Hi,
Your questions were of interest to me. Mty experience is in color imaging on
film and color accuracy on CRT screens and in print. If your question hasnąt
been answered already, Iąd be glad to contribute what I have learned if I
can understand your needs better. Since I have no experience in your fields
of expertise, perhaps you could answer a few questions.
What is your goal in color accuracy to represent collected wavelengths
additively, or is it to merge color data collected from several screens onto
one where different values may be differentiated? Is color assigned
accurately by wavelength through the software, or is it subjective to the
perception of each viewer and their CRT? What color is assigned to
wavelengths beyond the visible spectrum? It seems that the problem as stated
needs clarification so we arenąt confused by the color model of the flow
process, and the color model of the final output. If that can be clarified,
a correlation can be established between them and the appropriate software,
probably Photoshop, can be utilized intelligently to give you a known and
repeatable color value for the intended output.
Don Schaefer
Boston, MA
Responding to the following:
Date: Thu, 15 Aug 2002 09:11:17 +0100
To: cyto-inbox
Subject: Compensation ... in microscopy
Hi mr,
In my experience with confocal & CCD work you cannot
compensate data collected after acquisition, although saying that
Molecular Dynamics used to sell Unix software called 'Imagespace'
that apparently/theoretically could do compensation - never tried it
myself as it seemed impossible to tell which part of the image data
needed compensating and which did not. The new generation of CLSM's
(e.g.Lecia SP2) allow collection of data of specific wavelength's
chosen through the software and if you collect the image on
sequential - basically for example collect UV excited, then 488 and
633 nm data and then merge the data. There is obviously stretch boxes
to adjust the image colours collected for each wavelength collected
but this process is very subjective and is not mathematical as in
flow, the nearest approximation is to try and remember very roughly
what the image looked like (very difficult with Cy5 as only part of
bright epitopes can be observed by eye) and adjust the colours
accordingly.
I hope this is of some help as I am more into flow than
microscopy, there is a US company called "Universal Pixel" that sells
Mac software which seemed very good, although I'm sure there are a
lot of other alternatives available. Mac's are a better platform for
doing this as a lot of PC software pixelates the images when trying
to enlarge the image.
Best Regards
Gary Warnes,
FACSLab,
Cancer Research UK
London, UK
mr wrote:-
We are starting a multicolor project by microscopy (please, don't
hurl any epithets at me: I'm a true-blue-flow guy... just dabbling a
little in the obscure arts).
Can someone recommend decent software (preferably Mac) that can do
basic image processing that can load multiple images (taken with
different filter sets), and then compensate them to give pure
fluorochrome images?
--
Dr. Gary Warnes
FACS Laboratory
Cancer Research UK
44 Lincoln's Inn Fields
London WC2A 3PX
P: 020 7269 3394
F: 020 7269 3100
mail to:gary.warnes@cancer.org.uk
Charity No: 1089464
OCTOBER IS BREAST CANCER AWARENESS MONTH
To find out more visit www.cancerresearchuk.org
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