SpotFinderZ

Overview

SpotFinderZ (from now on simply SpotFinder) detects round, usually diffraction-limited spots inside bacterial cells outlined with MicrobeTracker and places them into the meshes structure produced by MicrobeTracker. The program is written in MATLAB and saves the data in the MicrobeTracker format by appending additional fields.

Buttons and controls

Main window of SpotFinder

Output

Normalization

Other controls

Adjustment mode controls

Output format

The output format is based on that of MicrobeTracker. It is saved as standard .mat files, which can be opened with MATLAB without running MicrobeTracker or SpotFinder, with all SpotFinder results appended into the cellList variable, and the parameters saved as the params variale.

The cellList variable is a MATLAB cell array with a hierarchical organization. It is an array of frames, each being an array of cells, each being a structure of fields describing one biological cell on one frame. Among those fields there is one called spots, which is produced by SpotFinder. The spots field has several subfields, listed below:

The parameters x, y, b, w, and h are obtained by fitting the spot to the function b+h·exp{-[(x0-x)2+(y0-y)2]/w2}, where (x0, y0) is the current point, the parameters correspond to the mentioned fields of the spots structure. The values of the l and d fields are calculated by projection of the spot center onto the cell coordinate system, the value of the magnitude field is calculated as πw2h.

Parameters

The typical values are shown for images obtained using a 100x objective at 0.064 μm/pixel resolution unless mentioned otherwise. The parameters from Max width and below are adjusted by training, usually there is no need for manual adjustment.

Usage example

In this example we use images of Caulobacter crescentus CB15N cells expressing CFP-ParB that localizes at the chromosomal origin of replication. The images (phase contrast and cfp fluorescence) are places in the \examples\spotfinder\ subfolder of the MicrobeTracker folder. The cells were outlined with MicrobeTracker, with the result saved in meshes.mat file in the same folder. To dislay the contours on top of the phase contrast images select the mentioned folder as the active MATLAB folder (otherwise the files will not be found) and type the following commands in MATLAB workspace:

   >>images = loadimageseries('phase_images');
   >>load('meshes')
   >>dispcellall(cellList,images)

This will display the image below. Note, the number of images will be the same as the number of original images. Therefore, you may want to limit it if the number of files is large (see dispcellall tool). Note, dispcellall creates new figures, so you do not neet to use the "figure" command.

Now run SpotFinder:

   >>spotFinderZ

And click "Adjust" button to modify the quality parameters for this image set. The program will prompt you for the input images folder (file) and the meshes file. Select cfp_images folder and meshes.mat file. In what follows, you have to go through several cells clicking on the "real" so that they are displayed in red and unclicking the "false" spots.

The navigation is performed by "Previous" / "Next" buttons of SpotFinder, "Stop" button will terminate the adjustment regime. "Update params" button is used to adjust the parameters and evaluate the spots on the next frames according to the newly set parameters. The number of cells to click on may be as small as just a few, but more cell will improve the quality of detection.

When you are done and have clicked "Stop" button, SpotFinder will return to the original view. Select now "Meshes" and "File" checkboxes to save the data at the end of processing into a file and click "Run" button.

The program will prompt for the file and folder names. Select the same input files and folders (will appear by default) and a different file for the output, for example meshes+spots.mat (make sure, the \examples\spotfinder\ folder is still the active folder in MATLAB). The program will process the images while displaying a progress bar and will save the data automatically. To view the results type:

   >>images = loadimageseries('cfp_images');
   >>load('meshes+spots')
   >>dispcellall(cellList,images,[],[],[],[],3,'circle')

Which will display the data indicating the spots with circles of the radius of 3 pixels:


Top pages: MicrobeTracker Tools, MicrobeTracker Suite.