When we collect BSE image in "Digitize Sample Positions On Image" window, save the image as BMP, and open the image from PictureSnap, the image is already automatically calibrated (2-points calibration).
Could you explain what data the system uses for automatically calibration the image:
information about central point coordinate and magnification of the image or something else?
Great questions.
The analog signal images acquired in Probe for EPMA in the Imaging and/or Digitize Image windows are calibrated based on the current (center) stage position and the magnification calibration which is stored in the probewin.ini file in the [Image] section as seen here for a typical JEOL instrument:
ImageInterfaceCalNumberOfBeamCalibrations="4"
ImageInterfaceCalKeV="20,20,15,15"
ImageInterfaceCalMag="200,400,400,1000"
ImageInterfaceCalXMicrons="454.5161,224.7639,226.9317,89.51028"
ImageInterfaceCalYMicrons="444.3432,222.1757,222.5682,89.19152"
ImageInterfaceCalScanRotation="0,0,0,0"
Note that the scan rotations were not calibrated.
I assume you are asking about this because when I was working with Ying Yu the other day I noticed that her Probewin.ini file only had a single magnification calibration at 400x. I mentioned to her that I suspect this was merely a nominal calibration and not calibrated specifically for her instrument.
To calibrate ones instrument, these image calibrations are performed using the Calibrate Electron Image calibration window which is accessed from the Imaging button window, which is in the Acquire! window. To perform these calibrations one should use a sample with a number of small but unique features which can be located in both the optical display and also visible in the acquired BSE or SE image. Once a suitable image has been acquired in the Imaging window, one should click on the little "@" button in the upper left of the image and the Calibrate Electron Image window is opened as shown in the previous post by myself.
In a perfect world, only a single calibration at a single magnification would be necessary. But we don't live in a perfect world!
I don't know the scan coil ranges on the JEOL 8200/8500 (where the magnification switches to different circuits), but ideally these different ranges are calibrated by your service engineer to obtain a smooth calibration as the magnification is changed. But since these hardware calibrations do drift out of adjustment over time, we have created this calibration adjustment capability in software. Here is an example from my SX100:
ImageInterfaceCalNumberOfBeamCalibrations="5"
ImageInterfaceCalKeV="15,15,15,15,15"
ImageInterfaceCalMag="1335,758,571,405,334"
ImageInterfaceCalXMicrons="282.546,498.7495,659.7579,926.9622,1118.275"
ImageInterfaceCalYMicrons="207.0935,376.3542,504.1712,705.2865,848.4153"
ImageInterfaceCalScanRotation="9.3,9.1,8.9,8.8,8.7"
The above example shows the scan rotation calibration changing as the magnification changes. This is not unusual, but what is a little unusual is that our scan coil is accidentally rotated about 9 degrees out of orthogonality. This scan rotation needs to be checked at each magnification using the edge of your sample holder to be sure you are orthogonal in X or Y. You'll also want to set the default scan rotation for your instrument (not available in the JEOL 8900) using this value in the [hardware] section of the Probewin.ini file:
ScanRotationPresent=1 ; non-zero = scan rotation interface present (Cameca SX100 and JEOL 8200/8500 only)
ScanRotation="9" ; default scan rotation value to send on startup (01-17-2011)
Then as mentioned above, acquire an image with two features on a diagonal (near two corners of the image) and proceed to locate the feature by moving your stage and then selecting the corresponding pixel using the mouse. Then clicking the Calculate Image Calibrate button followed by the Save Image Calibration to Probewin.ini if the calibration looks good.
Remember, to check your scan rotation for each image acquisition!
Generally one should calibrate two magnifications for each scan coil range. I've asked one of our consultants to write up a procedure for this process but this post should provide you with the general idea.
When your magnification values are sufficiently calibrated one should be able to utilize the Digitize Image feature and obtain accurate stage positions from your acquired images, with the possible exception of very low magnifications which exhibit significant distortion on most instruments.
Also be sure to utilize a slow scan speed for these magnification calibrations- generally the same slow scan speed you utilize for your Digitize Image acquisitions.
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