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Dear Oliver,

I was also confused by the literature on Evans Blue fluorescence. Strangely, the best combination for the optics on a good old MRC-1024 was to use an excitation of 568 nm and a 585 nm dichroic. This allowed visualizing motoneurons retrogradely labeled with Evans Blue that was previously injected in the muscle. See the following papers for some images:

Nguyen et al. (2004) J. Physiol. 556, 203-219
Nguyen and Kleinfeld (2005) Neuron 45, 447-457

Maybe the fluorescence depends on where Evans Blue is bound. Also, I could not get any two-photon images with Evans Blue with the same specimens, owing possibly to the symmetrical structure of Evans Blue that would preclude multiphoton excitation.
 
Hope this helps.


Quoc Thang Nguyen
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On Nov 5, 2013, at 12:08 PM, Oliver Biehlmaier wrote:

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> 
> Thanks for the links and answers! That helps.
> We might also go for measuring with a spectrophotometer...
> Cheers,
> Oliver
> 
> ----------------------------------------------------------------
> 
> Oliver Biehlmaier, PhD
> 
> Head of Imaging Core Facility 
> 
> Biozentrum (Kragenbau, Room G1054)
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> 
> Am 05.11.2013 um 17:30 schrieb Chris Tully:
> 
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>> 
>> Oliver,
>> 
>> I would highly recommend asking around among your local colleagues for someone who has a fluorescence spectrophotometer, and measure the curves yourself.
>> 
>> Alternatively, I have read this article because I do not have access to it at the moment but according to Google it does have a UV-Vis absorbance spectrum.
>> 
>> Chris
>> 
>> On 11/5/2013 10:45 AM, Oliver Biehlmaier wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>> 
>>> Dear listers,
>>> 
>>> I have a question regarding Evans Blue:
>>> 
>>> I have a user that is doing standard 3ch-labelings on a confocal microscope (DAPI, Alexa488, Alexa 568). Now she is having some sections from mice that have been injected with Evans Blue.
>>> When imaging these new samples, she wants to use the same 3ch approach as before but she does not want to see any fluorescence from the Evans Blue.
>>> I tried to find out what the excitation and emission spectrum of Evans Blue is but I could not find any clear information. E.g. one publication specifies the spectrum of Evans Blue as follows: excitation at 620 nm, emission at 680 nm (Saria A, Lundberg JM. Evans blue fluorescence: quantitative and morphological evaluation of vascular permeability in animal tissues. J Neurosci Methods. 1983 May;8(1):41-9. PubMed PMID: 6876872.) Others claim that it fluoresces with excitation peaks at 470 and 540 nm and that it has its emission peak at 680 nm. I tend towards the first spectrum but I did not find any other confirmation for it.
>>> 
>>> Does anyone of you guys know the exact spectrum of that dye?
>>> This would be great!
>>> 
>>> Cheers,
>>> Oliver
>>> 
>>> ----------------------------------------------------------------
>>> 
>>> Oliver Biehlmaier, PhD
>>> 
>>> Head of Imaging Core Facility
>>> 
>>> Biozentrum (Kragenbau, Room G1054)
>>> University of Basel
>>> Klingelbergstrasse 50/70
>>> 4056 Basel
>>> Switzerland
>>> 
>>> Office:	+41 (61) 267 20 73
>>> Lab:        +41 (61) 267 22 50
>>> Email:     [log in to unmask]
>>> http://www.biozentrum.unibas.ch/imcf
>>> http://microscopynetwork.unibas.ch
>>> ----------------------------------------------------------------
>> 
>> 
>> -- 
>> *Chris Tully*
>> Principal Consultant
>> Image Incyte, LLC
>> 240-475-9753
>> 
>> 
>>    Image Incyte, LLC
>> 
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