Of course, glad to help out.
(1) create an image of the capillary bed, as shown in my initial post. I got this by averaging 4-5 slices in the XYT stack where the fluorescence in the capillaries was at/near its peak
(2) threshold this (Image>Adjust>threshold). Try “Auto” as a starter. When satisfied that capillaries are selected (the upper portion of the pixel intensity histogram), hit Apply
(3) Run Analyze Particles. I set the following parameters in Analyze: size = 10.0 to infinity, circularity 0.0-1.0, Show Outlines, Include holes, Add to Manager (other items can also be checked…not sure it matters)
(4) This generates an ROI manager with multiple fragments of the capillary bed selected. I suppose one can mess around with various parameters to select the entire capillary bed as one unit, but I don’t know how to do this easily. Multiple little segments were okay for me
(5) open up the original stack (XYT) with the ROI Mngr still open and check the “show all” box on the ROI manager. This should superimpose outlines (ROIs) on top of your capillary bed in the stack
(6) in ROI mngr, select More>Multi-measure (make certain that in Analyze>Set measurements you have checked “mean gray value”)
(7) this generates a table of mean pixel intensities within each of the capillary fragments for every slice in your stack
(8) copy/paste the table into Excel and plot the average of all the fragments to get a picture of what went on in the entire capillary bed
To measure the appearance of dye in the surrounding tissue, repeat all of the above except at item #2, change the threshold to select the dark pixels in the image (the bottom part of the histogram). This selects for the regions outside the capillaries.
I got something that looks like (I was using FITC as the fluorescent dye. The initial peak is immediately after injecting FITC):
Hope this helps. If you need more specifics, give me your email address and I will send a more complete, annotated protocol.