a protective coat of 30 Â 30 µm with a thickness of 1 µm would take 15–20 min
with 1 nA FIB current. The thickness of deposition with the same FIB current will
be less over a larger area. If while performing the protection coat, the FIB is milling
rather than depositing, lower the current and/or reduce the size of the area.
Create a rough trench to expose the cross-section imaging surface (Fig. 5.6f and
h, T ). The imaging surface will be perpendicular to the sample top surface as
illustrated in Fig. 5.6f, h. Generally the trench is created with 15–30 nA milling
currents using a trapezium shape (Fig. 5.6h, T ). Draw a trapezium for the first
rough mill so that there is some distance between the protective coat and trench
(Fig. 5.6h, T ). The depth of the trench (Fig. 5.6h, D) is dependent on the size of the
imaging FOV. The rule of thumb is to create a trench that is three times deeper than
the aimed imaging depth. For example, a cell that is 10 µm in depth, would need a
milled trench that is 30 µm deep. The height of the trench (Fig. 5.6h, H ), which
together with the depth determines the slope of the trench, should not shadow the
imaging surface. In this example, this would be roughly 40 µm high (Fig. 5.6h, H ).
When working with complex tissue it may be possible to avoid the trench by
trimming the sample at the microtome so that the imaging surface is already
exposed (Fig. 5.6a–e) [37, 75, 79, 80]. The last stage of sample preparation is the
final polish (Fig. 5.6h, P, outlined in white) of the imaging cross section. Typically
the polished volume has the same width and depth as the rough trench, but only
1–3 µm in height and uses a smaller current (2–7 nA). There should be some
overlap with the trench so that a wall is not created between the trench and newly
polished surface.
Make the final adjustments to the SEM (lower keV, EsB detector, brightness and
contrast, focus, astigmatism and wobbler) and check the coincidence point. SEM
imaging parameters and FOV are chosen to optimize for the best SNR and resolution. Select the area for the acquisition, where the FIB will mill thin slices at the
chosen thickness. For an isotropic resolution of 8 nm then each slice would be
8 nm in z over the 30 µm ROI and the pixel size would also be 8 nm. FIB currents
in the range of 600 pA-2 nA are used for this step. The sample is now prepared for
the final mill and image acquisition.
Allow any thermal or mechanical drift to dissipate by leaving the room for
around 2 h [75]. Further operations are managed remotely. Check the positioning
of the milling area, and ensure there is a small overlap with the previous mill
(polishing) so that a wall is not created. Start mill and image with a low dwell time
(1–3 µs) over the chosen FOV and when it is milling uniformly slowly increase the
dwell time until the required image quality is achieved with consistent milling. This
should occur provided that the sample is mounted securely and time has been given
to stabilise. If it is not, then first adjust the image acquisition time. The best results
are obtained when the imaging time is similar to milling time [34]. Data collection
is limited by the slow speed of SEM image acquisition and to a lesser extent the ion
beam milling [34]. Imaging a 10 µm (in the milling direction indicated by black
arrow in Fig. 5.6f, h, and i) portion of a cell with xy FOV of 20 Â 20 µm, dwell
time 10–12 µs per pixel at 3D isotropic resolution of 8 nm takes approximately
24 h using Atlas 3D [34]. For FIB-SEM, the balance between the two beams and
140
R. I. Webb and N. L. Schieber
with 1 nA FIB current. The thickness of deposition with the same FIB current will
be less over a larger area. If while performing the protection coat, the FIB is milling
rather than depositing, lower the current and/or reduce the size of the area.
Create a rough trench to expose the cross-section imaging surface (Fig. 5.6f and
h, T ). The imaging surface will be perpendicular to the sample top surface as
illustrated in Fig. 5.6f, h. Generally the trench is created with 15–30 nA milling
currents using a trapezium shape (Fig. 5.6h, T ). Draw a trapezium for the first
rough mill so that there is some distance between the protective coat and trench
(Fig. 5.6h, T ). The depth of the trench (Fig. 5.6h, D) is dependent on the size of the
imaging FOV. The rule of thumb is to create a trench that is three times deeper than
the aimed imaging depth. For example, a cell that is 10 µm in depth, would need a
milled trench that is 30 µm deep. The height of the trench (Fig. 5.6h, H ), which
together with the depth determines the slope of the trench, should not shadow the
imaging surface. In this example, this would be roughly 40 µm high (Fig. 5.6h, H ).
When working with complex tissue it may be possible to avoid the trench by
trimming the sample at the microtome so that the imaging surface is already
exposed (Fig. 5.6a–e) [37, 75, 79, 80]. The last stage of sample preparation is the
final polish (Fig. 5.6h, P, outlined in white) of the imaging cross section. Typically
the polished volume has the same width and depth as the rough trench, but only
1–3 µm in height and uses a smaller current (2–7 nA). There should be some
overlap with the trench so that a wall is not created between the trench and newly
polished surface.
Make the final adjustments to the SEM (lower keV, EsB detector, brightness and
contrast, focus, astigmatism and wobbler) and check the coincidence point. SEM
imaging parameters and FOV are chosen to optimize for the best SNR and resolution. Select the area for the acquisition, where the FIB will mill thin slices at the
chosen thickness. For an isotropic resolution of 8 nm then each slice would be
8 nm in z over the 30 µm ROI and the pixel size would also be 8 nm. FIB currents
in the range of 600 pA-2 nA are used for this step. The sample is now prepared for
the final mill and image acquisition.
Allow any thermal or mechanical drift to dissipate by leaving the room for
around 2 h [75]. Further operations are managed remotely. Check the positioning
of the milling area, and ensure there is a small overlap with the previous mill
(polishing) so that a wall is not created. Start mill and image with a low dwell time
(1–3 µs) over the chosen FOV and when it is milling uniformly slowly increase the
dwell time until the required image quality is achieved with consistent milling. This
should occur provided that the sample is mounted securely and time has been given
to stabilise. If it is not, then first adjust the image acquisition time. The best results
are obtained when the imaging time is similar to milling time [34]. Data collection
is limited by the slow speed of SEM image acquisition and to a lesser extent the ion
beam milling [34]. Imaging a 10 µm (in the milling direction indicated by black
arrow in Fig. 5.6f, h, and i) portion of a cell with xy FOV of 20 Â 20 µm, dwell
time 10–12 µs per pixel at 3D isotropic resolution of 8 nm takes approximately
24 h using Atlas 3D [34]. For FIB-SEM, the balance between the two beams and
140
R. I. Webb and N. L. Schieber