Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
So, it seems difficult to photobleach FITC at 543 nm, I mean, these are
data for the pure molecule in solution, right pH etc.... I consider the
second column as a sort of cross-section related data. Is it correct?
Now the problem is what does it happen to the fluorophore in a real
life!?
Alby
On 24 giu 2004, at 17:22, Mcnamara, George wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> 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
> [log in to unmask]
> 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
> -----------------------------------------------------------------------
> -
> --------------------------
>
>
------------------------------------------------------------------------
---------------------------
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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