Greater DOF with secondary electron imaging is largely a matter of working distance--defined as the distance (in mm) from the objective lens to the top of the sample being imaged. Of course, the lenses in this type of instrument are electromagnetic (not glass) lenses, and can effect different crossover (focus) points based on current supplied to the lens coils.
The longer the WD, the greater the DOF, (but this entails other tradeoffs as with every operating parameter). Of course, this is a familiar principle to any photographer; the closer you move to an object, the shallower the DOF is.
The WD I used for this shot was 28mm, which is considered very long. I also use a tilt of around 30 degrees. This adds an additional sense of depth. If you were trying to convey the three dimensionality of a sphere, or ping pong ball for example, the worst way to photograph it would be from directly above. Better to come in obliquely from the side.
The protozoa (protists is a better word) that live in the guts of lower termites are often very large, and this presents a challenge for DOF. This one in question is about 40 microns long, but others can be up to 300 microns long. We beleive that they have evolved large size in order to engulf the relatively large wood fragments that make their way to the hindgut after being chewed by the termites jaws.
Focus stacking is something I've never tried, but for some large cells, I've taken multiple images with different portions in focus. If someone can point me to a tutorial for focus stacking, in Photoshop (I use CS2) I would appreciate it!
Thanks very much for this explanation. Never worked with or read about this kind of equipment before so I still didnít quite get it. Due to that I asked to Mr. Google who provided the following reference. Of course there could be a lot of different SEMs but the key details that caught my eye were:
ďThe scanning electron microscope has many advantages over traditional microscopes. The SEM has a large depth of field, which allows more of a specimen to be in focus at one time.Ē http://www.purdue.edu/rem/rs/sem.htm#2
My next question is: does a SEM require a light source? The shading on the image you displayed is so delicate it made me wonder how one could position one or more lights to produce the result on such a small object.
We believe that they have evolved large size in order to engulf the relatively large wood fragments that make their way to the hindgut after being chewed by the termites jaws.
Any time a case for selection can be exemplified that is pretty cool! I guess itís a completely different discussion but I have to ask: Are only the larger protists found in adult termites? Do they grow in size as the termite does?
This is all way outside of my experience, so I hope you donít mind some questions.
WRT focus stacking, there are a few here who have a lot of experience with focus stacking software. I think that kind of technology might be very useful for this kind of work.
Remember us when you get a Nobel for your future work! <big toothy grin>