Tutorial for count

Program options summary

Program should be invoked from the command line. If you do not know what is the command line, I cannot help you much. Try G Under Linux or Mac you should run a program called Konsole, under Windows go to the Menu Start, find Run and type cmd. In both cases a window will appear, and this is the place where you can type some commands. What commands? - this is beyond the scope of this tutorial, see Download and Installation for more details. But to run the program count, you will always need to write:

> count.exe OPTIONS FILES

where FILES is one or more BMP files (with an extension *.bmp). To specify a single file, just type its name. To process more than one file, you can either type their names separated by spaces, or type something like *.bmp which means that all BMP files in the current directory shall be analyzed.

OPTIONS means one or more of the following options, all separated by spaces

• -b NUM   sets the background level to NUM, range 10...250 (default: 60). This option allows to remove the photo background, usually caused by some non-specific staining.
• -S NUM   sets the cutoff for the spot size to NUM. The value NUM is measured in pixels and has the meaning of the smallest bright spot on the picture at the preprocessing stage, which is taken for subsequent calculations (separation of cells and counting). It is not the smallest cell size - it would be so only if all cells were separated.
• -D NUM   gives the minimal distance between cells, measured in pixels. If some cell centers lie closer that NUM, they are merged together at the final stage of the processing, and counted as a single cell.
• -m MODE   tells which channel should be counted. The following numbers are allowed: 1=red, 2=green, 4=blue, 3=yellow (red&green), 5=purple (red&blue), 6=navy (green&blue), 7=all channels (gray).
• -i   make a histogram: number of cells versus their brightness. Normally it is disabled. The histogram is saved to a file starting with _, then goes the name of the picture followed by .dat. It consists of two columns: brightness and the number of cells, separated by TAB.
• -w NUM   gives explicitely the average diameter of cell in pixels - normally it is calculated by the program during some preprocessing and used for counting cells. Sometimes it helps to change it (see examples below).

Now some more options, but not very useful:

• -s NUM   like -S, but the value NUM is measured in terms of the average diameter, and hence is related to the value given with the parameter -w. Default: 0.5.
• -d NUM   like -D, but again it is a relative value to the average diameter. Default: 0.3.
• -p NUM   isolation factor, controls the width of the function used for blurring the picture. Default value: 1. Use it only occasionally, when other options fail.

All these options can be listed by typing count.exe -help .

Examples

Example 1

Let us start with an example of a very good staining of nuclei in dorsal spinal cord, shown already in How does it work section. When we invoke the program without any options (only the filename is given):

> count.exe pic1.bmp

the following message will be displayed:

PROGRAM count: which channel? (7=all, +1=r,+2=g,+4=b)

and the execution stops until the user will type something. The program asks which channel should be analyzed. To have only green channel, type 2 and press Enter. If we would like to count cells which are simultaneously green and red, we should type 3 (because it is 1+2). Similarly, it should be 6 for green-blue cells (2+4). If we decide to have only green and type 2, the program will display:

only channel G
pic1.bmp : av.size=5.72121 cells=633
end

and we will get the following picture:

# number 	diameter 	pixels 	av. bright. 	name

633		5.72121		18212	25	        pic1.bmp

The same output can be produced simpler, when the channel is given explicitely when invoking the command:

> count.exe -m 2 pic1.bmp

where the number 2 means as before that only the green channel should be taken into account. When the option -m is given, the program does not ask about the channel and starts immediately. To calculate the number of red cells, we type:

> count.exe -m 2 pic1.bmp               (now press enter, and see...)
PROGRAM count: only channel R
pic1.bmp : av.size=5.51128 cells=463

end

and the picture looks now a bit different:

Example 2

Now we shall face with something more complicated: we wish to count yellow (green+red) cells in the following picture:

Example 3

Now I shall demonstrate how some options affect the program behavior. Our source file is shown below and we want to count blue cells: