ConfoCounter

QUICK START

This software allows counting of objects in 3D volume represented by a series of 2D optical sections acquired by a confocal microscope. Counting of cells in such 3D stack means identification of cells on individual sections and rounting their topmost positions. As this process is time-consuming, it is possible to count only the cells inside a selected subvolumes (stereological counting frames). If we know what fraction of the whole volume represent our "probes" we can estimate the total count (see. optical disector principle for details).

1. Stack menu item: Load stack opens a multipage tif file or a series of indexed image files. Stack browsing can be controlled by several ways (Slider on the left side, mouse wheel etc. - see table above). Counted Results can be archived in file using Load and Save functions. Image effects controls the way how images are displayed (blur, brightness, R, G, B colors). Region of interest (ROI) is initially set to cover almost the whole image (without safety border). User can Outline own ROI clicking the polygon vertices (followed by its smoothing if required). If Use frames is checked, then stereological probes are used. Each probe is defined in XY plane by counting frame parameters (Size, Distance, initial Random position). In Z-direction is probe limited by red and green lines on the slider representing its lower and upper levels.
2. Measurement menu item manages counting. All counted cells are registered in a table and displayed with the color corresponding to its class. The relevant content of the table can be copied into the clipboard and used in external program (e.g. Excel). After processing of all cells on the current Home level, the Next level increments the home position This process continues level-by-level until the home level reaches the top level (red line on the slider). Preview defines the pop-up preview window invoked after clicking the cell position (Figure 1). In Two clicks mode the user clicks the terminating position 'B' on preview window. The color of clicked position is used to filter all other colors (with sensitivity given by Threshold If both positions 'A' and 'B' are inside the counting frame and inside the permitted range of levels, the cell is counted. One click mode uses mouse button release in a new position instead of the second click. Drawcontains a group of checkboxes that determine the style of drawing of cells labels and circles outlining cells
3. View3D menu item offers the 2D view on the left side and 3D view on the right one. Mouse wheel applied on the right side Rotates 3D view according to selected axis. Draw checkboxes show ar hide axes or probes. The real values table contains data recalculated according to calibration constants from Settings. Spacing extends z-axis f better visual impression in 3D.
4. Settings menu item contains Random number generator test testing purposes. The cells positions are generated in random positions inside the ROI. Test allows comparison of generated and estimated values (the shuld be equal in ideal case). Calibration table allows manual enter of calibration data for real values calculations. These data can be automatically obtained from input file header if available. For instance ImageJ "Bioformats" plug-in saves images along with calibration data.

Figure 1 Pop-up window which appears on the clicked position 'A'. It shows subsequent levels where the user can identify and click the level where the cell terminates ('B').

STRATEGY

Various strategies are possible using combination of mouse, keyboard and gaze tracker (optional).