Re: Advice for low-mag, long-term imaging of live beasties?

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

Check for high autofluorescence with some plates and coatings and
agarose before you go into building a microscope...

There is a decent amount of background, from the agarose and nematode
media, as well as from the lawn of bacteria to feed the worms. It's
not a deal-breaker though -- the imaging works OK on our dissection
scopes with fluorescence illumination, and on the low mag settings of
our research scopes.

So why am I trying to build a new (and vaguely cheapo) scope if the
lab I'm in has real ones? Well, once I validate the assay, I'd like to
have dedicated equipment so that I can observe the worms 24/7.

I'm looking into high-intensity LEDs for both white light and blue
light for GFP fluorescence excitation: Lumiled Luxeon K2s (for white)

Why do you need fluorescense? they express gfp?

GFP and eventually perhaps some longer-wavelength fluorophores too.

you can buy LEDs sorted for wavelength with spectra of less than 10
nm. You will not need excitation filtes, but emission-filters and -
depending on your microscope- beamsplitters. They cost peanuts except
for UV (which you obviously don't need.) Look at LEDs from nichia.

It looks like the Lumiled Luxeon line of LEDs is much brighter (5-10x
lumen output?) than the nichias, both color and white. But that's just
based on web specs... have you ever used any of these Luxeons? The
data*** gives about a 25 nm FWHM around 470 nm for the blue LED:
http://www.lumileds.com/pdfs/DS34.pdf

For a lark, check out this page for a cool hack with the Luxeons to
cheaply add the ability to visually screen for GFP worms to a
stereomicroscope:
http://130.15.90.245/gfp_stereoscope.htm


As camera I would recommend a pixelfly QE fromwww.pco.com
This camera is very sensitive and offers the fastest shutter-speeds
one can buy. They offer a SDK. Depending on the NA and resolution of.
your objectives it maybe has not sufficient pixels, you have to figure
out this before...

I'm definitely looking at the pixelfly. It's a nice camera, but not
too cheap, and perhaps more camera than I really need.
(E.g. cooling is probably pointless since with living worms the
integration times can't be too long, so low dark noise is not needed.)

Out of curiosity, I was looking around at cameras like Point Grey's
(which have microManager support via the IIDC spec). The Grashopper
has an option to use the same (?) CCD that's in the pixelfly QE, but
the price is around 1/6th. The FPS is similar, so I expect that the
price difference is that the PCO camera has vastly less noisy A/D
electronics. What else does one need to be cautious about when looking
at cheaper cameras?

Similarly, Apogee has some interesting cameras, one with again the
same ICX285 chip, cooling, and somewhat inferior read circuitry (8 FPS
in a mode that's probably pretty noisy, 1 FPS in a fairly clean mode).
But approx half of what the Pixelfly runs.

PCO Pixelfly QE
http://www.pco.de/pco/php/products/index_1-en__01030101&view=detail&cam=55.html
Point Grey Grasshopper
http://www.ptgrey.com/products/grasshopper/index.asp
Apogee A285
http://ccd.com/ascent_a285.html

Other than just getting demo versions of all of these, how might one
evaluate which is appropriate? Especially given that they all seem to
have the same CCD.


Which leaves the microscope body, which is where I'm most unsure. I

you need a lens, a stand, a tube and a camera.
Last year I built an inverted microscope to put into an incubator and
shoot dividing cells. I took an old upright Leitz with a 25x at 160mm
fixed length, put it on top, removed the tubus, used a mirror to get
the light into the camera outside of the incubator and that's it.
Transmitted light and DF were no problem...

Cool. This sounds do-able, certainly. Did you use any relay optics to
get the intermediate image (at 160mm) onto the CCD? Or did you just
put the sensor directly at 160mm?

Did you also include a condensor, etc., for the transmitted light?

before you buy parts you should consider the steering, so maybe check
for compatibility with µManager (which brings me back to that Zeiss,
didn`t you mention a zeiss z-drive?...) And you will maybe need an
other camera? And an xy-stage that is supported by any other software?

I'm probably going to build the steering myself from one of the new,
"friendly" microcontroller packages like the Arduino or the Make
Controller Kit and some linear slides from Velmex. (Either stepper
motors or closed loop servomotors+rotary encoders.) At an order of
magnitude less expensive than pre-built solutions, and with about the
same potential precision, this is a cool option. As for a stand, I
inherited an older motorized velmex slide built into a stand for a
Wild M5 stereomicroscope. Could just remove the M5 and put on
something else, and then tie that motor into the controller too...

Thanks for your help and advice!

Zach
.