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I agree here with Jens: if you see some articles, in high impact factor
journals, showing images of a double-staining where the overlay of green and
red show nice yellow, I tend to wonder strongly if what's shown is indeed
true colocalisation. I mean, I've seen images where I was really wondering
if the original single channels were not oversaturated!
Though I also experienced the opposite: when having a nice image, for sure
not saturated, the final (pdf) print turns out to convert the colors in such
a way that they appear ór over- ór under-saturated. Simply because of
converting RGB to CMYK. There are so many different color palettes lately
that one doesn't know which one to use anymore... But it has a strong
effect on your proof of colocalisation! Of course other techniques will
help you showing expression of a certain molecule next to another one, but
still.
I always tend to grab for the confocal microscope when colocalisation is
requested, often two different optical sections of a histological section
will superimpose in 2D on a epi-fluorescent microscope and thus give an
impression of colocalisation, however they seem not to colocalise at all
when scanning in three dimensions. And even then one should take care of
the correct pinhole settings, z-stack-interval, detector gain etc. to
acquire correct images.
Colocalisation imaging, I believe it's something standards should be set up
for and requested by the journals...
With best regards,
Sven Terclavers
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Rietdorf, Jens
Sent: 08 February 2007 14:40
To: [log in to unmask]
Subject: Re: [CONFOCAL] colocalisation without software
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Dear Marc,
In my opinion this is the central issue for all the discussions on quality
of microscope pictures, standardisation, etc. As long as the scientific
journals accept microscopy at that level, it will be very hard to improve.
For coloc I tend to show students the following: If I increase contrast in
one channel, so it becomes completely saturated, I receive 100% coloc as
judged by yellow pixels.
The recent tutorial of S. BOLTE & F. P. CORDELIČRES (mentioned by John
Oreopoulos) discusses many aspects.
And of course it doesn't tell about protein interactions and if
regards, jens
---
Dr. Jens Rietdorf
head of microscopy unit
Novartis Foundation
Friedrich-Miescher-Institute, wro1066.2.32
Maulbeerstr.66, CH-4058 Basel, Switzerland
phone +41(61)69-75172 mobil +41 798284737
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Marc Thibault
Sent: Mittwoch, 7. Februar 2007 22:05
To: [log in to unmask]
Subject: colocalisation without software
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Hi all,
It seems that in many papers from biologists or chemists, and i'm talking
high impact factors journals, colocalisation of two elements is is often
assumed by simple color superposition (ex: red and green fluoresce yellow
when colocalising), while microscopists (many physisists I suppose) seem to
need a more complex software-based confirmation.
Is it ok, when using high end equipment and corrected objectives (apochromat
with high NA for ex.), to assume colocalisation by color superposition,
especially when fluorophore are confined to small volume entities, like
lysosomes ?
Thanks
Marc
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