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Dear Listserv,
Aryeh Weiss posted FITC data from Martin Wessendorf at
http://optics.jct.ac.il/~aryeh/Spectra/ASCII/
FITC-XScan & FITC.gp values for various laser lines (normalized to peak)
are:
458 nm ... 0.296
476 nm ... 0.53
488 nm ... 0.888
495 nm ... 1.0
514 nm ... 0.284
532 nm ... 0.246
543 nm ... 0.012
Aryeh's site has data for many other fluorophores.
I did not see what emission wavelength this was recorded at.
With respect to Sarah's comment that it would be better to post excitation
than absorption data - I encourage measuring and posting both. I think that
Molecular Probes, as well as Chroma Technology, deserve kudos for posting as
many spectra as they have. Carl Boswell and I have had excellent success in
getting numerical spectral data from many other vendors, including Omega
Optical, Evident Tech, and QDots. I've also on occasion used Un-Scan-It to
convert graphs to numbers. I strongly encourage all researchers to publish
your numerical data, either as supplementary information at the journal
site, or on your own web site. There are also XML standards efforts such as
SpectroML, and NIH/NCBI's PubChem site will serve up spectra as well as HTS
data for the 100K+ compounds they are aiming for.
Enjoy,
George
George McNamara, Ph.D.
Imaging Scientist
Congressman Julian Dixon Image Core
The Saban Research Institute of Childrens Hospital Los Angeles
4650 Sunset Blvd., MS 133, SRT 1016
Los Angeles, CA 90027
323-669-2548 voice
323-251-8878 cell
323-668-7921 fax
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http://shelia/image Image Core Web (CHLA Intranet)
\\ntapps49\home Image Core User Home (Intranet)
\\ntapps49\appnotes Image Core Application Notes (Intranet)
http://www.childrenshospitalla.org/research.html CHLA Saban Research
Institute
http://home.earthlink.net/~geomcnamara Personal web site
http://home.earthlink.net/~mpmicro Microscopy web site
http://www.mcb.arizona.edu/ipc/fret/default.html Boswell & McNamara
Fluorescence Spectra Site
"Old soldiers never die; they just fade away." - D. Macarthur.
"Old antibodies die; please throw them away." - GM.
-----Original Message-----
From: Locknar, Sarah A [mailto:[log in to unmask]]
Sent: Thursday, June 24, 2004 7:05 AM
To: [log in to unmask]
Subject: Re: FITC and ALEXA 555 ...
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi-
I'm not an expert on FITC excited state manifolds, but I DO know a lot
about polyene excited states which could shed some light on the matter.
This may be kind of hard to follow without an energy-level (Jablonski)
diagram, but here goes:
1. Polyenes have a low-lying excited state (2Ag) which is "forbidden"
for absorption. This would be observed as a very small red peak to the
right of the main absorption band. Of course the size of the peak
depends on how well the polyene obeys the symmetry approximation.
2. Emission from 2Ag is also forbidden, but the long-wavelength
emission tail on FITC suggests that there may be some emission from this
state or an equivalent one.
3. Absorption spectrum is not equivalent to excitation spectrum. It
really irritates me that Molecular probes publish absorbtion spectra
instead of excitation spectra.
4. What I suspect is that the emission spectrum shown on the Molecular
Probes website is from excitation at 495 nm. That way, emission from
the low-lying 2Ag state is small because it only occurs when there is
internal conversion from 1Bu (allowed) to 2Ag. Internal conversion
efficiencies are usually highly dependent on solvents which tend to
break symmetry in the chromophores and change the overlap between the
states.
5. If one did an excitation spectrum using emission at 565 nm or so
(where there looks to be a small hump in excitation), I wouldn't be
surprised to find a higher wavelength (i.e. greater than 495 nm) peak
corresponding to the direct ground-state to 2Ag transition which was
essentially invisible with absorption.
6. The upshot is that not all emission is completely relaxed (obeying
Kasha's rule). In fact, for polyenes (like the Cy-dyes), MOST of the
emission is non-Kasha emission from the 1Bu state. The implication is
that there may also be small amounts of excitation and emission from 2Ag
that are not evident in the Molecular Probes spectra. There also may be
transitions to other triplet states from 2Ag, or other photochemistry
occuring from the 2Ag state (in polyenes, photoisomerization is presumed
to occur through the 2Ag state for example) both of which can cause
"photobleaching".
I think fluorescein is pretty well studied, so an inspection of the
literature should reveal whether there are any low-lying excited states
to worry about. This all just points out that things are rarely as
simple as Molecular Probes would have you believe.
Sarah
------------------------------------------------------------------------
---------------
Sarah Locknar, Ph.D.
Director, Neuroscience COBRE Imaging / Physiology Core
College of Medicine, University of Vermont
E015 Given Building
89 Beaumont Ave.
Burlington, VT 05405
802-656-0413
802-656-8704 (fax)
------------------------------------------------------------------------
---------------
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Alberto Diaspro
Sent: Thursday, June 24, 2004 8:53 AM
To: [log in to unmask]
Subject: Re: FITC and ALEXA 555 ...
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
On 24 giu 2004, at 14:17, Martin Wessendorf wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Were both dyes present in the same domain--e.g., in the same cell (and
> cell compartment)? If so, is the Alexa 555 bleaching as well?
>
> Martin Wessendorf
>
> Aryeh Weiss wrote:
>
>> Alberto Diaspro wrote:
>>
>>> in order to check some possible FRET between FITC and ALEXA 555,
>>> when using bleaching of acceptor as method, we found that 543 nm
>>> bleached FITC as well. Did you ever experienced such a behaviour?
>>>
>>
>> We have observed this also. I had been meaning to repeat the
>> experiment because I was very surprised, but it you also observed
>> this then I have more confidence that it is real.
> --
> Martin Wessendorf, Ph.D. office: (612) 626-0145
> Assoc Prof, Dept Neuroscience lab: (612) 624-2991
> University of Minnesota Preferred FAX: (612) 624-8118
> 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
> Minneapolis, MN 55455 E-mail: [log in to unmask]
>
>
------------------------------------------------------------------------
---------------------------
Alberto Diaspro, Department of Physics, University of Genoa
Via Dodecaneso 33, 16146 Genova, Italy
facsimile +39-010314218 - voice +39-0103536426/480/309
URL: http://www.lambs.it
http://www.wiley.com/WileyCDA/WileyTitle/productCd-0471409200.html
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