Posted by
Wendy Salmon on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Online-fingerprinting-on-Zeiss-510-META-basic-questions-tp7582451p7582472.html
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopyPost images on
http://www.imgur.com and include the link in your posting.
*****
Dear Anders,
This is a common type of sample challenge I see in my core. Based on my experience, I think your time will be better spent optimizing your sample preparation rather than figuring out the linear unmixing. As you are experiencing and Julio states, unmixing is often more challenging than it sounds. Additionally, by splitting the signal into the small bins for the spectrum you make a huge sacrifice in SNR.
What type of sample optimization have you done? Spending a week on sample optimization can save you weeks of instrument and processing time as well as make conclusions more obvious.
In case you haven't already tried them, here are a few adjustments (in order) that help my users who have similar problems:
Autofluorescence:
There are a lot of previous discussions on autofluorescence in the list archive so I won't go into details, but the two main things to consider are fixation --fresh paraformaldehyde versus formalin is transformative-- and clearing or quenching agents
Bleedthrough:
Ideally your sample will have similar intensities in each channel. If you are staining with antibodies or dyes, there are a couple strategies to achieve this:
- First, for channels that are bright, play the "fluorescence limbo"--see how low you can go.
- If that is not possible (such as if the bright channel is a fluorescent protein and the dim channel is low abundance), you can try amplifying a dim antibody with TSA amplification (note: not good for quantifying intensity, which is a bag of worms unto itself).
Finally, if adjusting the staining conditions and autofluorescence isn't sufficient, you can measure the bleedthrough in standard imaging (using single-channel samples) and correct for it in the post processing. For more details on this, check the archives or let us know.
Best,
Wendy
Wendy Salmon
Light Microscopy Specialist
Whitehead Institute for Biomedical Research
W.M. Keck Imaging Facility
9 Cambridge Center, Rm 447
Cambridge, MA 02142
------------------------------
Date: Fri, 1 Aug 2014 01:12:13 +0200
From: Anders Lunde <
[hidden email] >
Subject: Re: Online fingerprinting on Zeiss 510 META basic questions
*****
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 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.
On Fri, Aug 1 , 2014 at 12:56 AM, Julio Vazquez <
[hidden email] > 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.
> *****
>
> 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