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See
Optical imaging to map blood-brain barrier leakage
http://www.nature.com/srep/2013/131101/srep03117/full/srep03117.html
(open access)
"EB dye fluoresces with excitation peaks at 470 and 540 nm and
an emission peak at 680 nm. The fluorescence of EB, coupled with the
sensitivity of an optical /in vivo/ two-dimensional planar fluorescence
imaging system (Maestro EX, Caliper Life Sciences, Inc., Hopkinton, MA),
that works on the principle of exciting fluorescent dye and recording
the intensity of the emitted fluorescence over a range of wavelengths,
allowed us to determine the extent of dye accumulation in various brain
locations in response to BBB disruption."
The methods mention making up the test dilutions in saline - so Jeremy's
point about EB vs EB:Albumin could affect your vs their results.
"A stock solution of EB dye (40 mg/ml in normal saline) was
diluted serially with saline to obtain dye solutions of desired
concentrations. A 5-?l aliquot of each dye solution was placed on a
filter paper disc 7 mm in diameter (Whatman Limited, Kent, UK); filter
papers were allowed to dry at room temperature for 1 hr. Discs were
imaged using the Maestro EX Optical Imaging System (Caliper Life
Sciences, Hopkinton, MA). Its blue filter was set between 500 and 720 nm
wavelengths for image acquisition, with 787 ms exposure time. The
near-infrared filter was set between wavelengths of 740 nm and 950 nm,
with 1183.04 ms exposure time. The blue filter captures any
autofluorescence and provides an outline of the sample; the
near-infrared filter visualizes EB. Total signal intensities of each
disc were measured by drawing regions of interest. To obtain a standard
plot using UV spectrophotometry, the stock solution of EB dye was
diluted with saline to obtain dye solutions of different concentrations."
For spectral unmixing, consider evaluating
http://www.mh-hannover.de/cellneurophys/poissonNMF/
George
On 11/5/2013 10:43 AM, Jeremy Adler wrote:
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>
> slightly off topic
> Evans blue is used to tag albumin but there is an equlibrium between albumin-bound and free Evans blue, so the presence of Evans blue suggests but is not proof that albumin-Evans blue has escaped from the vasculature.
> ________________________________________
> From: Confocal Microscopy List [[log in to unmask]] on behalf of Oliver Biehlmaier [[log in to unmask]]
> Sent: 05 November 2013 16:45
> To: [log in to unmask]
> Subject: Excitation and Emission of Evans Blue
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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>
> 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
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> Office: +41 (61) 267 20 73
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>
--
George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/26/
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