9. To calibrate the diffraction stigmator, adjust the C2 lens intensity. A fully condensed beam sits at a crossover point: adjusting
the C2 intensity in either direction should result in an expansion. The beam should expand and collapse concentrically as a
nearly circular ellipse. Shape distortions should be corrected by
adjusting the console multifunction knobs while the stigmator
adjustment icon is active.
10. While the XF416 detector is capable of full-chip readout at
nearly 50 frames/s, not all applications warrant this readout
speed. In particular, certain applications such as shutter-free
data collection may benefit from slower readouts that limit the
accumulation of readout noise. Likewise, spatial subsampling
or binning of the collected frames can enhance signal over
noise. The collection strategies described here rely on 2Â2
binning of full-frame diffraction images, but larger binning
can be employed if desired, with a caveat. Binning reduces
sampling of the reciprocal lattice and therefore constrains measurements, as lattice spacings shrink and reflections risk being
overlapped. This is also influenced by the selected camera
length as described in the following note.
11. The ideal detector distance places the finest resolution reflections at or near the edge of the detector, maximizing the
collection of reflections at both coarse- and fine-resolution
shells. Crystals with larger unit cell dimensions will exhibit a
more compact reciprocal lattice and require longer detector
distances to prevent overlapping spots from affecting
integrated intensities, which is influenced by detector sampling
of the reciprocal lattice.
12. Visual obstruction of the sample by grid bars, other crystals, or
debris is commonplace at high tilts, effectively limiting the
upper and lower bounds of rotation. Keep in mind that as the
x- and y-axes approach the stage’s outer boundaries, high-angle
tilts become more likely to cause physical collisions between
the microscope and the sample holder.
13. Coordination of stage rotation with diffraction data collection
requires intervention. The software module controlling stage
rotation can be accessed through the Temspy dialog box,
under dialogs, CompuStage. The parameters for saving
recorded movies can be set by accessing the Recorder Settings
dialog box from the toolbar menu. Video capture occurs by
pressing the camcorder icon on the EMMenu4 user interface
after having set a desired integration time per frame. A second
icon (red record button on the EMMenu4 user interface)
allows for storage of the captured video frames when pressed
as video is being captured.
MicroED of Small Macromolecules
343
the C2 intensity in either direction should result in an expansion. The beam should expand and collapse concentrically as a
nearly circular ellipse. Shape distortions should be corrected by
adjusting the console multifunction knobs while the stigmator
adjustment icon is active.
10. While the XF416 detector is capable of full-chip readout at
nearly 50 frames/s, not all applications warrant this readout
speed. In particular, certain applications such as shutter-free
data collection may benefit from slower readouts that limit the
accumulation of readout noise. Likewise, spatial subsampling
or binning of the collected frames can enhance signal over
noise. The collection strategies described here rely on 2Â2
binning of full-frame diffraction images, but larger binning
can be employed if desired, with a caveat. Binning reduces
sampling of the reciprocal lattice and therefore constrains measurements, as lattice spacings shrink and reflections risk being
overlapped. This is also influenced by the selected camera
length as described in the following note.
11. The ideal detector distance places the finest resolution reflections at or near the edge of the detector, maximizing the
collection of reflections at both coarse- and fine-resolution
shells. Crystals with larger unit cell dimensions will exhibit a
more compact reciprocal lattice and require longer detector
distances to prevent overlapping spots from affecting
integrated intensities, which is influenced by detector sampling
of the reciprocal lattice.
12. Visual obstruction of the sample by grid bars, other crystals, or
debris is commonplace at high tilts, effectively limiting the
upper and lower bounds of rotation. Keep in mind that as the
x- and y-axes approach the stage’s outer boundaries, high-angle
tilts become more likely to cause physical collisions between
the microscope and the sample holder.
13. Coordination of stage rotation with diffraction data collection
requires intervention. The software module controlling stage
rotation can be accessed through the Temspy dialog box,
under dialogs, CompuStage. The parameters for saving
recorded movies can be set by accessing the Recorder Settings
dialog box from the toolbar menu. Video capture occurs by
pressing the camcorder icon on the EMMenu4 user interface
after having set a desired integration time per frame. A second
icon (red record button on the EMMenu4 user interface)
allows for storage of the captured video frames when pressed
as video is being captured.
MicroED of Small Macromolecules
343