Larry, welcome aboard.
Vibration is by far the most likely source of your problems. At 10X it only takes about 1 micron = 0.001 mm of movement, at the end of the lens, to give you a 2-pixel smear back at the sensor.
The safest approach, especially when you're just getting started, is to illuminate with electronic flash set on low power so as to get especially short light pulses. Most consumer-grade flash units will produce light pulses around 1/5000 second if you set them down to 1/16 power. Such short effective exposure time is a powerful method of freezing out whatever vibration you might have.
If you're using continuous illumination, then you should definitely be
shooting in Live View too. The reason is that when you shoot in Live View, there's no mechanical vibration at start of exposure, only at end when it hardly matters. The official phrase is EFSC -- Electronic First Shutter Curtain. See
http://www.krebsmicro.com/Canon_EFSC/ for discussion and illustration of why this is important.
One more thing about microscope images: they're never as sharp as a good landscape lens will produce, simply because of diffraction. A 10X NA 0.25 objective has an effective aperture of f/20, and you probably know that setting a landscape lens on f/20 will produce images that are not very sharp. They can be improved a lot by strong sharpening in Photoshop or whatever, but compared to say f/5.6, f/20 will always come out not looking so good. Your 20X Mitutoyo is probably NA 0.42, which means that it will be running at effective f/24. (The formula is Feff = magnification/(2*NA).) This level of unsharpness will be easily visible when viewing actual pixels, but it absolutely should not be visible in images scaled to 640 pixels as you've done here.
And finally, just to cover the base: be sure you have that 200 mm rear lens set on infinity focus. Having it focused anywhere else will introduce some aberrations that will degrade image quality a little bit. Again, this effect is small and would not be seen in the resized images that you're showing here.
Other than vibration and those other minor effects described above, the only thing that can really go wrong is a damaged objective. If you bought new then they're probably OK, but some significant fraction of used objectives have been dropped or banged hard enough to knock lens elements out of alignment. The damage can be completely invisible on the outside, manifesting only in a degraded image. But since you have two lenses, and they're both producing blurred results, lens damage is not likely the source of the problem.
One last thing about Live View: studying a Live View image at maximum magnification can give you some good clues about where things are going wrong. If an in-focus image looks sharp in Live View but a lot less sharp when captured, then the capture is probably degraded by vibration. And of course if the Live View image itself is moving around, that will tell you that your system is picking up vibration from the environment, not just creating its own from moving parts.
--Rik
tag:vibration
