Do you want to measure the vascular surface or the vascular cross section area?

For the former you need to use stereology or 3D acquisitions. What type of data do you have?

Have you considered to use ImageJ? If you can draw around the edges of your capillaries with the free hand selection tool, you can then use the "measure" tool (ctrl+m), it will give you the area (expressed in pixels, you will have to convert it).

You need to be careful to consider the random orientation of vessels as they travel through tissue, but if you want to estimate total capillary length in a volume, Stereo Investigator (from Microbrightfield) is a great software package that has a probe called Space Balls. This probe creates a virtual hemisphere within your tissue to control for changes in orientation, and may be used on virtual microscopic images (e.g. from a Nanozoomer digital microscope) or in conjunction with a moving stage microscope linked with the computer system. Please keep in mind that the sections need to be adequately thick and the sections you choose need to adhere to requirements for unbiased quantification (i.e. random starting section, every nth section, etc.). The software can help you figure that out, too.

ImageJ is easy to use and you can convert pixels to respective square units. 1 mm² = (1412 pixels) * (1412 pixels) = 1,993,744 pixels

Be careful! The actual area will depend on the magnification and the camera you use!

If I am not wrong, think it does not matter with ImageJ software, if you take a fixed area the number of pixels remains constant. for example if you have an area of 1mm2, the number of pixels is the same. If you use higher magnification, you might see the object bigger but the area is still 1mm2 and pixels are constant. The advantage of imageJ is that it calculates the number of pixels. Anyway, thanks for the suggestion! I had the same doubt when I was doing the quantification.

Indeed, it is partially right. With your setup, if you use the same camera and the same magnification, 1 pixel will always give you the same area (1um2 for example). Let's say that you measure in your picture an object of 16 pixels, you will have a final area of 16 um2. If you change the resolution (it depends mostly of your camera), the area of your object, expressed in pixels, will change but, of course, you object still measure 16 um2. So to reflect this change, you have to change the scale (Analyse menu, set scale). If you change the magnification but not the resolution of your picture, you will also have to change the scale factor. Hope this will help, this is a bit difficult to explain.

Thank you all my dear colleagues . I did it with Image J and it worked

Hi Hadi

Can you please provide me with the protocol you used for the quantification of the capillaries using ImageJ for capillaries quantification.

please see below link 

https://www.researchgate.net/post/What_is_the_most_accurate_way_to_quantify_capillaries_in_a_mouse_brain_section.

thanks 

Sweilem