Posted by
Julio Vazquez on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Online-fingerprinting-on-Zeiss-510-META-basic-questions-tp7582451p7582454.html
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Yes, you get fairly strong reflections over the parts of the spectrum that overlap with the laser lines (at least we did with our confocal). However, if I remember correctly, in the case where you use multiple excitation lines in the same track, these will be collected (you can select the band for acquisition, but can not turn OFF individual bins during image acquisition, although you can tell software to ignore them later on). This is why it is preferable to do the online fingerprinting with a single excitation line, so you don't have to worry about this. If you decide to do it with four tracks, i.e. one per channel, then you would need four spectra (one for each dye acquired with its own specific laser line), and you may also need to collect spectra for the autofluorescence/background, so maybe four more spectra (the background may be different for different excitations). Now my memories of the 510 and linear unmixing are a bit rusty, so I can't be of much help for the actual procedure of collecting four channels in multi-track mode with online fingerprinting. For instance, I don't remember if the Zeiss software has a direct way of handling multi-track unmixing, but I am guessing that there should be no special issue with treating each dye separately. For example, you can use 488 excitation and extract the Alexa 488 signal from the background, and repeat this for all other dyes. Then you rebuild your four channel image from the extracted channels. However, it may be possible to do that a bit more directly. For that, depending on where you are located, you may be able to get a hold of one of Zeiss' applications specialists, and they could guide you through the procedure, or you could contact their support in NY if you're in the US (1-800-509-3905). They also have some info here:
http://zeiss-campus.magnet.fsu.edu/articles/spectralimaging/index.htmlJulio
On Jul 31, 2014, at 4:12 PM, Anders Lunde wrote:
> Dear Julio,
>
> 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,
> autofluorescence, and possibly excessive bleed through due to large
> difference in concentration of fluorophores, and I want to examine
> posibillities for eliminating this. I also use a ImageJ plugin to analyze
> the final images, and the plugin does much better with a better signal to
> noise ratio (which I hope I can get by removing some background
> autofluorescece).
>
> By blocking laser lines, do you mean that the bins (10nm) that are directly
> illuminated by a laser should be turned off?
>
> And yes, I mean that I dont have a 4 line dichroic mirror.