Dear Joachim,

You could try back scatter (reflectance) imaging if you have access to a laser scanning confocal. There is no need to add a fluorophore, and many cell types show a surprising amount of back scatter. In back scatter imaging the light is bounced back up through the illuminating lens - this would allow you to irradiate your samples  with the ionizing radiation from the other side without blocking the illumination used for imaging.

Regards, Alan.

BIOCON, specialists in confocal microscopy         ABN: 46 196 908 558
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Dr. Alan R. Hibbs                                                                        FAX: 61 3 8660 2290	


-----Original Message-----
From:   Joachim Walter [SMTP:[log in to unmask]]
Sent:   Friday, December 22, 2000 9:51 AM
To:     [log in to unmask]
Subject:        unusual brightfield setup

Dear all,

we are struggeling with designing a microscope setup that lets us
observe cells in front of a source of ionizing radiation. Practically
this means that one side of the cell chamber is blocked by the outlet of
the source and one cannot fit in a condensor and an objective for
transmission (e.g. phase contrast) imaging. On the other hand we want to
avoid staining the cells for fluorescence microscopy to keep photodamage
down. Therefore I have these two questions:

+ Does anyone know of a contrast technique replacing phase contrast that
works with illumination and detection from the same side?

+ Alternatively it might be possible to couple light into the source and
illuminate the cells from the back side (obtaining a transmission
setup). However, due to geometrical constraints the light would have to
be almost parallel, when it gets to the cells, i.e. the light is coupled
into the source through a small window (~ 1cm) and reflected to the
outlet by a 45° mirror (with a hole in the middle to pass the
radiation). The outlet again is <~ 1cm in diameter and at some distance
to the window, so that one would have to use collimated light. Can
anybody suggest how to get a good contrast for transmission images with
these constraints?

I hope I made the problem clear. If not, please just ask. Any suggestion
to this is welcome.

Thanks, Joachim



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  Joachim Walter, Dipl. Phys.
  Institut für Anthropologie und Humangenetik der LMU München
  AG Cremer
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