Advice for low-mag, long-term imaging of live beasties?
- From: Zach <zpincus@xxxxxxxxx>
- Date: Wed, 19 Dec 2007 11:53:31 -0800 (PST)
Hello all,
As a follow up to my questions about the Wild M5, here are some more
details about what I want to do with this all...
I want to image nematode worms (they're about 1mm full grown) as they
live out their lives in individual wells of a 96-well microtiter plate
(6 mm diameter). I'll want to capture bright-field and fluorescence
images over their 2-4 week lifespans. The resolution/magnification
doesn't need to be particularly high, either. If an adult worm fills
half of the field of view, that's plenty large for me -- perhaps too
large! The worms will be crawling around on agarose pads in the wells,
so the imaging has to be from above, which will require something like
5 or 6 mm or working distance at least.
This will be eventually computer-controlled with a home-made X-Y
positioning system (you can get XSlide linear drives from Velmax with
a good degree of precision, especially for this application, along
with motors, motor controllers, and rotary encoders, for an order of
magnitude less than any assembled motorized stages), and I hope to
recycle a much older Velmax slide that's currently mounted to a Wild
M5 stereomicroscope for use as a z-motor to control the height of the
scope/camera apparatus (and thus focus). Or maybe I'll control stage
height -- whatever is lighter-weight will be made to move.
I'm looking into high-intensity LEDs for both white light and blue
light for GFP fluorescence excitation: Lumiled Luxeon K2s (for white)
and Vs (for colors) look like they'll fit the bill, and there are
reports on the web of successful use of these for GFP excitation in
similar setups with nematodes. The main reason is that since I want to
be taking images pretty frequently for weeks at a time, traditional
arc lamp systems would just chew through (expensive) mercury bulbs.
Failing these, I'm thinking about tungsten/halogen bulbs. The LEDs are
cool because they have very narrow emission spectra, and probably
wouldn't even need an emission filter.
In terms of the camera, finding something that's appropriate to take
low-res images of moving fluorescent worms (relatively dim, but
visible to the eye) is a bit of a challenge. I think that some of the
cameras in the $1-2k range from Apogee might be OK, depending on the
noise at their fast readout speeds. Point Grey also has some very
inexpensive cameras with the same sensors... These might be too slow/
noisy, though, so I'm also looking at some mid-range ones from Cooke
QImaging and the like.
Which leaves the microscope body, which is where I'm most unsure. I
have the afore-mentioned Wild M5, which is a nice scope, but not
telecentric (as pointed out), which might make quantittive imaging /
morphometry studies trickier than necessary. I could also imagine
buying a long-working-distance 10x objective from Edmunds or
somewhere, and building up a dedicated scope from scratch. (Basically:
objective, tube, diachroic mirror, tube lens, relay lens? [is that
necessary here?] and c-mount camera.) Or I could build a macro-system
with various macro lenses instead of objectives. Or the Nikon AZ100 in
the "system integration" configuration looks interesting indeed.
Finally, I could buy a full-on upright microscope and build out from
there.
Clearly I'd like to keep costs down, since (a) this project might not
bear fruit, and (b) the project has some pretty modest requirements,
optically.
Does anyone have any thoughts or recommendations about this quirky
project? I think I could pull it off, but getting some opinions from
those who know better whether this is a fool's errand or not might be
really helpful!
Thanks everyone so much for your time,
Zach Pincus, PhD
Molecular, Cellular and Developmental Biology
Yale University
.
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