3.6.2 Collect Atlas
Switch to the “Atlas” tab and collect a low-magnification atlas of
the entire grid, starting from the center and spiraling out. This will
take about 10 min and the atlas should be used for all long-range
navigation of the grid. Right-click on any location on the atlas and
select “Move Stage Here”. The red cross-hair that indicates current
stage location will move to the new location on the atlas. Navigate
to and store the coordinates for a large hole in the carbon support.
Setting the dose will require imaging through a vacuum with
nothing in the beam path.
3.6.3 Set Eucentric
Height
Move to a grid square near the center of the grid, or near the center
of the grid-region where data will be collected. Then, in the
“Search/Template” mode, locate and center a visible feature.
Turn on the stage wobbler and adjust the stage Z-axis on the
control panels until lateral translations in the object are minimized
as the stage wobbles.
3.6.4 Grid Square
Exploration
Using the atlas, identify grid squares to further examine if they
contain neurons (see Fig. 4a, b). In the “Preparation” tab, select
the “Overview/Positioning” imaging mode from the drop-down
menu. Load the saved settings by clicking the “Set” button. Navigate to the center of each grid square of interest and collect an
image. This setting should produce an image of the entire grid
square, giving a broad overview of what the cells on the grid surface
look like. Save the stage coordinates for easy navigation back to
these locations at the next higher magnification.
3.6.5 Search
for Target Areas
In the “Preparation” tab, select the “Search/Template” imaging
mode from the drop-down menu and click “Set”. This magnification should be set to display the largest area of the grid possible,
where features of the cell are still identifiable as target areas for data
collection. For a neuronal branch, ~10 μm of neurite length, mitochondria, and other subcellular organelles can be used as target
guides (see Fig. 4c). Insert the objective aperture, if needed. Use the
stage coordinates saved during grid square exploration to revisit
specific grid squares of interest. Examine them in live view using the
K2. Move around the grid square using minimal exposure time and
dose to locate cellular regions of interest. Center these areas quickly
and save the stage coordinates (see Note 12). Repeat until each
target has been identified and locations saved.
3.6.6 Select Specific
Targets for Data Collection
In the “Preparation” tab, select the “Exposure” imaging mode
from the drop-down menu and click “Set”. This imaging mode
should be set to the data collection parameters. Recall the first stage
position saved as a target coordinate and switch to live view. Briefly
center the target and stop the live view to prevent further electron
dosing. If necessary, acquire an image for a final assessment of the
target region with better signal-to-noise. Centering targets at the
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Ryan K. Hylton et al.
Switch to the “Atlas” tab and collect a low-magnification atlas of
the entire grid, starting from the center and spiraling out. This will
take about 10 min and the atlas should be used for all long-range
navigation of the grid. Right-click on any location on the atlas and
select “Move Stage Here”. The red cross-hair that indicates current
stage location will move to the new location on the atlas. Navigate
to and store the coordinates for a large hole in the carbon support.
Setting the dose will require imaging through a vacuum with
nothing in the beam path.
3.6.3 Set Eucentric
Height
Move to a grid square near the center of the grid, or near the center
of the grid-region where data will be collected. Then, in the
“Search/Template” mode, locate and center a visible feature.
Turn on the stage wobbler and adjust the stage Z-axis on the
control panels until lateral translations in the object are minimized
as the stage wobbles.
3.6.4 Grid Square
Exploration
Using the atlas, identify grid squares to further examine if they
contain neurons (see Fig. 4a, b). In the “Preparation” tab, select
the “Overview/Positioning” imaging mode from the drop-down
menu. Load the saved settings by clicking the “Set” button. Navigate to the center of each grid square of interest and collect an
image. This setting should produce an image of the entire grid
square, giving a broad overview of what the cells on the grid surface
look like. Save the stage coordinates for easy navigation back to
these locations at the next higher magnification.
3.6.5 Search
for Target Areas
In the “Preparation” tab, select the “Search/Template” imaging
mode from the drop-down menu and click “Set”. This magnification should be set to display the largest area of the grid possible,
where features of the cell are still identifiable as target areas for data
collection. For a neuronal branch, ~10 μm of neurite length, mitochondria, and other subcellular organelles can be used as target
guides (see Fig. 4c). Insert the objective aperture, if needed. Use the
stage coordinates saved during grid square exploration to revisit
specific grid squares of interest. Examine them in live view using the
K2. Move around the grid square using minimal exposure time and
dose to locate cellular regions of interest. Center these areas quickly
and save the stage coordinates (see Note 12). Repeat until each
target has been identified and locations saved.
3.6.6 Select Specific
Targets for Data Collection
In the “Preparation” tab, select the “Exposure” imaging mode
from the drop-down menu and click “Set”. This imaging mode
should be set to the data collection parameters. Recall the first stage
position saved as a target coordinate and switch to live view. Briefly
center the target and stop the live view to prevent further electron
dosing. If necessary, acquire an image for a final assessment of the
target region with better signal-to-noise. Centering targets at the
34
Ryan K. Hylton et al.