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That's right I think, but maybe a little to perfect for some of us? For
those looking at very small objects, I'd say yes, 250nm can make a
difference, but I believe that for those working in the bio-medical sector,
like me, 250nm is a step too far in defining if there's colocalisation or
not.
Here I am talking for myself, so I'm interested in knowing other's opinions,
because it's very well possible I'm completely wrong!
I look at eg. Zebrafish & bloodvessels & cells and the "smallest"
colocalisation I want to know about is if on the cellular membrane one
receptor is present (let's say VEGFR3) and one molecule has bound or not (eg
VEGF-D). Isn't it than enough to image the cell (with optimal settings) in
Z and look through the z-stack to figure out if both different stained
molecules are present at the same level? Or can there still be a difference
in the 250nm-scale and should I really go to FRET? Because, if there is no
binding, only one color will be present...
Another example that I already had to scan here in the lab are
differentiating stemcells: at a certain moment some will, others won't,
start expressing a certain marker. If I stain one molecule known to be
present in a stemcell, to mark a stemcell and no other cells, and transfect
the cells with eg. GFP in such a way that when the marker is expressed, also
GFP is expressed, can I not show that
1. I have a stemcell (the others are not stained) and
2. this stemcell is expressing the marker and the other not
by showing that both colors are present in that cell, again while looking at
the X- & Y- and Z-axis?
Sven Terclavers
-----Original Message-----
From: John Oreopoulos [mailto:[log in to unmask]]
Sent: 08 February 2007 16:25
To: [log in to unmask]; Confocal Microscopy List
Cc: Christopher Yip
Subject: Re: colocalisation without software
This is a good thread, and I'd like to ask an additional question:
Even if one determines that two images show perfect colocalization
(as determined by some quantitative co-efficient), this result is
still limited by diffraction, correct? All that can be said for two
colocalized fluorophores is that there is a high PROBABILITY they are
separated by a distance that is ~250 nm or less. The only way to
determine the distance (0-250 nm) would be to use FRET imaging or
some super-resolution technique like STED or structured illumination
which has a better resolution below the diffraction limit, right?
John Oreopoulos, BSc,
PhD Candidate
University of Toronto
Institute For Biomaterials and Biomedical Engineering
Centre For Studies in Molecular Imaging
Tel: W:416-946-5022
On 8-Feb-07, at 9:24 AM, Sven Terclavers wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> 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
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> 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
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> 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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