(A) Workflow for unsupervised grid navigation and imaging. SmartScope handles specimen exchange, atlas acquisition, regions of interest (ROIs) identification, classification, and selection. It then …
Steps carried out by SerialEM (Mastronarde, 2005) are shown in purple and steps carried out by SmartScope are shown in blue. Steps that can be modified within the sessions setup or during the …
(A) The hole grouping function takes the coordinates of the holes in a square, the maximum grouping radius in microns and a minimum group size. The algorithm uses the distance matrix between the …
SmartScope uses a database server (MariaDB), a web application with a web server and a Worker that runs the main processes. Each component can be installed on different computers and communicate …
Sample images show the performance of the square (A) and hole (B) detectors applied to gold (left) and carbon (right) grids. (A) Automatic detection of squares and classification into six different …
Atlas (A) and Windows (B) where manually picked and annotated. The annotated dataset was augmented using a combination of rotations, translations, contrast variations, magnifications, and flip …
Screening rates with and without beam-image shift (BIS) were 1.0 and 0.7 holes per minute, respectively (RANSAC regression). The red arrow indicates the time of specimen loading and start of atlas …
(A) Atlas of the specimen (left), typical micrograph (center) with some particles picked (purple circles) and 2D classes of POLG2 (right). (B) Resulting map of POLG2 colored by local resolution …
(A) Screening of the specimen used for POLG2 data collection. Sample images from screening are shown. Data can be labeled from the webUI as a way to keep track of the quality. The holes will be …
POLG2 (EMD-25764) | |||
---|---|---|---|
No tilt | Tilted | Combined | |
Hardware | |||
Microscope | Talos Arctica (Thermo Fisher) | ||
Detector | K2 summit (Gatan Inc) | ||
Data collection and processing | |||
Magnification | 45,000 | ||
Voltage (kV) | 200 | ||
Electron exposure (e-/Å2) | 50 | ||
Defocus range (µm) | 1.2–1.8 | 1.4–1.6 | 1.2–1.8 |
Tilt angle (°) | 0 | 30 | |
Pixel size (Å/pixel) | 0.932 | ||
Movie No. | 3029 (70%) | 1282 (30%) | 4311 |
Symmetry imposed | C2 | ||
Final particles | 99,189 (78%) | 28,641 (22%) | 127,860 |
Map resolution (unmasked) | 3.7 | 4.3 | 3.7 |
Map resolution (masked) | 3.5 | 3.9 | 3.4 |
FSC threshold | 0.143 | ||
Data collection statistics | |||
Setup time* (min) | 60 | ||
Throughput (movies/hr) | 117.9 | ||
Throughput last hour (movies) | 130 |
Data collection times are calculated as the time needed from grid loading to the collection of the first 50 high-magnification targets minus the time required for these 50 targets to be acquired.
Parameter names and their description. This is presented as a form on the web interface. These parameters can also be updated during the imaging process.
Parameter name | Description |
---|---|
General session parameters | |
Session name | Name of the microscopy session |
Group | Group name of the microscopy session. Usually the principal investigator’s name |
Microscope | Microscope being used for the session |
Detector | Detector being used for the session |
Collection parameters | |
Atlas X | Number of tiles for the Atlas acquisition in X axis (default: 3) |
Atlas Y | Number of tiles for the Atlas acquisition in Y axis (default: 3) |
Square X | Number of tiles for the window acquisition in X axis (default: 1) |
Square Y | Number of tiles for the window acquisition in the Y axis (default: 1) |
Squares num | Number of windows to be selected for high-magnification imaging (default: 3) |
Holes per square | Number of targets per windows to be selected for higher-magnification imaging. If 0 is entered, all targets will be selected and data collection mode will be enabled (default: 3) |
BIS max distance | Beam-Image shift grouping radius in microns (default: 0) |
Min BIS group size | Smallest Beam-Image shift group size to be considered (default: 1) |
Target defocus min | Lower end of the defocus range for rolling defocus in microns (default: –2) |
Target defocus max | Higher end of the defocus range for rolling defocus in microns (default: –2) |
Defocus step | Step by which the defocus is varied between each target group (default: 0) |
Drift crit | Drift threshold to be met during the drift settling procedure before proceeding with high-magnification imaging. Use –1 to disable (default: –1) |
Tilt angle | Tilt angle to use for high-magnification imaging. Works with BIS enabled (default: 0) |
Save frames | Whether to save the movie frames or return aligned sum (default: Saving enabled) |
Zeroloss delay | Time delay in hours for zero loss peak refinement. Only useful if the microscope has an energy filter. Use –1 to deactivate (default: –1) |
Offset targeting | Enable random targeting off-center to sample the ice gradient and carbon mesh particles. Automatically disabled in data collection mode (default: enabled) |
Offset distance | Override the random offset by an absolute value in microns. Can be used in data collection mode. Use –1 to disable (default: disabled) |
Autoloader (1 per grid) | |
Name | Name of the grid |
Position | Position in the autoloader |
Hole type | Grid hole spacing type (i.e. R1.2/1.3) |
Mesh size | Grid mesh size and spacing (i.e. 300) |
Mesh material | Grid mesh material (i.e. carbon or gold) |
These settings serve as guidelines and will need to be adapted for different hardware combinations. Updated versions of this table can be found at github.com/NIEHS/SmartScope.
Example 1 | Example 2 | |
---|---|---|
Instrument | ||
Microscope | Talos Arctica | Titan Krios G4 |
Detector | Gatan K2 Summit | Gatan K3 |
Energy filter | - | Gatan BioContiuum |
Low Dose Presets | ||
Search | ||
Magnification | 210 | 580 |
Pixel size (Å/pixel) | 196 | 152 |
Mode | Linear | Counting |
View | ||
Magnification | 2600 | 8700 |
Pixel size (Å/pixel) | 16.1 | 10.1 |
Mode | Linear | Counting |
Focus/record | ||
Magnification* | 36,000 | 81,000 |
Pixel size (Å/pixel)* | 1.19 | 1.08 |
Mode | Counting | Counting |
Full grid montage presets | ||
Magnification | 62 | 135 |
Pixel size (Å/pix) | 644 | 654 |
Mode | Linear | Counting |
These are the presets that are used for screening. They can be changes to suit the requirement for the specimen.
Feature type | Small | Cracked | Dry | Contaminated | Good | Partial |
---|---|---|---|---|---|---|
Without augmentation | 65.7% | 71.6% | 77.9% | 46.7% | 77.7% | 45.0% |
With augmentation | 73.4% | 76.5% | 79.4% | 50.41% | 81.2 % | 45.5% |
All grids were collected using a K2 detector. The default parameters for screening mode are a 9-tile atlas, 3 squares and 3 holes per square.
Total specimens = 981 | min | max | mean | median | standard deviation |
---|---|---|---|---|---|
Squares sampled | 0 | 7 | 2.5 | 3.0 | 1.3 |
Holes sampled | 0 | 33 | 8.0 | 9.0 | 5.0 |
Time spent on specimen (min) | 3.0 | 56.3 | 21.0 | 20.9 | 8.4 |
All grids were collected using a K2 detector. The default parameters for screening mode are a 9-tile atlas, 3 squares and 3 holes per square.
Total specimens = 772 | min | max | mean | median | standard deviation |
---|---|---|---|---|---|
Square sampled | 1 | 7 | 3.0 | 3.0 | 0.9 |
Hole sampled | 1 | 33 | 9.4 | 9.0 | 4.0 |
Time spent on specimen (min) | 9.1 | 56.3 | 23.0 | 21.7 | 6.7 |
Start times are at the start of specimen loading in the column. All grids were imaged using a K2 detector.
Total specimens = 58 | min | max | median | mean ± SD |
---|---|---|---|---|
Total micrographs | 565 | 8333 | 1626 | 2155±1502 |
Holes per hour | 51 | 141 | 100 | 100±22 |
Data collection setup time (min)* | 6 | 80 | 32 | 34±17 |
Setup time per 1000 micrographs (min) | 4 | 55 | 17 | 19±11 |
Data collection times are calculated as the time needed from grid loading to the collection of the first 50 high-magnification targets minus the time required for these 50 targets to be acquired.