http://confocal-microscopy-list.275.s1.nabble.com/Online-fingerprinting-on-Zeiss-510-META-basic-questions-tp7582451p7582453.html
Thank you for your answer. As you say the imaging is running relatively ok
in normal mode. But I have had some problems with high background,
posibillities for eliminating this. I also use a ImageJ plugin to analyze
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> Hi Anders,
>
> From our experience with a 510 META a few years ago, I would say that the
> benefit of online fingerprinting would be primarily for separating two
> labels that are too close spectrally to be separated with the conventional
> emission filters. In your case, I have a strong feeling that you will be
> better off collecting your four channels the normal way on the standard
> detectors. These will probably give you a better signal to noise, and
> therefore better data. You can always verify the registration of the
> channels with some fluorescent beads. In terms of image quality (noise),
> normal PMTs will probably be better. The META with online fingerprinting
> might have a benefit with regard to channel registration if you could image
> your four channels at once (in a single track), but if you need to
> multi-track, then the benefit is gone.
>
> More specifically to your point, we haven't done unmixing with multiple
> laser lines. You could either do it in a single track, four channels at
> once ( so you would need to collect spectra for each dye with all lasers
> ON), but you would need to block the laser lines and collect rather narrow
> bands (for example, if you have a 543 laser, your "green" channel will be
> pretty narrow). You seem to say you don't have a four line dichroic, so
> this means you would need to do two tracks at a minimum. How you combine
> them would depend on your choice of dichroics. You could maybe do DAPI/Cy3
> and 488/Cy5, so that laser lines and emission bands don't overlap too much,
> and you would collect four spectra, one for each dye, under the conditions
> that you will be imaging them (for example, spectrum of Cy3 with 405 and
> 543 laser ON, if you will be imaging DAPI and Cy3 at the same time). I
> don't remember how the unmixing would work in multi track mode. But again,
> I see no obvious reason why you wouldn't just image your samples in the
> normal (emission filter/standard P<MT) mode. The imaging will be much
> simpler, and the data quality probably better.
>
>
> Julio Vazquez
> Fred Hutchinson Cancer Research Center
> Seattle, WA 98109
>
>
http://www.fhcrc.org/en.html>
> --
>
>
>
> On Jul 31, 2014, at 2:47 PM, Anders Lunde wrote:
>
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> > To join, leave or search the confocal microscopy listserv, go to:
> >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> > Post images on
http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Dear Confocal List,
> >
> > Im a PhD student trying to do some colocalization anlysis on multiple
> > transcription factors in mouse neural tissue using uimmunofluorescence.
> To
> > get as clean data as possible I want to utilize the "online
> fingerprinting"
> > capabillity of our Zeiss 510 META confocal microscope.
> >
> > I understand that the first step is to generate reference samples for
> each
> > fluorophore. So far so good. But what I am confused about is that in most
> > tutorials etc. it seems that online fingerprinting is explained in
> samples
> > where you only use one laser-line to excite all your fluorophores. For
> > example: I plan on doing online fingerprinting with Hoechst, Alexa 488,
> > Cy3, and Cy5. There is really no practical way to use the same laser to
> > exite both Hoeckst and Cy5. So optimally I would like to use 4 different
> > lasers in sequential scanning for the 4 different fluorophores. Is this
> > compatible with online fingerprinting? In my mind I guess this means that
> > for all reference samples I have to obtain reference spectra of each
> > fluorophore with all 4 lasers? So that means 4*4=16 reference spectra (+
> > background and autofluorencese references)? Moreover, I want to do this
> on
> > Z-stack samples, but that should be possible, right?
> >
> >
> > Another question: While it is important to use the same dichroic mirrors
> > and settings for the reference samples and real sample imaging, this will
> > not be possible if I want to image 4 fluorophores, because (as far as I
> > remember) the dichroic mirror is only optimized for 3 wavelengths so I
> have
> > to switch when going from Hoechst to, say, Cy5. However, this issue might
> > be solved if it turns out that I only need to generate 4 reference
> spectra,
> > and not each for each laser line (4*4=16), due to me misunderstanding the
> > concept properly.
> >
> > Lastly, while it is important to use the same dichroic mirrors and other
> > settings for the reference samples and real sample imaging, can digital
> > offset and detector gain be adjusted when imaging real samples? If not it
> > might be difficult to get the grey values withing optimal ranges as there
> > is some natural variability between samples.
> >
> > Best regards.
>