Excitation and Emission of Evans Blue

[Oliver Biehlmaier-2]

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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
Klingelbergstrasse 50/70
4056 Basel
Switzerland

Office: [phone]
Lab:        [phone]
Email:     [hidden email]
http://www.biozentrum.unibas.ch/imcf
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----------------------------------------------------------------

[Cromey, Douglas W - (dcromey)]

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Vendor's cube recomaendations
http://www.microscopyu.com/articles/fluorescence/filtercubes/yellow/yellowhome.html
http://www.lifetechnologies.com/us/en/home/life-science/cell-analysis/cellular-imaging/cell-imaging-systems/evos-led-light-cubes/evos-dye-compatibility.html 

See DB53: http://tdl.libra.titech.ac.jp/z3950/eespdf/10106030/V0134I01/00005492/00005492.pdf

I guess this question has come up here before: http://lists.umn.edu/cgi-bin/wa?A2=ind9805&L=CONFOCALMICROSCOPY&P=14267

From Histo-net: http://www.histosearch.com/histonet/Oct98A/Re.EvansBlueotherfluoresc.html

Hope this helps.
Doug

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Associate Scientific Investigator
Dept. of Cellular & Molecular Medicine, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

office:  AHSC 4212         email: [hidden email]
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Oliver Biehlmaier
Sent: Tuesday, November 05, 2013 8:46 AM
To: [hidden email]
Subject: Excitation and Emission of Evans Blue

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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
Klingelbergstrasse 50/70
4056 Basel
Switzerland

Office: [phone]
Lab:        [phone]
Email:     [hidden email]
http://www.biozentrum.unibas.ch/imcf
http://microscopynetwork.unibas.ch
----------------------------------------------------------------

[Chris Tully-2]

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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: [phone]
> Lab:        [phone]
> Email:     [hidden email]
> http://www.biozentrum.unibas.ch/imcf
> http://microscopynetwork.unibas.ch
> ----------------------------------------------------------------

--
*Chris Tully*
Principal Consultant
Image Incyte, LLC
240-475-9753


      Image Incyte, LLC

<http:%5C%5Cwww.ImageIncyte.com> [hidden email]
<mailto:[hidden email]>

[Jeremy Adler-4]

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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 [[hidden email]] on behalf of Oliver Biehlmaier [[hidden email]]
Sent: 05 November 2013 16:45
To: [hidden email]
Subject: Excitation and Emission of Evans Blue

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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
Klingelbergstrasse 50/70
4056 Basel
Switzerland

Office: [phone]
Lab:        [phone]
Email:     [hidden email]
http://www.biozentrum.unibas.ch/imcf
http://microscopynetwork.unibas.ch
----------------------------------------------------------------

[Oliver Biehlmaier-2]

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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)
University of Basel
Klingelbergstrasse 50/70
4056 Basel
Switzerland

Office: [phone]
Lab:        [phone]
Email:     [hidden email]
http://www.biozentrum.unibas.ch/imcf
http://microscopynetwork.unibas.ch
----------------------------------------------------------------




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: [phone]
>> Lab:        [phone]
>> Email:     [hidden email]
>> http://www.biozentrum.unibas.ch/imcf
>> http://microscopynetwork.unibas.ch
>> ----------------------------------------------------------------
>
>
> --
> *Chris Tully*
> Principal Consultant
> Image Incyte, LLC
> 240-475-9753
>
>
>     Image Incyte, LLC
>
> <http:%5C%5Cwww.ImageIncyte.com> [hidden email] <mailto:[hidden email]>

[Quoc Thang Nguyen-2]

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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
[hidden email]



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

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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)
> University of Basel
> Klingelbergstrasse 50/70
> 4056 Basel
> Switzerland
>
> Office: [phone]
> Lab:        [phone]
> Email:     [hidden email]
> http://www.biozentrum.unibas.ch/imcf
> http://microscopynetwork.unibas.ch
> ----------------------------------------------------------------
>
>
>
>
> 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: [phone]
>>> Lab:        [phone]
>>> Email:     [hidden email]
>>> http://www.biozentrum.unibas.ch/imcf
>>> http://microscopynetwork.unibas.ch
>>> ----------------------------------------------------------------
>>
>>
>> --
>> *Chris Tully*
>> Principal Consultant
>> Image Incyte, LLC
>> 240-475-9753
>>
>>
>>    Image Incyte, LLC
>>
>> <http:%5C%5Cwww.ImageIncyte.com> [hidden email] <mailto:[hidden email]>

[George McNamara]

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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:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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 [[hidden email]] on behalf of Oliver Biehlmaier [[hidden email]]
> Sent: 05 November 2013 16:45
> To: [hidden email]
> Subject: Excitation and Emission of Evans Blue
>
> *****
> 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: [phone]
> Lab:        [phone]
> Email:     [hidden email]
> http://www.biozentrum.unibas.ch/imcf
> http://microscopynetwork.unibas.ch
> ----------------------------------------------------------------
>
>    

--



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/

[Steffen Dietzel]

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On 05.11.2013 21:47, Quoc Thang Nguyen wrote:
>
> 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.
>

Multi-photon excitation worked nice on our system, with fluorescence in
the 665 nm channel. So it works in principle. I seem to remember that
excitation was around 830 nm but am not sure about it.

Steffen

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
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[jmvdwin]

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Dear,

no personal experience but see:

Li JL, Goh CC, Keeble JL, Qin JS, Roediger B,
Jain R, Wang Y, Chew WK, Weninger W, Ng LG.
Intravital multiphoton imaging of immune
responses in the mouse ear skin. Nat Protoc 2012;7:221-234
http://www.ncbi.nlm.nih.gov/pubmed/22240584

p223 - quote

Table 1 | List of vessel-labeling agents.
Vessel-labeling agentsAdvantagesDisadvantages
Evans blue dyeInexpensive
Bright
Relatively nontoxic
Good contrast over a wide range of excitation wavelengths (720-990 nm)
Sharp emission peak at ~680 nm
Obvious visual cue to the success of an
injectionLeakage into mouse ear interstitium
end quote

Sincerely.

JM

At 14:52 6/11/2013, you wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>On 05.11.2013 21:47, Quoc Thang Nguyen wrote:
>>
>>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.
>
>Multi-photon excitation worked nice on our
>system, with fluorescence in the 665 nm channel.
>So it works in principle. I seem to remember
>that excitation was around 830 nm but am not sure about it.
>
>Steffen
>
>--
>------------------------------------------------------------
>Steffen Dietzel, PD Dr. rer. nat
>Ludwig-Maximilians-Universität München
>Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
>Head of light microscopy
>
>Mail room:
>Marchioninistr. 15, D-81377 München
>
>Building location:
>Marchioninistr. 27,  München-Großhadern

Jean-Marie.

Jean-Marie Vanderwinden, M.D., Ph.D.
Directeur de Recherche F.N.R.S. / Research
Director, National Fund for Scientific Research (Belgium)

Université Libre de Bruxelles (ULB)

ULB Neuroscience Institute - Neurophysiology lab
http://www.ulb.ac.be/medecine/neurophy/

Light Microscopy Facility http://limif.ulb.ac.be/

Postal address:
Laboratoire de Neurophysiologie (Prof. S.N. Schiffmann),
Faculté de Médecine, Campus Erasme, CP 601,
Université Libre de Bruxelles,
808 route de Lennik, B-1070 Brussels, Belgium.

(: Secretary: +(32).2.555.42.30, Direct: +(32).2.555.69.88
7: +(32).2.555.41.21,
. [hidden email]

Skype: Jean Marie Vanderwinden
12voip / netappel: jeanmarievanderwinden  

[Quoc Thang Nguyen-2]

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Dear all,

If Evans Blue peaks at ~ 680 nm I was probably not looking at it with the right optics on our 2-P scope. Forget what I said about Evans Blue not visible under 2-P.

Quoc Thang Nguyen
[hidden email]



On Nov 6, 2013, at 7:13 AM, Jean-Marie Vanderwinden wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear,
>
> no personal experience but see:
>
> Li JL, Goh CC, Keeble JL, Qin JS, Roediger B, Jain R, Wang Y, Chew WK, Weninger W, Ng LG. Intravital multiphoton imaging of immune responses in the mouse ear skin. Nat Protoc 2012;7:221-234
> http://www.ncbi.nlm.nih.gov/pubmed/22240584
>
> p223 - quote
>
> Table 1 | List of vessel-labeling agents.
> Vessel-labeling agentsAdvantagesDisadvantages
> Evans blue dyeInexpensive
> Bright
> Relatively nontoxic
> Good contrast over a wide range of excitation wavelengths (720-990 nm)
> Sharp emission peak at ~680 nm
> Obvious visual cue to the success of an injectionLeakage into mouse ear interstitium
> end quote
>
> Sincerely.
>
> JM
>
> At 14:52 6/11/2013, you wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> On 05.11.2013 21:47, Quoc Thang Nguyen wrote:
>>>
>>> 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.
>>
>> Multi-photon excitation worked nice on our system, with fluorescence in the 665 nm channel. So it works in principle. I seem to remember that excitation was around 830 nm but am not sure about it.
>>
>> Steffen
>>
>> --
>> ------------------------------------------------------------
>> Steffen Dietzel, PD Dr. rer. nat
>> Ludwig-Maximilians-Universität München
>> Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
>> Head of light microscopy
>>
>> Mail room:
>> Marchioninistr. 15, D-81377 München
>>
>> Building location:
>> Marchioninistr. 27,  München-Großhadern
>
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