Fluorescence lifetime of the fluorescein

[Ihalainen Teemu Olavi]

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Dear list,

Im sorry to bother you all with so trivial matter.

Does somebody have a good reference for the fluorescence lifetime of the
free fluorescein in different pH environments (aqueous solutions)? I
would really appreciate your help.

Best,
Teemu Ihalainen

--
Teemu Ihalainen, Ph.D.
Swiss Federal Institute of Technology (ETH) Zurich
ETH Hönggerberg
Laboratory for Biologically Oriented Materials
Wolfgang-Pauli Str. 10, HCI E417
CH-8093 Zürich
Tel.: [phone]

[George McNamara]

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Hi Teemu,

See figure 1 of:

Physiol Meas. 2005 Aug;26(4):N9-12. Epub 2005 Apr 15.

Sodium fluorescein as a retinal pH indicator?
Hammer M, Schweitzer D, Richter S, Königsdörffer E.

Department of Ophthalmology, University of Jena, Bachstr. 18, 07740 Jena,
Germany. [hidden email]

Abstract
Retinal neovascularization is a symptom associated with various diseases
revealing ocular fundus manifestation. Often, these neovascularizations
originate from retinal hypoxia. A concomitant phenomenon of hypoxia is
acidosis. To recognise this would permit the identification and treatment of
hypoxic fundus areas long before first vascular modifications are seen. Thus,
the goal of this investigation was to elucidate whether sodium fluorescein
could be used as a retinal pH indicator. Sodium fluorescein solution was diluted
in PBS (ratio: 1:150,000). The pH was varied from 6.5 to 8.6 by
supplementation of HCl or NaOH, respectively. The fluorescence was excited
by a pulsed diode laser (wavelength: 446 nm, pulse width: 100 ps) and
detected by time-correlated single photon counting (TCSPC) technique. A
least-squares fit of the measured fluorescence decay versus time by an
exponential function results in the fluorescence lifetime. Ten measurements
were taken at each pH for statistical analysis. The dependence of the
fluorescence lifetime on the temperature and the concentration of sodium
fluorescein was investigated in the same way. The fluorescence lifetime was
found to rise from 3.775 ns to 4.11 ns with increasing pH (6.5 to 8.6).
However, the gradient decreases with increasing pH. We found highly
significant differences (Student's t-test, P<0.0005) of the fluorescence
lifetimes for pH values with a mean difference of 0.125 at pH<7.65 whereas
the differences were still significant (P<or=0.02) at pH>7.65 and mean pH
differences of 0.2. The fluorescence lifetime was independent of the
temperature (22 degrees C to 37 degrees C) and the concentration of sodium
fluorescein (dilution 1:150,000 to 1:2000). The fluorescence lifetime of sodium
fluorescein depends on the pH but not on temperature and concentration.
Thus, the discrimination of areas with retinal acidosis should be possible by
combination of the TCSPC technique with scanning laser ophthalmoscopy.
Further investigations have to clarify whether the accuracy of the
measurement at the fundus in vivo is sufficient.

PMID: 15886427

and references cited.


You might also find useful information in this paper on fluorescein's closely
related Oregon Green 488 (or perhaps the other JPCA articles by Orte ...
Boens on this fluorophore):

J Phys Chem A. 2005 Feb 10;109(5):734-47.

Absorption and emission study of 2',7'-difluorofluorescein and its excited-state
buffer-mediated proton exchange reactions.
Orte A, Crovetto L, Talavera EM, Boens N, Alvarez-Pez JM.

Department of Physical Chemistry, University of Granada, Cartuja Campus,
Granada 18071, Spain.

Abstract
2',7'-Difluorofluorescein (Oregon Green 488) is a new fluorescein-based dye,
which has found many applications, above all in biochemistry and
neurosciences, and its use has become very popular in the last years. In
recent years, we have been investigating the excited-state proton exchange
reactions of fluorescein and the effect of suitable proton acceptors and
donors which promote these reactions. The excited-state proton transfer
reactions may appreciably influence the fluorescence results when using these
dyes. We present steady-state emission evidence that acetate buffer species
promote an excited-state proton transfer between neutral, monoanionic, and
dianionic forms of 2',7'-difluorofluorescein. The time course of the excited
species in this reaction was characterized through time-resolved fluorescence
measurements, and the kinetics of the reaction was solved by using the global
compartmental analysis. A previous identifiability study on the compartmental
system set the conditions to design the fluorescence decay surface. This is
the first experimental system, studied within this kinetic model, solved under
identifiability conditions through global compartmental analysis. The recovered
rate constant values for deactivation were 2.94 x 10(8) s(-1) for the
monoanion and 2.47 x 10(8) s(-1) for the dianion, whereas the rate constant
values of the buffer-mediated excited-state reaction were 9.70 x 10(8) and
1.79 x 10(8) M(-1) s(-1) for the deprotonation and protonation, respectively.
With these values, a pK(a) = 4.02 was obtained. In this work, we additionally
provide an absorption study, including acid-base equilibria, determination of
ground-state pK(a) values (1.02, 3.61, and 4.69), and recovery of molar
absorption coefficients of every prototropic species, including absorption and
NMR evidence for the existence of three tautomers in neutral species. Steady-
state emission spectra of 2',7'-difluorofluorescein in aqueous solution are also
described, where the strong photoacid behavior of the cation is noteworthy.

PMID: 16838941

Lakowicz also published papers on SNARF and other fluorescein analogs, and
might have material in his Principle of Fluorescence Spectroscopy book.

On Wed, 1 Dec 2010 17:30:49 +0100, Teemu Ihalainen
<[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear list,
>
>Im sorry to bother you all with so trivial matter.
>
>Does somebody have a good reference for the fluorescence lifetime of the
>free fluorescein in different pH environments (aqueous solutions)? I
>would really appreciate your help.
>
>Best,
>Teemu Ihalainen
>
>--
>Teemu Ihalainen, Ph.D.
>Swiss Federal Institute of Technology (ETH) Zurich
>ETH Hönggerberg
>Laboratory for Biologically Oriented Materials
>Wolfgang-Pauli Str. 10, HCI E417
>CH-8093 Zürich
>Tel.: [phone]