Try this:

  1. Load up your image and do a section on both of the channels that you want sections from.
  2. Draw a section cursor on one of the channels
  3. right-click on the channel tab on the left and select "Copy cursor"
  4. right-click on the other channel tab and select "Paste cursor"

The cursors should be in exactly the same position in both of the channels allowing you to see a section in exactly the same area.  If you need to plot them on the same plot, you will need to export the section data to a text file and plot it up in Excel or something.  The export function can be found by right-clicking on the plot.

--Bede

  • | Post Points: 12
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tadayyon replied on Thu, Jan 27 2011 12:13 PM

Perfect! Thank you very much.

Seyed

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