lipophilic dye fare red with narrow excitation peak

[Thomas Horn]

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Hello all,
we are currently using DiD to label a structure on a micro fluidics device. However, the cells that we need to visualize do contain mCherry. Problem is that the absorption/excitation curve of DiD has a long tail "on the left" so that 561 excites it very well. As the DiD fluorescence is very strong we do get a lot of Signal in the mCherry channel when exciting with a 561nm laser -  even if we use a small band pass on the lower emission spectrum of mCherry. The DiD signal is very strong versus a weak mCherry so that spectral unmixing  is problematic.

To cut a long story short: can someone recommend a lipophilic   dye that is good for 647 excitation but is not excited by 561nm.

Kind regards,
Thomas




Dr. Thomas Horn,
The Single Cell Unit, U1.46
Department of Biosystems Science and Engineering (D-BSSE)
Swiss Federal Institute of Technology Zurich (ETH)
Mattenstrasse 26
CH 4048 Basel
Switzerland
Phone: [phone]
mail: [hidden email]

[John Oreopoulos]

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Hi Thomas,

Since the lipophilic probes like DiI, DiO, and DiD usually absorb and emit light strongly compared to mCherry (as in your case), perhaps you could switch over to the longer wavelength probe DiR (no commercial interest):

https://products.invitrogen.com/ivgn/product/D12731?ICID=search-product

This probe has a much lower absorption at 647 nm, but I bet you'll still be able to get plenty of signal to form an image with the same laser line. The emission is further out in the red around 780 nm, so you'll need a different emission filter (your existing filter might work if it's a long-pass), but the bleedthrough into your mCherry channel should be much more reduced since the absorption at 561 nm is less than 5%.

You didn't mention what type of imaging system you're using. If it's a laser scanning confocal with a PMT, you might need to play with the settings a bit. If it's a camera-based system (like a spinning disk confocal), you should have no problems since most CCDs can still detect out in that wavelength range. Don't bother trying to look for the probe in the eyepieces, though!


Cheers,


John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2012-10-22, at 10:46 AM, Horn Thomas wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello all,
> we are currently using DiD to label a structure on a micro fluidics device. However, the cells that we need to visualize do contain mCherry. Problem is that the absorption/excitation curve of DiD has a long tail "on the left" so that 561 excites it very well. As the DiD fluorescence is very strong we do get a lot of Signal in the mCherry channel when exciting with a 561nm laser -  even if we use a small band pass on the lower emission spectrum of mCherry. The DiD signal is very strong versus a weak mCherry so that spectral unmixing  is problematic.
>
> To cut a long story short: can someone recommend a lipophilic   dye that is good for 647 excitation but is not excited by 561nm.
>
> Kind regards,
> Thomas
>
>
>
>
> Dr. Thomas Horn,
> The Single Cell Unit, U1.46
> Department of Biosystems Science and Engineering (D-BSSE)
> Swiss Federal Institute of Technology Zurich (ETH)
> Mattenstrasse 26
> CH 4048 Basel
> Switzerland
> Phone: [phone]
> mail: [hidden email]

[George McNamara]

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DiR
abs max 750 nm ... about 20% of max at 633 nm
em  max 780 nm ... FW10% is 740 to 850 nm
Not all detectors count photons in the near infrared. Not all filters
are blocked in the NIR.

http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html
(The U arizona spectra Viewer has bad trace for DiR emission)

note some "alternation" (I think they should have written "alteration")
of spectra of DiI and DiD by confocal laser illumination was reported in:

    Michal Majkowski, Pawel Póda, Julita Kulbacka, Jolanta  Saczko,and
    Aleksander F. Sikorski


            Alternation of Fluorescent Spectra of Membrane Markers DiI
            C18(3) and DiI C18(5) Evoked by Laser Illumination

    J Histochem Cytochem October 2012 60: 789-791, first published on
    July 21, 2012 doi:10.1369/0022155412457572

    http://jhc.sagepub.com/content/current


Personally, I would not worry about the effect(s).


George




On 10/22/2012 10:46 AM, Horn Thomas wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello all,
> we are currently using DiD to label a structure on a micro fluidics device. However, the cells that we need to visualize do contain mCherry. Problem is that the absorption/excitation curve of DiD has a long tail "on the left" so that 561 excites it very well. As the DiD fluorescence is very strong we do get a lot of Signal in the mCherry channel when exciting with a 561nm laser -  even if we use a small band pass on the lower emission spectrum of mCherry. The DiD signal is very strong versus a weak mCherry so that spectral unmixing  is problematic.
>
> To cut a long story short: can someone recommend a lipophilic   dye that is good for 647 excitation but is not excited by 561nm.
>
> Kind regards,
> Thomas
>
>
>
>
> Dr. Thomas Horn,
> The Single Cell Unit, U1.46
> Department of Biosystems Science and Engineering (D-BSSE)
> Swiss Federal Institute of Technology Zurich (ETH)
> Mattenstrasse 26
> CH 4048 Basel
> Switzerland
> Phone: [phone]
> mail: [hidden email]
>
>