If you want to open gating plot you can click on the 'New (all cells)' item in the 'Gating' menu. Then scatter chart for original post-segmentation FCS will be opened below previous opened scatter charts.
You can select any column as x/y axes in corresponding combo boxes.
Log10 / Arcsinh scales
Also you can set logarithmic or arcsinh scale for both axes by clicking on corresponding radio buttons
t-SNE is calculated by the CODEX Processor and it can be opened in MAV gates. Select tsne_x and tsne_y for X and Y respectively, using linear scaling.
To view t-SNE results via Gating interface then you should click on the 'Open' item in the 'Gating' menu
Selecting FCS file wth T-SNE results
Then choose FCS file that contains T-SNE results. By default it is located at the base FCS folder.
Change X axis to tSNEX
When scatter chart is opened, change X and Y axes to tSNEX and tSNEY.
T-SNE results on scatter chart
For creating a gate you are able to choose between three drawing options: rectangle, polygon, oval. Then you can draw a gate on the scatter chart with a mouse cursor.
Create new gate
After releasing the mouse a new gate will be added to the gating table. Each gate has a name, color and can be selected or not. Also you can deleted gate by clicking on the 'trash' button.
Note: if you want to erase some gate, you can click on this gate and then press 'DEL' key. If you want to cancel gate creation, you can press 'ESC' key.
Create a population
Any changes on the gate will be affected to the population. For example:
- Setting a new color
- Changing the gate bounds
- Moving gate from one position to another
- Deletion of gate will lead to deletion of related population
Create a child plot
You can create a gating hierarchy by adding child scatter plot to any selected gate.
Then we are able to see a new scatter chart for the current gating on the right of parent scatter chart. These scatter charts are connected with an arrow, which shows the parent-child relationship. If you change shape/position of the parent gate, then these changes will be propagated to the downstream gating plots.
Disabled options for the parent gate are:
- Changing axis
If you close a scatter chart panel, then this and it's child plots (hierarchy) will be also closed
You can change scatter chart scales by entering new min and max x/y in corresponding text fields and then clicking on the '+lens' button.
You can always return to default scales by clicking on the '-lens' button.
Coloring the plot
You can color the points on the scatter charts by density / marker / population. By default all scatter charts are colored by density with rainbow color scheme where blue means lower density and red means greater density.
Color points by marker
If you will color points by any marker then points will be colored by rainbow color scheme where blue means lower marker value and red means greater marker value.
Color points by population
Coloring by population means that the points will be colored by selected populations colors. All other points, that not belong to any selected population, will be colored with gray color.
All regions mode
All regions mode
If you want to see the cells from the all regions in one plot you can click on the 'All regions' button. In this mode you can't change region and all gates that you will draw will contain cells from all regions.
Color points by region
When 'all regions' mode is active you are able to select coloring option 'By region'. After that, all points in the scatter chart will be painted with the color of the corresponding region. All points that contain cells from many regions will be colored in gray color.
Save gating plots
Copy gating plot to the clipboard
You can copy gating any plot to the clipboard by clicking on the corresponding button.
Export gating plot
Also you can click on the 'Export' button and then choose destination directory for exported PNG file.
Save/copy gating hierarchy
For saving whole gating hierarchy you can click on the 'Copy' and 'Save' items in the 'Gating' menu.
Saved gating hierarchy
Save as scheme
Another option is to save whole gating hierarchy as a scheme. This option gives you the next advantages:
- 1.Gating hierarchy will be save to the GatingScheme JSON file.
- 2.You will be able to open the saved GatingScheme JSON file in any experiment. In this case: a. Only gating plots for existing markers in the current experiment will be opened. b. Not opened plots will remain in the GatingScheme JSON file. c. Gates will be in the same positions in which they were saved, even if the markers in the current experiment have different intensities. (Gate bounds are normalized, see example)
- 3.You will be able to update the opened GatingScheme JSON file.
Gating scheme saved in the U54.Nolan experiment
The same gating scheme opened in the Human.Tonsil experiment
There is a lock scales button in each scatter chart panel. If you click on this button, then current range will be applied to all existing plots. And as long as the 'lock scales' feature is enabled, any range that you will specify will be applied to all existing plots. Even if you change some axis, locked range will stay.
If you want to see frequency of some column, then you can select 'Frequency' column for the Y axis. It will activate histogram mode.
In this mode you are able to select kernel smoothing with predefined options for kernel size.
Also you are able to select Column chart option. Column chart differs from the common histogram only by number of bins.
If you want to zoom some are in the gating plot, then you should draw a selection using right mouse button. After that, scatter chart range will be updated with the range of the selection you drawn. You can always return to the default zoom level by right click.