Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi,

As a biologist I feel that I also should give some remarks on the use of
confocal microscopes and colocalisation.

Looking at a lot of confocal images, and having extensive experience with
molecular biologists wanting to collect some nice picture to illustrate their
paper, I regularly have the impression that a lot of confocal is done as an
improved digital camera set-up. The pixel size and the resolution or Nyquist are
frequently not fullfilled or are impossible to reach due to lack of image
intensity of lack of photons.

In the end, if resolution is decreasing, all objects colocalize. Therefor I
think that an essential information for pixel colocalisation is pixel resolution
and this should be further implemented with more mathemathical data.
Be aware that due to low light levels, you can run out of photons (statistical
noise) and than I guess colocalisation at that moment is quite difficult to give
reasonable information. We than need FRET or any other interacting proof.

Bye

Pattrick Van Oostveldt

Quoting Kevin Braeckmans <[log in to unmask]>:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi Marc,
>
> A pixel is observed in a yellow color when there is an equal amount of green
> and red intensity in both channels. The signal in any channel depends (at
> least) on detector gain settings, integration time, laser intensity,
> concentration of the respective dyes, confocal aperture and cross talk.
> Image post processing can also influence the absolute intensity levels.
> Moreover, perceiving something as yellow is determined by the eye-brain
> mechanism of the observer and is subjective (not to mention the influence of
> the monitor or printer with which the images are observed). So, clearly, the
> observation of a yellow color is not a very reliable and reproducible
> measure for any serious colocalisation analysis.
>
> Assuming that at least a suitable correction was made for cross-talk (which
> is likely not the case), at most the conclusion can be drawn that 'some
> colocalisation' was observed as indicated by the presense of a yellow color.
>
> Sometimes, this is enough to know. But usually, and especially physicists
> like that (I am one of them ;-), one would like to draw some quantitative
> information from the images, which requires more careful image analysis and
> an objective algorithm to decide for colocalisation.
>
> I guess we all read from time to time papers where the use of microscopy
> images is at least questionable (most likely unintentional by the authors,
> just because of a lack of proper knowledge). Especially if the main focus of
> the paper is biology or chemistry, by submitting it to journals specialised
> in those fields, there is a good chance not to encounter referees that are
> knowledgeable in microscopy or image analysis. With todays microscopy
> systems, anyone can shoot pretty pictures and make a nice color overlay.
>
> Best regards,
>
> Kevin
>
> > -----Oorspronkelijk bericht-----
> > Van: Confocal Microscopy List
> > [mailto:[log in to unmask]] Namens Marc Thibault
> > Verzonden: woensdag 7 februari 2007 22:05
> > Aan: [log in to unmask]
> > Onderwerp: 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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