I'm new to this type of analysis and am unsure of the identification of everything in this image (see attached). This is a 100x image.
Is there a clear picture of a phagocytosing macrophage? Schwann cells that have detached from their myelin sheaths?
I'm wondering if I can only quantify degenerating fibers and perhaps use EM for cell analysis...
This is actually a pretty good image, although it would be easier to interpret in color since the lipid in the myelin looks black while proteins in the cytoplasm are more of a dark blue. There are lots of degenerating axons in this photograph. To me, they have a very obvious crumpled appearance. I can see a close grouping of 4 large prominent degenerating myelinated axons in the upper right quadrant of the photograph. (The ones that look like squashed spirals.) Just below this group of degenerating axons is part of a phagocytic cell (it could be a macrophage or a Schwann cell) engulfing several bundles of myelin debris. The nucleus of the phagocytic cell is not visible in this section. There are also 2 1/2 nice looking intact myelinated axons int the upper right quadrant (thick black bands surrounding pale centers containing a few small dark mitochondria) and lots of unmyelinated axons (small pale circles). Also there is one presumptive Schwann cell cell not attached to an axon in the lower center of the image that looks almost like a sperm in this section and another one with tree processes in the upper left quadrant. (These are just the cells where the nucleus is visible in the sections. A lot of the other process are probably Schwann cells as well but could be parts of macrophages or fibroblasts.) You cannot however distinguish between Schwann cells that have become detached from their axons and those which never were myelinating unless you catch them right in the act. EM makes it much easier to count unmyelinated axons and to look at the state of any remyelinating fibers as well as to more convincingly identifying some other cell types by their organelle and cytoskeletal compositions. However, in both light and EM sections, you can only see a part of the structure so cell type identification can be somewhat ambiguous. For speed of counting, light microscopy is usually easier. By the way, I have generally found it easier to count the numbers of spared intact myelinated and unmyelinated fibers than to count degenerating fibers, since by definition, degenerating axons tend to be lost. Good luck.
Thank you for your response! You mention that the unmyelinated axons are small and pale, but what about the ones that are slightly darker and roughly the same size (upper left quadrant)? They don't have that swirly degenerating myelin pattern and they seem to have a small nucleus. Additionally, can you identify that very white circular mass in the upper left corner?
The two dark medium sized, nearly circular, objects that that I see in the upper left quadrant are nuclei. The dark irregular spots near the centers of each are the condensed chromatin of the nucleoli. These are almost certainly quiescent Scwhann cells. The appearance of a cell's nucleus will change depending on what the cell is doing. These cells have very little cytoplasm surrounding their nuclei and are not doing a lot of protein synthesis for making new cell membrane or phagocytizing cell debris. Therefore the chromatin is in a more condensed arrangement bound up with histones and is therefore darker than in more active cells.
Thanks again. One final question: When measuring axon/fiber diameter, is it customary to measure from the widest or narrowest part of the axon? One example would be that whole myelinated axon on the right side of the image.
Well I suppose it would make the most sense to find the area and then work back to the diameter. Nevermind then!
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