Dear friend Khawla Saif

When using the threshold tool in Fiji, there are several methods available to set the threshold value, including manual, default, and auto methods. The manual method involves dragging the threshold bar to the desired value, while the default method uses a predetermined algorithm to set the threshold value automatically. The auto method uses a range of algorithms to determine the threshold value based on image statistics.

To ensure proper thresholding, it is important to choose the appropriate method for the specific image being analyzed. In general, the manual method may be more appropriate when the image has clear contrast and intensity differences between the foreground and background, while the auto method may be more appropriate when the contrast and intensity differences are not as clear.

Additionally, it may be helpful to use image pre-processing techniques, such as image smoothing and background subtraction, to improve the contrast and enhance the image features before thresholding.

Here are some references that may be helpful in setting the proper thresholding method using Fiji ImageJ:

- Fiji User Guide: https://imagej.net/Fiji/Documentation

- ImageJ Wiki: https://imagej.net/Welcome

- Image Processing with ImageJ by Johannes Schindelin et al.: https://imagej.net/ImgLib2-Book

I am not quite sure if you are already doing this, but in the thresholding step you will have to press enter/return after entering each individual number ie. type 130, hit enter, click over to the other box, type 255 and hit enter again.

If you type 130 then 255 and then hit enter, the 130 will essentially be ignored.

Dear Máté Nászai

Thank you for the response, i tried your way and i got same values either ways. but i i knew my mistake i should click invert to get the right value.