Elizabeth, I have processed images for many years. What I have found is that each situation is unique. As any photographer will tell you, it is all in the lighting. I have used ImageJ and plugins. These are great programs but tend to be limited and do not lend themselves easily to batch processing such as with analysis of many images in a movie. Photographer friends inform me that Photoshop is useful. Personally, I have never gone that route. I have always gone to the basics and written my own image analysis programs. Python and modules such as OpenCV and PIL are incredibly powerful and allow for multiprocess, programmatic image analysis that is customizable for your specific needs. I am fluent in Python, so this route is obvious to me. The internet has thousands of image analysis examples. If you do not program, then you have a barrier. My suggestion is to find a colleague conversant in Python. A simple, custom image analysis routine can be written in several hundred lines of code.

Dear Elizabeth,

I think to sputter iridium on the surface is a good idea to compare the reflectivity of the leafs (I am no biologist, only physicist). To get equal reflectivities, the thickness of the film must compare with the wavelength. So shorter the wavelength, so thinner the film and so higher the local resolution. It may be, that fine details are after evaporation no longer visible.

Another problem is the sputter direction. It can be (me seems in your case), that certain parts stay unsputtered. It is necessary to rotate the leaf whereas the sputtertime. Otherwise remain dark parts and the problem only reduces.

With Regard

R. Mitdank