Excitation spectrum with excitation to S2 or higher?

[Steffen Dietzel]

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Dear all,

I guess all of us who are teaching fluorescence microscopy came to the
point where they mentioned excitation to S2 and higher states, and that
this shows in the excitation spectrum. I was wondering whether anybody
knows of an example where a specific higher energy state (S2, S3, S4...)
can be assigned to a specific second maximum in the excitation spectrum.

Some spectra have pretty nice local maxima, like Texas Red around 550
nm, or at least shoulders like TRITC around 520 nm. But is this S2 or S3
or even higher? The problem arises because if S2 has only a little more
energy than S1 the two peaks will not be resolvable.

So I was hoping that somebody may know of an example where the shoulder
or secondary maximum can be assigned to a specific state. Preferably of
a widely used fluorochrome, but anything is welcome. If it should come
from a citable source, even better.

Best

Steffen

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[James D. Manton]

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Dear Steffen,

Azulene is a nice example, with the article 'Control of the
Photophysical Properties of Polyatomic Molecules by Substitution and
Solvation:  The Second Excited Singlet State of Azulene' by Tétreault et
al. (doi.org/10.1021/jp984407q) having a nice figure (1) of the
absorption spectrum of 1,3-difluoroazulene showing the S0 − S3, S0 − S2
and S0 − S1 transitions as nicely separated regions.

Best wishes,
James

[Alex Asanov]

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Hi Steffen,

Porphyrins with their S0-->S2 Soret bands stronger than S0-->S1, and vibronic bands clearly dependent on the chelated  metal and substitutes at porphyrin ring, are definitely the champions and excellent objects for your purpose, I guess. There is vast literature on fluorescence  of porphyrins. E.g. http://www1.lasalle.edu/~prushan/Abs%20and%20Fluor%20of%20TPPH2.pdf.

Best regards,
Alexander N. Asanov, Ph.D.
President,  TIRF Labs
[hidden email]
www.tirf-labs.com   www.TIRFmicroscopy.com



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
Sent: Tuesday, May 29, 2018 3:41 AM
To: [hidden email]
Subject: Excitation spectrum with excitation to S2 or higher?

*****
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*****

Dear all,

I guess all of us who are teaching fluorescence microscopy came to the
point where they mentioned excitation to S2 and higher states, and that
this shows in the excitation spectrum. I was wondering whether anybody
knows of an example where a specific higher energy state (S2, S3, S4...)
can be assigned to a specific second maximum in the excitation spectrum.

Some spectra have pretty nice local maxima, like Texas Red around 550
nm, or at least shoulders like TRITC around 520 nm. But is this S2 or S3
or even higher? The problem arises because if S2 has only a little more
energy than S1 the two peaks will not be resolvable.

So I was hoping that somebody may know of an example where the shoulder
or secondary maximum can be assigned to a specific state. Preferably of
a widely used fluorochrome, but anything is welcome. If it should come
from a citable source, even better.

Best

Steffen

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de


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[Iain Johnson]

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Steffen:

The absorption peak of fluorescein (or FITC, or whatever you choose to call
it) at about 320 nm is the S0 to S2 transition.  The best way to determine
this (for fluorescein or any other fluorphore) is to look at the
fluorescence polarization excitation spectrum.  This because the S0 to S2
absorption transition is typically orthogonal to the S0 to S1 transition
that generates the fluorescence emission. In the case of fluorescein (and
other similar dyes such as rhodamines), the S0 to S1 transition is parallel
to the long axis of the aromatic ring system.  S2 to S0 is perpendicular to
the ring system.  So excitation into S2 gives rise to negative fluorescence
polarization, whereas S0 to S1 excitation gives positive fluorescence
polarization.

Iain

On Tue, May 29, 2018 at 8:10 AM, Alexander Asanov <[hidden email]
> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Steffen,
>
> Porphyrins with their S0-->S2 Soret bands stronger than S0-->S1, and
> vibronic bands clearly dependent on the chelated  metal and substitutes at
> porphyrin ring, are definitely the champions and excellent objects for your
> purpose, I guess. There is vast literature on fluorescence  of porphyrins.
> E.g. http://www1.lasalle.edu/~prushan/Abs%20and%20Fluor%20of%20TPPH2.pdf.
>
> Best regards,
> Alexander N. Asanov, Ph.D.
> President,  TIRF Labs
> [hidden email]
> www.tirf-labs.com   www.TIRFmicroscopy.com
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Steffen Dietzel
> Sent: Tuesday, May 29, 2018 3:41 AM
> To: [hidden email]
> Subject: Excitation spectrum with excitation to S2 or higher?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear all,
>
> I guess all of us who are teaching fluorescence microscopy came to the
> point where they mentioned excitation to S2 and higher states, and that
> this shows in the excitation spectrum. I was wondering whether anybody
> knows of an example where a specific higher energy state (S2, S3, S4...)
> can be assigned to a specific second maximum in the excitation spectrum.
>
> Some spectra have pretty nice local maxima, like Texas Red around 550
> nm, or at least shoulders like TRITC around 520 nm. But is this S2 or S3
> or even higher? The problem arises because if S2 has only a little more
> energy than S1 the two peaks will not be resolvable.
>
> So I was hoping that somebody may know of an example where the shoulder
> or secondary maximum can be assigned to a specific state. Preferably of
> a widely used fluorochrome, but anything is welcome. If it should come
> from a citable source, even better.
>
> Best
>
> Steffen
>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Biomedical Center (BMC)
> Head of the Core Facility Bioimaging
>
> Großhaderner Straße 9
> D-82152 Planegg-Martinsried
> Germany
>
> http://www.bioimaging.bmc.med.uni-muenchen.de
>
>
> ---
> This email has been checked for viruses by Avast antivirus software.
> https://www.avast.com/antivirus
>