Could you please provide one example image and describe what you want to measure in it?

Hi, what exactly are you trying to measure? Deposition area? Amount of tissue occupied by tissue?...

It will depend of what you want to measure, do you have a stack of images (a volume) or do you have only one image. If you have a volume or an area you want to quantify volume and not intensity. If you quantify intensity it will be dependent of your threshold, there could be sections of collagen that were not well stained that won't be analyzed. When you quantify by volume it is better since you can set the background noise as your threshold. Now it will depend on how you adquired your images. If you used a confocal microscopy you can detect collagen using second harmonic detects mostly collagen type I as far as I can remember. You can quantify the volume using imageJ. With picrosirius red you can detect collagen type I and type III. You need to use a filter and it will have red color for type I and green color for type III. You can detect an increase by splitting the channels in red, green and blue and analyze individually the volume. To have a better comparison you would need to quantify normalizing by volume using the same area independent of the thickness.

I hope it helps you or someone else that is having the same issues.

Francisco I. Ramirez-Perez