Matthew,
You have an interesting problem here. To start with since you are
looking for an absorber, not a fluorochrome, you MUST use transmitted
light! Next, since you have many other potential absorbers in the
light path (e.g. melanin and lipofusin) You will likely need to take a
background shot of the cells prior to adding the macular pigment as a
reference for comparison to the post treatment images. This then
implies that you will need to grow the cells on a substrate that can
be re-registered so that you can image the same set of cells before
and after treatment. Or, depending on the time scale of the treatment
and the available equipment (heating stages, etc.) it may be useful to
due the experiment on the microscope stage.
Now, for the filters... You will likely need to use a very narrow
band filter to insure that you are only looking at 465nm light. I
would start but putting this filter in the light path before the
sample. You may find that you need to use a very bright light source
(HG, Xe or metal halide) to get much light through such a setup. The
pre-treatment image should reveal any "native" absorbers in the 465nm
band. Hopefully they are not too common.
Analysis: Since you are looking for changes in light that is
absorbed, I would approach this by inverting both images, and then
subtracting the pre treatment image from the post-treatment image (or
using the pre-treatment image as the background for a background
subtraction operation). Reinvert the result and the macular pigment
particles should show up as dark spots in the image. You may also
find it useful to take a full spectrum brightfield image for the cells
(pre-treatment) to make localization of the macular pigments relative
to the cells easier.
--Chris
Chris Tully
Microscopy and Image Analysis Expert
[hidden email]
240-888-1021
http://www.linkedin.com/in/christullyOn Fri, Feb 13, 2009 at 6:58 AM, Matthew Pearson
<
[hidden email]> wrote:
> Hi everyone,
>
> I was wondering whether anyone has had any experience of imaging macular
> pigments in live cells using either light/confocal microscopy.
>
> I have a colleague who wants to image live ARPE-19 cells in culture which
> they have added macular pigment crystals to, to see if the cells take them
> up. The macular pigment apparently absorbs light at 465nm but they do not
> emit any. ARPE-19 cells are also naturally heavily pigmented with melanin
> and lipofusin. So i am wondering A. whether the macular pigment will be
> visible either by light or confocal microscopy and B. We basically want to
> see where this pigment goes in the cell or if indeed it enters the cell at
> all.
>
> I would appreciate any advice people have on this subject. I have only seen
> papers of macular pigments being imaged in vivo using a laser scanning
> ophthalmoscope...
>
> Thanks for your help!
>
> Matt Pearson.
>
>
> --
> Imaging Technician
> University College London
> EC1V 9EL
> 020 7608 6857
>
[hidden email]
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