Filters (like flow cytometry)
Creating gated populations can help you create filtered populations to answer things like:
- How many large (area >1SD above the mean) and round (eccentricity > 1SD above the mean) organoids do I have? What percent are they? How does their average intensity compare to the broader population?
- What's the average size and count of double positive cells for CD8 and CD4?
There are multiple tools you can use to create gated populations. Simply click on the Create filter to see these tools.
You can gate populations manually by using the Rectangle, Oval, or Freeform tools.
After selecting Rectangle tool, gate around desired population to create.
Once you selected the population, you need to name that population and it will automatically be saved under population tree.
GFP+ population created by rectangle tool.
The By Statistics gating tool allows you to gate populations based on standard deviation or threshold.
When you select By Statistics, a new window will appear where you can toggle between Standard Deviation and Simple Threshold gating.
Standard Deviation gating
For Standard Deviation gating, you can specify how many standard deviations (SD) from the mean you want to use. For the SD value, you need to specify the desired settings for how the SD value should be applied. Choose the less than (<) or greater than (>) sign and specify either above or below the mean.
Lastly, you need to define what metrics (channel, cell, nuclear size, etc.) to use for gating.
Simple Threshold gating
For Simple Threshold gating, you need to specify the threshold and decide if you want the population Below or Above the threshold. Lastly, you need to define what metrics (channel, cell, nuclear size, etc.) to use for gating.
This gating tool is used to select a specific image(s) you want to look at.
Currently, Biodock does not support overlays for scatterplots with over 60,000 objects.
For every population you created, you can overlay them with a specific color. Hover over three dots (...) on the right side of the population and click on Overlay and select the color.
After you select a specific color, the gated population appears colored in the graph.
Once you created a population with gating tools (refer to creating populations with filters), and overlay them with a specific color, the image viewer will also reflect this color overlay.
In the image below, the image viewer shows the red outlines for the GFP+ population created in the gating tool.
The red outlines are the same GFP + population from the population tree.