Re: Advice for low-mag, long-term imaging of live beasties?
- From: heini <buergers@xxxxxxxxxxxxxxxxxxxxxxxxxxxxx>
- Date: Thu, 20 Dec 2007 01:38:21 -0800 (PST)
Hi Zach,
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...
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?
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.
As camera I would recommend a pixelfly QE from www.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...
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...
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!
The project is not quirky at all, some people do so with a motorized
Zeiss and an incubator on the xy-stage.
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?
Thanks everyone so much for your time,
Zach Pincus, PhD
Molecular, Cellular and Developmental Biology
Yale University
.
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