[3DEM] microscope indicated defocus values
John Minter
jrminter at rochester.rr.com
Wed May 23 15:40:55 PDT 2007
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Paul Chipman asked how different labs handle the defocus calibration on
CM-series microscopes. Our lab has an older CM-20 with the latest version of
the software available from FEI. I had noticed that the nominal defocus
steps did not change with accelerating voltage. The deviations between
nominal defocus and true defocus are greater at higher accelerating
voltages. To make a long story short, I used several software packages
(Gatan's HREM plug-in, SPIDER scripts, EMAN's CTFIT, and Niko Grigorieff's
CTFIT3) to measure the defocus values at 200kV for a wide range of defocus
values (using 768 nm nominal increments with Step 5). All the software
analyses gave consistent results, so I used the Gatan values for further
analysis because they were faster to measure. A plot of measured defocus (in
nm) as a function of nominal defocus gave a monotonic curve with a slight
deviation from linearity. I used the JMP statistical software to fit a
second order polynomial though the data. The polynomial fit was cumbersome
to use in practice.
Over the range of defocus I was most interested, it was easier to use the
Gatan HREM software to calibrate the defocus step per change in DAC units. I
then wrote a custom low-dose DigitalMicrograph script to permit focus in the
CM "focus mode", changing the defocus by a specified amount, and then
switching to exposure mode, recording the image, and setting the whole
system back to the focus mode and zero defocus. Because I like to focus at a
higher magnification than that used for recording, the script permits a
constant defocus offset to correct for that. The source for this script is
included below. Please feel free to share suggestions and improvements.
number FocusToDac( number nDefocusMicron)
{
// Compute the number of DAC steps to achieve defocus
// Calibration of 3.889±0.038 nm/ct verified 13-Feb-06
number nFocusDacCalib = 0.003889; // [µm] focus change per DAC Unit
number nDacUnits; // step size in DAC units
nDacUnits = Round( nDefocusMicron / nFocusDacCalib);
return nDacUnits;
}
number SetSearchMode()
{
number nRet;
number nSearchKey = 6;
number nPresses = 1;
nRet = CMPressSoftKey(nSearchKey, nPresses );
return nRet;
}
number SetFocusMode()
{
number nRet;
number nFocusKey = 5;
number nPresses = 1;
nRet = CMPressSoftKey(nFocusKey, nPresses );
return nRet;
}
number SetExposeMode()
{
number nRet;
number nExposeKey = 4;
number nPresses = 1;
nRet = CMPressSoftKey(nExposeKey, nPresses );
return nRet;
}
void TakeLowDoseImage(void)
{
number nHardware = 1; // set to > 0 if we really have a camera
number nExposureTime, nTop, nLeft, nCcdSize;
number nDefocus, nDacUnits;
number dMag;
number nOffset;
nExposureTime = 1;
nTop = 0;
nLeft = 0;
nCcdSize = 2048;
number bRet = GetPersistentNumberNote("TakeLowDoseImage:Defocus",
nDefocus);
if (!bRet)
{
nDefocus = -1.0;
}
bRet = GetPersistentNumberNote("TakeLowDoseImage:Offset", nOffset);
if (!bRet)
{
nOffset = 0.0;
}
bRet = GetPersistentNumberNote("TakeLowDoseImage:Exposure",
nExposureTime);
if (!bRet)
{
nExposureTime = 1.0;
SetPersistentNumberNote("TakeLowDoseImage:Exposure", nExposureTime);
}
bRet = GetNumber( "Enter desired defocus", nDefocus, nDefocus );
if (bRet)
{
SetPersistentNumberNote("TakeLowDoseImage:Defocus", nDefocus);
nDacUnits = FocusToDac( nDefocus- nOffset);
bRet = SetExposeMode();
// let's double check the mag
dMag = CM( );
SetPersistentNumberNote("Microscope Info:Indicated Magnification",
dMag);
EMChangeFocus(nDacUnits);
image imgResult := SSCGainNormalizedAcquire( nExposureTime, nTop,
nLeft, nCcdSize, nCcdSize );
ShowImage(imgResult);
EMChangeFocus(-nDacUnits);
bRet = SetFocusMode();
TagGroup tg = imgResult.ImageGetTagGroup();
SetNumberNote( imgResult, "Microscope Info:Defocus_µm", nDefocus );
tg.TagGroupGetTagAsNumber( "Microscope Info:Defocus_µm", nDefocus );
Result("The image was recorded with a defocus of " + nDefocus + "
[µm].\n" );
}
return;
}
TakeLowDoseImage();
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