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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

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On Jul 31, 2014, at 2:47 PM, Anders Lunde wrote:

> *****
> 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.