The graph window is the starting point for data analysis in FlowJo. By default, FlowJo presents you with a biaxial plot, presented as a pseudocolor plot. Pseudocolor, indicating that there density information provided, as well as the fluorescence intensity of each one of the dots. For example, in this graph; the red events correspond to the most dense, where the cells are most dense. The blue events are where the cells as most sparse.
On the bottom X axis, is a drop down menu that allows you to select other parameters. Similarly, on the Y axis, on the left hand side, you can change the Y axis parameters by clicking on the drop down menu, and choosing a different parameter. You can even change this plot, to make it into a histogram, by selecting the Y axis parameter list, and selecting histogram from the bottom option.
In addition, the X axis and Y axis parameters, you will notice there are two T buttons that flank the drop down menus. The T button will allow you to rescale the axis data. For example, when I click the T button, the drop down menu presents linear access, log, reset, or customize axis.
I can immediately transform the data that are plotted on the X axis in this case, by clicking on the linear access option. This will convert the data to a linear scale. Similarly, if I click on log access, this will plot the data on a logarithmic scale. If I click the reset button, this will return the data to the original scaling preferences. In this case, FlowJo presents fluorescent data on a biexponential scale.
If we click the customize axis choice, this will take us to a transform window, where we can play around with the scaling; adding extra negative space, or cutting off extra positive space. We can also control the area around zero.
You can always change the plot type in FlowJo fairly quickly. Instead of looking at a pseudocolor plot, down at the bottom of the graph window you'll notice this options menu. By clicking on it, you will expand the menu options. Inside, there will be several options to choose the different types of graph, to change the foreground and background colors, and also to make some adjustments to smoothing and the presentation of the graph. For example, we can change this over from a pseudocolor plot to a contour plot. Contour lines can be adjusted by simply clicking on the accompanying drop down menu, and increasing or decreasing the amount of contour lines present.
In addition, we have density plots, which give us information about the density of events simply by indicating, the darker colors here, indicating where events are most dense, and the lighter colors where fewer events are presented. In addition, we have a zebra plot, which is really a hybrid between the density plot and the contour plot. It used the contour lines as well as the shading, to give you an idea about where the cells are most dense.
In addition, we have the dot plot, which by the way, has the highest resolution. We can push the numbers of events that are presented in a dot plot at any time, by simply clicking inside the box and typing the value in. In this case, I increase the number of events presented to 20,000. Notice the dot plot doesn't really give us too much in the way of density information, but it does have very high resolution, in that we can see every last cell that was present in this particular sample.
Last, we have two univariate plot options. We have a histogram, as I showed you earlier, or the cumulative distribution function. Let's go ahead and change our graph back to a pseudocolor plot. We'll collapse the options drop down menu, and change the pseudocolor X axis back to forward scatter.
In the upper left of the graph window, you'll notice that we have several different buttons. Each button corresponds to a different graph type that you can use to draw a region of interest around the events in the graph space. For example, we have a rectangle tool that will draw a rectangle or a square around the events of interest. An ellipse tool, or a polygon tool.
Polygon tool will allow you to create an odd shaped gate, with as many vertices as you desire; stopping at each point to create a new vertex. If I double click on the last vertex, or match the final vertex up to the initial vertex, I'll get prompted for a name for this particular gate. In our case, FlowJo autogenerates the name of this gate. We can choose a different name, if we so desire. In my case, I might label this cells.
You'll notice as soon as the gate is created and named, the gate name will appear in the graph window above or within the gate that you have just created. In addition, you'll see a number. The number corresponds to the percentage of events that are contained within that gate, relative to all of the events that are presented within this graph space.
In addition to the polygon tool, we have a freehand tool, which will allow you to create an odd shaped gate. You just have to mouse over the region of interest, and essentially drag your mouse around the screen. We also have an auto-gating tool, which is kind of a neat tool, when you consider that it follows the density of events. Essentially, if we were looking at a contour plot, you would notice that this gate forms a line or forms a gate on the next concentric ring of contour lines. Stop at the point that you want to create the gate, and again, give it a name. I'll go ahead and cancel this for the time being.
In addition, we have quad gates. You have straight, perpendicular quads off towards the left. Then we also have spider quads and curly quads. The spider quads will allow you to create obtuse or acute angles, with each one of the gates. You can move the legs by simply grabbing the base, and moving them up or down along each axis.
Let's go ahead and get rid of these quad gates that we just created. The arrow keys in the upper right hand side, you'll notice there are two that are bent; one pointing to the left, the other to the right. These are the undo and redo buttons. The block arrows, in green, allow us to advance through the samples either forward or backward. If we happen to have a set of dependent gates, the green up or down arrow keys will actually guide us through the hierarchy. We'll discuss more about the hierarchy in a later session.
The tools in the graph window are present on the bottom left hand side. The first one is the 3D viewer. When you click this button, this will allow you to see your events in three dimensions: X, Y, and Z axis. You can, of course, export or save these options as a PNG, a TIF, an SVG, et cetera. There are other plot types here that you can play around with.
Next to the 3D viewer is another button, with what appears to be two windows overlaid with one another. This button allows you to duplicate the graph that you're looking at. There are two other drop down menus that we haven't mentioned yet.
The first one is the active gate option. If we click this drop down menu and expand it, we have to first select the gate that we're interested in modifying. The active gate options allow us to change the nature of the gate. The first option here, where it says events inside; if we actually toggle this off, what this will do is create a quick NOT gate.
The magnetic gate choice causes the gate to snap to the centroid of the cells. Wherever the cells are most dense, this gate will always snap to the centroid. This is a useful tool, if you notice that from sample to sample, your cells move a little bit to the right, or a little bit to the left. This type of a gate will always nudge itself to the right or to the left, following that dense cluster.
In addition, we have the ability to tint these gates, if you would like. You can change a different color. The last sets of tools that you have off to the right here, these allow you to proportionally increase or decrease the size of the gate that you have chosen.
Finally, you can use the statistics drop down menu to add statistics to a particular population. In our case, maybe we want to add statistics to this child population, known as our cells. Simply click on the statistics drop down menu to expand it, then click the sigma button. This will present you with a prompt, that on the left hand side contains all the statistics that are present in FlowJo, and a list of parameters that you can associate those statistics with.
For example, I might choose the geometric mean for this population of cells, with respect to the CD8 parameter. I can go ahead and click the add button, and this will add the geometric mean directly into the little drop down statistics menu in the graph window, as well as add that statistics node in the workspace. I have two places to reference this.