of GIS platinum as a longer deposition time at a further distance. The deposition stage position/coordinates can be
adjusted to allow long/short deposition times or can be
adjusted to be centered between the two grids such that both
the left and right grids are coated at the same time.
11. We have found that judging appropriate lamella thickness and
when to stop milling requires some experience and iterative
comparison between the SEM and TEM to “calibrate” the
user. One helpful exercise is to make a lamella, estimate its
thickness at the SEM, then measure the actual thickness from
a tomogram.
12. To manually find the beam-coincidence point, tilt to the
desired milling angle and go to a low mag view for both SEM
and FIB. Find your target in both SEM and FIB, and center the
target in the FIB view using XY moves. Switch to SEM view
and move the stage in Z so that the target is about halfway
closer to the center of the SEM field of view (e.g., if the target
was 100 μm away from the center of the SEM, move the stage
in Z so that the target is now about 50 μm away). Switch to the
FIB view and recenter the target using XY moves. Switch to
SEM and check positioning. Reiterate between XY moves in
FIB view and Z moves in SEM view until the target is centered
in both SEM and FIB. Alternatively, the proprietary MAPS
software package from TFS allows correlation between fluorescent, SEM, and TEM images. Additionally, MAPS may be used
as a microscope control program. This software package has
the ability to calculate eucentric height for a given point on a
sample, which should be very close to the beam-coincidence
point. To do so, add a lamella object, then go to its mapping
position. Select “Calculate Eucentric Height” which will ask
you to keep the object centered at several tilts. The specific
internal calibration for accurate eucentricity should be determined during software installation and kept for all users.
13. This apparent drift is not due to physical specimen movement,
but due to slight movement in the beam due to sample interaction. Especially at high FIB currents, the sample may acquire
charging that influences the FIB [48].
14. Step milling improves lamella stability by forming a gradual
transition from the unmilled material to the lamella. Without
step milling, the lamellae are more prone to developing cracks
at the edge where they connect to the bulk material.
15. The FIB view has a shallow depth of field, which can be
observed when the front of the lamella is in sharp focus, but
the back of the lamella is out of focus. This leads to heterogeneity in milling efficiency over the surface of the lamella. At the
TEM, this can be observed where the back of the lamella is
Practical Approaches for Cryo-FIB Milling
79
adjusted to allow long/short deposition times or can be
adjusted to be centered between the two grids such that both
the left and right grids are coated at the same time.
11. We have found that judging appropriate lamella thickness and
when to stop milling requires some experience and iterative
comparison between the SEM and TEM to “calibrate” the
user. One helpful exercise is to make a lamella, estimate its
thickness at the SEM, then measure the actual thickness from
a tomogram.
12. To manually find the beam-coincidence point, tilt to the
desired milling angle and go to a low mag view for both SEM
and FIB. Find your target in both SEM and FIB, and center the
target in the FIB view using XY moves. Switch to SEM view
and move the stage in Z so that the target is about halfway
closer to the center of the SEM field of view (e.g., if the target
was 100 μm away from the center of the SEM, move the stage
in Z so that the target is now about 50 μm away). Switch to the
FIB view and recenter the target using XY moves. Switch to
SEM and check positioning. Reiterate between XY moves in
FIB view and Z moves in SEM view until the target is centered
in both SEM and FIB. Alternatively, the proprietary MAPS
software package from TFS allows correlation between fluorescent, SEM, and TEM images. Additionally, MAPS may be used
as a microscope control program. This software package has
the ability to calculate eucentric height for a given point on a
sample, which should be very close to the beam-coincidence
point. To do so, add a lamella object, then go to its mapping
position. Select “Calculate Eucentric Height” which will ask
you to keep the object centered at several tilts. The specific
internal calibration for accurate eucentricity should be determined during software installation and kept for all users.
13. This apparent drift is not due to physical specimen movement,
but due to slight movement in the beam due to sample interaction. Especially at high FIB currents, the sample may acquire
charging that influences the FIB [48].
14. Step milling improves lamella stability by forming a gradual
transition from the unmilled material to the lamella. Without
step milling, the lamellae are more prone to developing cracks
at the edge where they connect to the bulk material.
15. The FIB view has a shallow depth of field, which can be
observed when the front of the lamella is in sharp focus, but
the back of the lamella is out of focus. This leads to heterogeneity in milling efficiency over the surface of the lamella. At the
TEM, this can be observed where the back of the lamella is
Practical Approaches for Cryo-FIB Milling
79