the “focus” or “search” mode, while diffraction patterns can be
recorded with the beam at crossover in “exposure” mode on an
FEI microscope.
7. Find an appropriate object on the grid to use as a reference and
fine-tune the specimen’s eucentric height by adjusting the zheight while wobbling.
8. Diffract from a target crystal by (a) inserting the SAA;
(b) inserting and adjusting the beamstop; (c) returning the
beam to crossover; (d) capturing an initial diffraction pattern
with an exposure ranging from 0.1 to 10 s (see Note 10);
(e) repeating steps a to d as necessary to (1) assess the resolution to which the sample appears to diffract and (2) diagnose
whether or not this particular crystal diffracts well enough to
warrant collection of a full dataset.
9. Verify if the detector distance is appropriate for sample and
adjust if necessary, using the magnification knob (see Note 11).
10. Fine-tune the z-axis height in overfocused diffraction mode
while tilting the sample between the upper and lower limits
planned for data collection. The crystal should always remain
within the SAA throughout the desired tilt range (see
Note 12).
11. To collect diffraction movies as the stage rotates from one high
angle tilt to the other, initiate data recording on the camera
while independently setting a stage trajectory at a desired
rotation rate (see Note 13).
12. Repeat step 11 for subsequent data collection from different
samples (see Note 14).
Subheadings 3.4–3.6 provide detailed instructions to execute
data processing with select software programs (Fig. 3).
3.4 Data Conversion
and Processing
1. Use a script such as tvips2smv to convert raw movie files from
native “.tvips” format (Tietz Video and Image Processing Systems, generic output from TemCam detectors) or other proprietary format into a series of images in standard
crystallographic format (e.g., SMV, with a “.img” suffix) readable by XDS. Before executing conversion, specify the oscillation range and detector distance used for the dataset in the
command line [13].
2. Visualize the images using Adxv to cycle through the converted
frames. Select an appropriate image to use for identification of
the beam center (see Note 15) Repeat this process on several
different images to obtain a more precise value.
3. Generate or download an appropriate XDS input template
(http://xds.mpimf-heidelberg.mpg.de/html_doc/xds_pre
pare.html).
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Chih-Te Zee et al.
recorded with the beam at crossover in “exposure” mode on an
FEI microscope.
7. Find an appropriate object on the grid to use as a reference and
fine-tune the specimen’s eucentric height by adjusting the zheight while wobbling.
8. Diffract from a target crystal by (a) inserting the SAA;
(b) inserting and adjusting the beamstop; (c) returning the
beam to crossover; (d) capturing an initial diffraction pattern
with an exposure ranging from 0.1 to 10 s (see Note 10);
(e) repeating steps a to d as necessary to (1) assess the resolution to which the sample appears to diffract and (2) diagnose
whether or not this particular crystal diffracts well enough to
warrant collection of a full dataset.
9. Verify if the detector distance is appropriate for sample and
adjust if necessary, using the magnification knob (see Note 11).
10. Fine-tune the z-axis height in overfocused diffraction mode
while tilting the sample between the upper and lower limits
planned for data collection. The crystal should always remain
within the SAA throughout the desired tilt range (see
Note 12).
11. To collect diffraction movies as the stage rotates from one high
angle tilt to the other, initiate data recording on the camera
while independently setting a stage trajectory at a desired
rotation rate (see Note 13).
12. Repeat step 11 for subsequent data collection from different
samples (see Note 14).
Subheadings 3.4–3.6 provide detailed instructions to execute
data processing with select software programs (Fig. 3).
3.4 Data Conversion
and Processing
1. Use a script such as tvips2smv to convert raw movie files from
native “.tvips” format (Tietz Video and Image Processing Systems, generic output from TemCam detectors) or other proprietary format into a series of images in standard
crystallographic format (e.g., SMV, with a “.img” suffix) readable by XDS. Before executing conversion, specify the oscillation range and detector distance used for the dataset in the
command line [13].
2. Visualize the images using Adxv to cycle through the converted
frames. Select an appropriate image to use for identification of
the beam center (see Note 15) Repeat this process on several
different images to obtain a more precise value.
3. Generate or download an appropriate XDS input template
(http://xds.mpimf-heidelberg.mpg.de/html_doc/xds_pre
pare.html).
336
Chih-Te Zee et al.