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After reconstructing microscopy data, the second aim of ProkaryMetrics is to provide the tools for quantitative analysis of the visualized communities. This section provides information on each of the tools provided by ProkaryMetrics and how to use them.

Data Display Panel

Before describing the available quantitative analysis tools, the data display panel needs to be introduced. This panel (normally hidden) is a simple text display that ProkaryMetrics uses to provide the user with numerical information relating to the performed analyses. For some tools this information is supplementary to the visual representation of the results, for other tools the data display panel is the only means to provide the results to the user.

To access the panel on a Windows machine, hold down the Ctrl button and press the D key. On a Mac, hold down Command (or apple) and press the D key.

Quick Reference

Windows: Ctrl-D

Mac OS: Apple-D (Command-D)

Volume Estimation

A useful method for understanding the amount of space occupied by a microbial community is to fit an ellipsoid (also known as a 3D ellipse, or a generalized sphere) to the community itself. ProkaryMetrics can use either recorded bacteria or, if you want a quick estimation of volume, any markers that are placed. In that second case, just toggle into Recording Mode and place markers on the reconstructed data near the extents of the community. Note that if you are doing this, ProkaryMetrics will provide a better estimation if the markers are not right up against the edge of the data, but somewhat inward a bit.

Performing the Fit

To actually perform the volume estimation, click on the Tools >> Volume Estimation >> Fit Ellipsoid menu option. The only requirement to using this tool is that you have at least nine (9) markers placed. This can either be actual markers (as in the second option from above) or recorded bacteria that required at least 9 markers to create. For example, if you have 3 bacilli (2 markers each) and one filament that required 3 markers, the recorded bacteria make up 9 markers in total. When you click the Record Bacterium button, ProkaryMetrics visually represents the bacterium as the bacillus-like shapes that appear, but internally represents the bacterium as a set of 3D points corresponding to the markers you placed.

When you select this menu option, ProkaryMetrics performs the necessary calculations and then displays a mostly transparent ellipsoid surrounding the data:


The data display panel provides additional numeric information as seen below:


ProkaryMetrics provides the user with additional quantitative information including a broad classification of the ellipsoid type (here Scalene because two of the three radii are essentially equal), the numeric volume of the fitting as well as that of the recorded bacteria, and finally the ratio of the two.

Toggle visibility

If you want to continue marking bacteria, the rendered ellipsoid will interfere with the cursor tracing the surface of the rendered image data. So to hide the ellipsoid, select the Tools >> Volume Estimation >> Toggle Ellipsoid Visibility. To see it again, simply click the menu item again.

Bacteria Orientation

Another interesting and useful bit of quantitative data is calculating how the bacteria are oriented within the 3D space that they occupy. For each bacterium, ProkaryMetrics calculates the main axis vector and compares that to the X, Y, and Z axes to get a number that describes how much the bacterium deviates from each of the axes. Those numbers are then used to color the bacteria, associating Red, Green, and Blue with the deviations from X, Y, and Z respectively.


ProkaryMetrics provides three coloring schemes to map X/Y/Z to colors: RGB, GBR, and BGR. The figure above shows the BGR coloring, and gives a good example of the utility in comparing orientations between data sets. In the community on the left, the bacteria are split between those with the major orientation component along the X (blue) axis and the Y (green) axis. Whereas with the community on the left, the clear majority of bacteria are aligned along the Y (green) axis. Note that a few of the bacteria have a signficant portion of their orientation in the Z axis, and they have a reddish component, making the bacteria either purple for those with the other major component along the X axis, or yellow for those with a major component along the Y axis.

Performing the Orientation Visualization

To color the bacteria using orientation information, as seen in the figure above, select the Tools >> Orientation >> Color Bacteria by Orientation submenu, and choose one of the available coloring schemes as listed previously. You can return the bacteria to their original colors through the View >> Image Layer Settings dialog box. Additionally, the orientation information can be exported to a CSV through the File >> Export menu.

Descriptive statistics can also be calculated for the overall orientation information. Select the Tools >> Orientation >> Calculate Orientation Stats menu item. The results are shown in the Data Display Panel and give the mean, median, standard deviation, and first and third quartiles.

Community Distance Information

The final analytic tool available is calculating the average distance among the community members. This calculation is meant to give a metric for comparing how densely packed the bacteria are within the community. The community distance currently has no visual aspect, but the results are output to the Data Display Panel. To perform the calculation, select the Tools >> Calculate Community Distance Stats menu item. The results give the mean, median, standard deviation, and first and third quartiles.