Article

Cite This: Acc. Chem. Res. 2019, 52, 110−118 pubs.acs.org/accounts

Photophysics at Unusually High Dye Concentrations

Hernán B. Rodríguez,† Martín Mirenda,§ M. Gabriela Lagorio,‡,∥ and Enrique San Román*,‡,∥
†
Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA, UNLP-CONICET), Facultad de Ciencias Exactas,
Universidad Nacional de La Plata, Diagonal 113 y 64 S/N, B1904DPI La Plata, Argentina
§
Gerencia Química, Centro Atómico Constituyentes, Comisión Nacional de Energía Atómica (CNEA-CONICET), Av. Gral. Paz
1499, B1650KNA San Martín, Buenos Aires, Argentina
‡
CONICET - Universidad de Buenos Aires, Instituto de Química Física de los Materiales, Medio Ambiente y Energía
(INQUIMAE), Ciudad Universitaria Pab. II, C1428EHA Buenos Aires, Argentina
∥
Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Química Inorgánica, Analítica y Química
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Física, Ciudad Universitaria, Pab. II, C1428EHA Buenos Aires, Argentina

*
S Supporting Information
Downloaded via UNIV DE GUANAJUATO on May 11, 2019 at 00:48:57 (UTC).

CONSPECTUS: The study of the interaction of light with systems at high dye
concentrations implies a great challenge because several factors, such as emission
reabsorption, dye aggregation, and energy trapping, hinder rationalization and
interpretation of the involved photophysical processes. Space constraints induce
dye interaction even in the absence of ground state stabilization of dimers and
oligomers. At distances on the order of 1 nm, statistical energy traps are usually
observed. At longer distances, excited state energy transfer takes place. Most of
these factors do not result in ground state spectroscopic changes. Rather,
fluorescence phenomena such as inner filter effects, concentration-dependent
Stokes’ shifts, and changes in quantum yields and decay times are evidenced.
Photophysical studies are commonly carried out at high dilution, to minimize
dye−dye interactions and emission reabsorption, and in the absence of light
scattering. Under these conditions, the physical description of the system
becomes rather simple. Fluorescence and triplet quantum yields become
molecular properties and can be easily related to ratios of rate constants. However, many systems containing dyes able to
fulfill specific functions, whether man-made or biological, are far from being dilute and scattering-free. The photosynthetic
apparatus is a paradigmatic example. It is clear that isolation of components allows gathering relevant information about
complex systems. However, knowledge of the photophysical behavior in the unaltered environment is essential in most cases.
Complexity generally increases when light scattering is present. Despite that, our experience shows that light scattering, when
correctly handled, may even simplify the task of unraveling molecular parameters. We show that methods and models aiming at
the determination and interpretation of fluorescence and triplet quantum yields as well as energy transfer efficiencies can be
developed on the basis of simple light-scattering theories.
Photophysical studies were extended to thin films and layer-by-layer assemblies. Procedures are presented for the evaluation of
fluorescence reabsorption in concentrated fluid solutions up to the molar level, which are being applied to ionic liquids, in which
the emitting species are the bulk ions. Fluorescence reabsorption models proved to be useful in the interpretation of the
photophysics of living organisms such as plant leaves and fruits. These new tools allowed the assessment of chlorophyll
fluorescence at the chloroplast, leaf and canopy levels, with implications in remote sensing and the development of
nondestructive optical methods.

1. INTRODUCTION dye aggregation, inner filter effects, self-quenching, and light

Upon performing experimental work on the photochemistry of
heterogeneous systems focused on singlet molecular oxygen
photosensitizers,1 we realized that general methods able to
quantify the efficiency of such materials were rather scarce. As
a first step, we explored the fluorescence of microparticles
containing dyes. Some methods originally used in the study of
inorganic phosphor films were indeed available. However, they
are relatively complex and require specially designed
integrating spheres.2 Furthermore, efforts to extract the
photophysics of dyes in their actual environment, affected by
scattering, were not undertaken in a comprehensive way. To
address the problem, we asked ourselves if introducing light
scattering in a systematic way, instead of avoiding or reducing
its effect, would simplify the task. The quantification of color in
light-scattering materials,3 an old problem from the paper,
textile, and coatings industry, gave us the answer in the form of
a model based on the rather simple Kubelka−Munk theory.4
Received: June 7, 2018
Published: November 28, 2018

© 2018 American Chemical Society 110 DOI: 10.1021/acs.accounts.8b00271

Acc. Chem. Res. 2019, 52, 110−118
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The Kubelka−Munk theory describes the radiation field
within ideal (Lambertian) light-scattering solids, that is, those
showing the same radiance irrespective of the observation
angle, in terms of two average radiation fluxes perpendicular to
the irradiated surface, traveling forward and backward relative
to the illumination beam. Based on this theory, a model for
reabsorption and reemission of fluorescence was developed for
light-scattering materials, which will be hereafter referred to as
the modified Kubelka−Munk (mKM; a summary of all
symbols and abbreviations is included in the Supporting
Information) model.5 This model can be applied to optically
thick solids (those showing negligible transmittance) such as
microcrystalline cellulose beads and other substrates in which
dyes and mixtures of dyes have been embedded.
Figure 1 shows at the left side an arbitrary light path in a
system of micrometric particles: an excitation photon is
Figure 1. An arbitrary light path within a system of microparticles, the
modified Kubelka−Munk model, and the schematic microstructure of
a plant leaf. Arrows: excitation in green; fluorescence in yellow.
scattered upon hitting a particle and absorbed by a second
particle, yielding an emitted fluorescence photon further
scattered until it eventually emerges at the surface (radiation-
less deactivation is omitted in the figure). The mKM model
principle is schematized roughly in the middle of the figure. It
incorporates the emitted light into the two-flux Kubelka−
Munk theory. The excitation beam is attenuated by absorption
and scattering yielding two opposite fluxes (not shown) within
the solid. Part of the absorbed radiation results in fluorescence
emission, which is also assumed to travel forward and
backward undergoing again absorption and scattering.
Reabsorbed emission yields two secondary fluorescence fluxes
and so forth. Most strikingly, the model has been successfully
applied to optically thick groups of intact plant leaves, whose
complex microstructure is depicted at the right side of the
figure.
Later on, we undertook the study of dyes included into spin-
coated polymeric and self-assembled polyelectrolyte thin films,
concentrated solutions, ionic liquids, and biological organisms
(chloroplasts, plant leaves, flowers, fruits, and a fluorescent
frog). The typical feature of these systems, whether devoid of
or affected by light scattering, is the presence of dyes in a wide
range of local concentrations, reaching in some cases 1 M.
Irrespective of the particular purpose of each project, a general
objective was to obtain information about the relevant
photophysical processes. In all cases, fluorescence was the
essential analytical tool, as intensity, spectrum, and lifetime
changes carry information about trivial and nonradiative
energy transfer, self-quenching and energy trapping.
Different models were conceived to correct observed
emission spectra in order to obtain reabsorption-free
fluorophore spectra in the actual environment in which they
are embedded. The already cited mKM model was applied to
highly concentrated artificial systems to obtain fluorescence
spectra and quantum yields devoid of concentration artifacts.
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This knowledge is relevant for the design of heterogeneous
photosensitizers, highly efficient in the capture and use of light
energy. Other developments allowed the study of emission
from fruits and fluorescent animals and the extrapolation of
plant fluorescence to canopies, leading in the latter case to a
powerful nondestructive analytical methodology for the remote
sensing of vegetation. We also dedicated efforts to the study of
reabsorption and reemission of fluorescence in concentrated
solutions of dyes and to unravel the role of concentration on
fluorescence and singlet oxygen photogeneration in thin films.
2. SPECTRUM, QUANTUM YIELD, AND EFFICIENCY
Most reference books recommend the acquisition of
fluorescence spectra at low absorbance, generally <0.05.6
Otherwise, inner-filter effects would result in an uneven
distribution of excitation and emission within the sample. The
example of a 1.3 × 10−4 M solution of rhodamine B in ethanol
is shown in Figure 2. It is clearly seen how the emission
Figure 2. Fluorescence from a 1.3 × 10−4 M ethanolic solution of
rhodamine B, path length 1 cm. The sample is illuminated from the
left side at different wavelengths. The red curve represents the fraction
of light absorbed by the sample, 1−10−A, where A is the absorbance at
the excitation wavelength.
volume shifts to the excitation side when absorbance grows. A
decrease in the measured fluorescence quantum yield upon
fluorescence reabsorption is found as well.
In practice, however, photoactive materials typically involve
pigment absorbances in excess of 0.05. The acquisition of
fluorescence from biological samples must be also performed
in environments in which concentration and, hence,
absorbance cannot be controlled. The measurement of
fluorescence spectra and quantum yields is particularly
troublesome under these conditions.
The quantum yield is defined as the number of events (e.g.,
emitted fluorescence photons, generated triplet states) divided
by the number of absorbed photons.7 In what follows three
different quantum yields will be distinguished:8 observed or
technical (Φ obs), corrected or “true” (Φ cor), and molecular or
at infinite dilution (Φ ∞ or simply Φ ). The quantity usually
measured, Φ obs (see Figure 3), may be affected in concentrated
solutions or solid media by reabsorption and reemission of
fluorescence and, eventually, by absorption of the solvent or
supporting material.
Our aim was to develop models to obtain Φ cor, a magnitude
devoid of fluorescence reabsorption and reemission artifacts.
Even after correction, the quantum yield might differ from Φ ∞
if dye−dye interactions cannot be neglected. The value of Φ ∞
can be expressed as a ratio of molecular rate constants of the
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Figure 3. Observed quantum yields. The quantity Np is the number of
incident photons at the excitation wavelength λ0; expressions within
parentheses at the bottom represent the fraction of incident photons
absorbed by the sample: A is the absorbance for transparent media,
and R and T are the reflectance and transmittance, respectively, for
light-scattering media, all quantities measured at λ0.
absorbing molecule in its actual environment.8 To relate Φ cor
and Φ ∞, theories for energy transfer and trapping were used
(see below). We developed instrumental techniques to
determine absolute quantum yields of light-scattering materi-
als, as observed fluorescence quantum yields, Φ F,obs, from
diffuse reflectance9 and Φ F,obs or observed triplet quantum
yields, Φ T,obs, from laser-induced optoacoustic spectroscopy10
measurements.
Unlike Φ obs, which refers to absorbed photons, the quantum
efficiency, η, of a photoactive material (called external quantum
efficiency in LEDs and solar cell technology) can be defined as
the ratio of the number of events and the number of incident
photons, Np (Figure 3). Both values are related by the fraction
of incident photons absorbed by the sample:
η = Φobs(1 − 10−A) (1)
for transparent media and
η = Φobs(1 − R − T ) (2)
for light-scattering samples. In principle, to maximize η the
fraction of absorbed radiation can be enhanced by increasing
dye concentration. However, molecular crowding reduces the
distance between chromophores and enhances molecular
interaction, leading to a loss of dye individuality and the
opening of radiationless excited state deactivation channels,
thus impairing photoactivity. The interplay between these two
opposite effects determines that maximum efficiency can be
obtained in a short interval of dye concentrations. The search
for enhancement of light collection, avoiding energy wasting,
requires understanding of the photophysics of dyes at
unusually high concentrations.
3. PHOTOPROCESSES IN HIGHLY ABSORBING
SYSTEMS
3.1. Reabsorption and Reemission of Fluorescence
11,12
More than 60 years ago, Birks handled emission of highly
absorbing samples from a probabilistic point of view as
resulting from an infinite set of reabsorption and reemission
events, obtaining
ΦF,obs
ΦF,cor =
1 − P + PΦF,obs (3)
where P is the reabsorption probability of an emitted photon.
An extended version of eq 3 is presented in the Supporting
Information (SI, eq 1). From the study of light-scattering
materials, we derived the following expression for P:5
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P= ∫λ f (λ)[1 − γ(λ , λ0)] dλ; ∫λ f (λ) dλ = 1 (4)
where f(λ) is the fluorescence spectrum of the dye devoid of
reabsorption and reemission effects (e.g., measured for a thin
layer of particles) normalized to unit area and γ(λ,λ0) is the
probability that an emitted photon at wavelength λ resulting
from excitation at λ0 escapes out of the sample. Equation 4
may be applied to any fluorescent material and even to
transparent solutions. The function γ(λ,λ0), which assumes
different expressions according to the nature of the system and
the way fluorescence is detected, depends on (a) the
concentration and the absorption coefficient of the emitter at
λ and λ0 and (b) the geometry of the whole arrangement,
implying a strong dependence of emission fluxes on the
position where emission takes place. The Supporting
Information contains a set of equations for the evaluation of
γ(λ,λ0) for optically thick light-scattering materials (SI, eq 3),
transparent media in a collinear excitation−emission arrange-
ment (SI, eq 12) and a biological light-scattering layer with
nonzero transmittance (SI, eq 10).
The function γ(λ,λ0) derived from the mKM model can be
used to correct the shape of the observed fluorescence
spectrum, fobs(λ,λ0), of optically thick solid light-scattering
samples:
fcor (λ) = fobs (λ , λ 0)/γ(λ , λ 0) (5)
Figure 4a illustrates how emission shifts to shorter wavelengths
and changes its band ratio as the correction is applied to an
optically thick layer of methylene blue on microcrystalline
cellulose.13 Excellent agreement is found between the
corrected spectrum, fcor(λ), and the experimental fluorescence
spectrum in conditions at which reabsorption is negligible.
At emission wavelengths for which γ(λ,λ0) ≈ 0, the result of
eq 5 is undetermined and calculation of fcor(λ) from fobs(λ,λ0)
is not possible. Instead, multiplying the experimental
fluorescence spectrum at infinite dilution by γ(λ,λ0), a
calculated spectrum is obtained that can be compared with
fobs(λ,λ0). Figure 4b shows results for a transparent solution of
9,10-diphenylanthracene in toluene. The excellent agreement
found shows that dye−dye interactions can be neglected.
Observed fluorescence spectra, fobs(λ,λ0), and quantum
yields, Φ F,obs, are useful for the characterization of luminescent
materials or studying fluorescence biosignals, because the
radiation that escapes from the system is the signal recognized
by a receptor (photodetector, living organism, etc.). Con-
versely, corrected and calculated magnitudes contain relevant
information for the understanding of the photophysical
processes taking place at the molecular or organelle level.
3.2. Light-Scattering Solid Particles
Reabsorption effects are particularly relevant for light-
scattering materials when absorption and emission spectra
substantially overlap. Figure 5 shows results obtained for cresyl
violet included into microcrystalline cellulose.9 It can be
observed that Φ F,obs decreases as dye concentration grows and
is larger near λ0 = 600 nm, where absorption is stronger. On
the other hand, Φ F,cor, coincident with Φ F,∞, is constant and
similar to the value found in solution of alcohols. This fact
shows that dye−dye interactions, and hence concentration
quenching, are negligible at all concentrations quoted in Figure
5b.
For dyes embedded in solids, concentration quenching is
generally ascribed to the formation of molecular aggregates.
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Figure 4. (a) Normalized fluorescence spectra and γ values of a light
scattering sample containing 3.39 μmol of methylene blue per gram of
microcrystalline cellulose, λ0 = 620 nm: observed spectrum (red),
corrected spectrum (dashed blue) obtained dividing the former by
γ(λ,λ0) (SI, eq 3, green), optically thick layer in both cases, and
spectrum of a thin layer of the same particles (black). Spectra were
measured in front-face geometry, detecting emission at 90° from the
incident beam. Adapted with permission from ref 13. Copyright 2006
Wiley. (b) Normalized observed fluorescence spectrum of a
transparent solution of 9,10-diphenylanthracene in toluene at infinite
dilution (black) and calculated spectrum for a 0.013 M solution
(dashed blue), obtained by multiplying the former by γ(λ,λ0) (SI, eq
12, green). The observed fluorescence spectrum at the same
concentration (red) is scaled for comparison. Spectra were measured
in transmission geometry, detecting emission at the opposite side of
excitation with a 0.2 cm path length, λ0 = 350 nm, A = 14 (calculated
from the known molar absorption coefficient). Adapted with
permission from ref 21. Copyright 2017 American Chemical Society.
However, when dye−support interactions are strong, ground
state dye−dye interactions are generally weakened. The
aggregation tendency is thus low and high concentrations
can be reached without spectroscopic detection of molecular
aggregates. Despite this, self-quenching attributed to excitation
energy trapping centers (statistical traps) still takes place.
Traps are formed by molecular pairs or multiplets interacting
in the excited state, brought together as a consequence of
molecular crowding. Energy trapping results either from the
direct absorption of excitation light by traps or by radiative
(fluorescence reabsorption) and nonradiative energy transfer
from monomeric dyes, presumably by Förster resonance
energy transfer (FRET). For the general case of an energy
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Figure 5. Observed (a) and corrected (b) fluorescence quantum
yields for light scattering samples of cresyl violet included into
microcrystalline cellulose as a function of the excitation wavelength.
Dye concentration (0.045 to 0.14 μmol g−1) increases from top to
bottom in panel a. The most concentrated sample is not shown in
panel b. Reproduced with permission from ref 9. Copyright 2004
American Chemical Society.
donor D and a nonemitting acceptor A included in a
Lambertian scatterer, Φ F,obs can be expressed as a function of
the probabilities of fluorescence reabsorption, fluorescence
escape, and nonradiative energy transfer (EDA) (SI, eq 8).
Upon correction for reabsorption and reemission of
fluorescence, EDA can be calculated as a function of
concentration (SI, eq 9):
ΦF,cor
E DA = 1 −
ΦF,∞ (6)
This is the case of rhodamine 6G adsorbed on microcrystal-
line cellulose shown in Figure 6, for which Φ F,cor/Φ F,∞ drops as
the amount of dye increases.14 Dye concentration is expressed
as a molecular surface density, σ. According to the specific
surface area of cellulose (60 m2 g−1), the dye concentration is 1
μmol g−1 for σ = 0.01 (molecules) nm−2. A model based on
Poisson statistics and FRET between monomeric dyes and
dimeric or multimeric traps allowed expression of EDA as a
function of a quenching radius, rQ, that is, the limiting distance
at which two dye molecules constitute a trapping center,
yielding rQ = 1.5 nm. Beddard and Porter have shown that
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xanthene dyes in microcrystalline cellulose evidenced the

formation of photoactive traps capable of yielding triplet states
by a charge-transfer assisted mechanism.20
3.3. Transparent Media
Figure 7 shows results obtained for transparent solutions of
9,10-diphenylanthracene21 in toluene and quinine bisulfate in

Figure 6. Φ F,cor/Φ F,∞ as a function of molecular surface density for

rhodamine 6G adsorbed on microcrystalline cellulose. Experimental
results (open circles) and model fittings (solid lines) considering self-
quenching by statistical traps with a quenching radius of 1.5 nm;
dynamic (pink area) and static (green area) quenching counterparts
are discriminated (see text). The line dividing both areas was obtained
considering only dynamic quenching, that is, disregarding the fraction
of excitation light absorbed by traps. Inset: energy flow diagram
illustrating photophysical processes for a system composed by
monomers (energy donors) and nonfluorescing statistical traps
(acceptors). Adapted with permission from ref 14. Copyright 2008
Wiley.

chlorophyll molecules in higher plants have to be on average 1

nm apart to prevent trap formation by orbital overlap.15
The photophysical processes so far described can be
interpreted by an energy flow diagram, where the monomeric
dye is the donor and statistical traps are the acceptors (see
inset in Figure 6). The observed fluorescence is a result of all
possible pathways in the diagram and their respective
probabilities. Quenching may be static if excitation light is
absorbed by preformed nonemitting traps, thus not affecting
fluorescence lifetimes, or dynamic if light is absorbed by
monomers and nonradiatively transferred to traps. Notice that
dynamic quenching does not imply diffusional processes but
excitation energy trapping mediated by FRET, leading to a
reduction in fluorescence lifetime.
Various ways have been envisaged to enhance solar cell
efficiencies, among them solar concentrators.16 The use of
molecules acting as antennae for the collection and transfer of
excitation energy to an effector center constitutes another
possible strategy aiming to broaden the absorption spectrum.17
Our approach allows the quantitative evaluation of energy
transfer efficiencies in D−A systems.13,18,19 It was demon-
strated that high energy transfer probabilities can be achieved
for randomly distributed dyes. Nonradiative transfer proba-
bilities up to 60% were obtained for rhodamine 101 (donor)
and methylene blue (acceptor) included into microcrystalline
cellulose, reaching an overall transfer probability of 80% when
reabsorption is considered.18
Reabsorption repopulates excited states, thus enhancing
triplet state or photoproduct formation. Corrections of
observed triplet quantum yields for fluorescence reabsorption
can be performed for light-scattering samples by using the
mKM model (SI, eq 5). Recently, the comparison between
corrected fluorescence and triplet quantum yields for different
114
Figure 7. Φ F,obs (red dots), Φ F,cor (blue dots), and Φ F,∞ (green lines)
as a function of dye concentration for (a) 9,10-diphenylanthracene in
toluene and (b) quinine bisulfate in acidic water solution. The upper
axis in both figures is the reabsorption probability. Panel a adapted
with permission from ref 21. Copyright 2017 American Chemical
Society. Panel b created with raw data extracted from ref 22.
acidic water22 (Φ F,cor was obtained from Φ F,obs applying SI eq
11). Apparently, both dyes behave in a similar way, as Φ F,obs
decreases strongly with concentration. However, for 9,10-
diphenylanthracene Φ F,cor ≈ Φ F,∞ over a three-order of
magnitude concentration range, meaning that self-quenching
does not take place. On the other hand, for quinine bisulfate
Φ F,cor ≠ Φ F,∞ at concentrations on the order of 10−3 M and
higher, pointing to the occurrence of self-quenching. Notice
that the reabsorption probability P is very small for quinine
bisulfate because absorption and emission spectra (not shown)
are widely separated. The opposite holds for 9,10-diphenylan-
thracene, for which the Stokes shift is smaller.
The aforementioned methodology can be applied to the
incipient field of fluorescent ionic liquids bearing cationic or
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Figure 8. Emission of the highly fluorescent ionic liquid BMIM3HPTS in BMIMCl. The system is excited from the right side with a blue laser (405
nm) in dilute (a) and concentrated (b) solutions and with a green laser (532 nm) in dilute (c) and concentrated (d) solutions (see text).
anionic chromophores.23 Figure 8 shows the emission of tris(1-
butyl-3-methylimidazolium) 8-hydroxypyrene-1,3,6-trisulfo-
nate (BMIM3HPTS).24 When diluted with the slightly
fluorescing ionic liquid 1-butyl-3-methylimidazolium chloride
(BMIMCl), a very strong blue fluorescence is observed for
excitation with 405 nm, arising from HPTS anions. Excitation
with green light at 532 nm does not yield any detectable
fluorescence. In concentrated solution, however, an unex-
pected orange fluorescence is observed when exciting with 532
nm. The nature of this unusual emission can be tentatively
assigned to the interaction of HPTS anions with BMIM cation
aggregates.25 There is much work to be done on these
materials, mainly considering that solutions of some dyes in
ionic liquids can be used in highly promising applications, as
mirrorless dye doped ionic liquid lasers.26 Since oxygen is
scarcely soluble in the ionic liquid, chromophores are generally
very stable in these media.
Highly concentrated systems such as transparent thin films
with very small optical paths are not always affected by
fluorescence reabsorption. Despite that, concentration can be
so large that statistical trapping takes place. Excitation energy-
trapping models for transparent systems27,28 can be applied to
describe their photophysics. An example is shown in Figure 9
for phloxine B included into poly(2-hydroxyethyl methacry-
late) (pHEMA) thin films,29 yielding rQ = 1.2 nm. The relative
Figure 9. Dependence of Φ F,obs (red circles) and Φ Δ,rel (blue circles)
with dye concentration for phloxine B included into pHEMA
nanometric thin films. The solid line is a model fitting considering
energy traps with a quenching radius of 1.2 nm. Graphical inset:
relative excited state generation efficiency as a function of dye
concentration; the dashed line is a tendency curve without physical
meaning. Picture inset: trap formation at short dye−dye distances.
Adapted from ref 29 with permission from the European Society for
Photobiology, the European Photochemistry Association, and The
Royal Society of Chemistry.
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singlet oxygen quantum yield, Φ Δ,rel, follows the same trend as
Φ F,obs, and the efficiency of singlet oxygen formation at the
surface reaches a maximum at ca. 0.02 M. Similar results were
obtained for rose bengal included into pHEMA, but in this
case, the formation of photoactive traps (rQ = 1.5 nm) capable
of yielding fluorescence and singlet oxygen was evidenced.30
According to the photoactive nature of traps, the dye
concentration at which the maximum efficiency is attained
(ca. 0.15 M) increases. Photoactive traps were also found for
rose bengal in microcrystalline cellulose.31,32
Dye concentrations of about 1 M were obtained for layer-by-
layer self-assembled thin films composed of rose bengal and
poly[diallyldimethylammonium] chloride (PDDA), showing
measurable fluorescence and singlet oxygen photogeneration
even at these extreme conditions.33 In this case, however, dyes
are not randomly located, as the unfolded conformation of
PDDA provides uniformly distributed charged sites for rose
bengal adsorption, thus leading to some degree of molecular
organization, which markedly reduces dye aggregation and
energy trapping.
3.4. Light-Scattering Biological Systems
Biological systems are strongly light-scattering materials usually
bearing elevated chromophore concentrations. The chlorophyll
fluorescence emerging from chloroplasts is currently used to
obtain information on photosynthesis and plant health through
remote sensing.34 The emission ratio red/far-red (680:730
nm) is used as an indicator of plant stress.35 However, the
observed fluorescence should be corrected for reabsorption
and reemission within the leaf. The correction factor γ(λ,λ0)
obtained from the mKM model (SI, eq 3) can also be applied
to optically thick groups of leaves.36,37 Figure 10 shows nicely
how the correction works.
When fluorescence from crops and forests is remotely
analyzed, an additional correction factor is needed, as leaf
fluorescence is further reabsorbed by the canopy. The mKM
model cannot be applied because the canopy is not optically
thick. Therefore, the system has to be modeled as a layer
(canopy) with a back face (soil) able to absorb and reflect
light. A suitable correction factor (SI, eq 10) was obtained
under the assumption of exponential attenuation of absorption
and fluorescence within the canopy (see Figure 11).38
Consequently, the sequential application of the two
corrections, that is, canopy to leaf and leaf to chloroplasts,
allows obtaining chloroplast information from the canopy
fluorescence spectrum.
The correction factors for canopies and transparent
solutions rely on similar arguments: absorption and emission
are exponentially attenuated, and emission profiles result from
the superposition of fluorescence originating at different layer
depths.
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Figure 10. Normalized fluorescence spectra of Ficus benjamina leaves and chloroplasts. The spectra of an optically thick group of leaves show
important fluorescence reabsorption. Once corrected, spectra are coincident with those of a thin layer of isolated chloroplasts, not affected by
reabsorption. Adapted from ref 36 with permission from the European Society for Photobiology, the European Photochemistry Association, and
The Royal Society of Chemistry.
Figure 11. (a) Photon fluxes involved in the interaction between light
and canopy. (b) Experimental canopy fluorescence spectrum (blue)
and retrieved fluorescence spectra at leaf (green) and chloroplast
(black) levels. I0 and IF stand for incident and fluorescence light
intensity; R, RB, and TL (variable or subscript) are total reflectance,
background (soil) reflectance, and canopy layer transmittance,
respectively; R′ is the reflectance of an optically thick group of
leaves. Adapted with permission from ref 38. Copyright 2018 Elsevier.
For multilayered fluorescent structures, it is frequently
worthwhile to evaluate the contribution of each layer to the
fluorescence observed for the whole system. An estimation of
the observed fluorescence spectrum may be obtained as the
sum of the fluorescence spectra of all layers, excited by the
incident light attenuated by the upper layers and filtered by the
slabs through which fluorescence is transmitted. This approach
was used to study the fluorescence spectra of kiwi39 and
eggplant40 fruits and the first fluorescent frog discovered
around the planet.41 While reabsorption models allow
calculation of the fluorophore spectrum free of artifacts due
to the high chromophore concentration, the present approach
seeks to reproduce the global observed fluorescence spectrum
from the quantitative contributions of each component in the
system. Figure 12 depicts the model layout for the kiwi fruit.
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Figure 12. Pictorial description of light fluxes for the kiwi fruit. I0,
intensity of the excitation beam at λ0; IF,pulp and IF,peel, fluorescence
intensities measured for the isolated layers; T(λ) and T(λ0), peel
transmittance at the emission and excitation wavelengths, respectively.
Adapted from ref 39 with permission from the European Society for
Photobiology, the European Photochemistry Association, and The
Royal Society of Chemistry.
The dilemma frequently arises whether fluorescence emitted
by living organisms has some signaling function or it is merely
a nonfunctional result of the chemical nature of tissues.35
Therefore, it is important as a first step to compare the number
of emitted photons (Φ F,obs times the amount of absorbed
photons) with the number of photons reflected by the material.
This treatment was applied to flower petals to demonstrate
that fluorescence cannot be considered an attraction signal
toward pollinators, to a blue-winged parrolet, showing
substantial sexual dichromatism in UV-induced fluorescence,35
and to a frog, whose fluorescence contributed up to around
30% to the optical signal.41 It has to be recalled that, during
studies of fluorescence as a biosignal, Φ F,obs instead of Φ F,cor
should be used because photons reabsorbed by the tissues fail
to come out.
IV. CONCLUSIONS AND OUTLOOK
The methodology so far developed allows for a quantitative
evaluation of the escape probability of emitted light and triplet
and photoproduct formation in different environments, such as
solutions, thin films, particulate solids, and biological
structures. It permits the retrieval of photophysical information
at molecular and organelle levels and constitutes a powerful
analytical tool for the design of efficient photoactive materials
as well as for the monitoring of biological organisms. The
design of biomimetic systems is another field to which the
approach discussed in this work can be applied.
Generally, concentration quenching imposes a practical limit
to the concentration of dyes. In solution, it is principally
DOI: 10.1021/acs.accounts.8b00271
Acc. Chem. Res. 2019, 52, 110−118
Accounts of Chemical Research
related to dye aggregation and diffusional processes, whereas in
solid materials it may be associated with the presence of
statistical traps. In the latter case, the quenching radius
represents a key parameter for the design of random and
nonrandom arrays of dyes in order to avoid cross-talk among
individual dye molecules, thus ensuring low losses by energy
trapping. On the other hand, trapping can be harnessed in
donor−acceptor systems, where donor molecules act as a light-
harvesting antenna.
For intact biological organisms, the described approach
allows the identification of the region where the fluorescence
originates and its contribution to the total observed emission.
It also enables the quantitative assessment of fluorescence in
optical biosignaling. Moreover, it permits the elimination of
reabsorption artifacts in order to obtain spectral information
connected with the physiological state of the organism,
relevant to nondestructive analytical methodologies and for
the growing field of remote sensing of vegetation.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.ac-
counts.8b00271.
Glossary of symbols and abbreviations and a set of
equations with appropriate references to fluorescence
reabsorption−reemission models (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: esr@qi.fcen.uba.ar.
ORCID
Hernán B. Rodríguez: 0000-0001-7766-6933
Martín Mirenda: 0000-0003-0478-5178
M. Gabriela Lagorio: 0000-0003-3761-6733
Enrique San Román: 0000-0002-1166-9928
Author Contributions
H.B.R., M.M. and M.G.L. contributed equally to this
manuscript.
Notes
The authors declare no competing financial interest.
Biographies
Hernán B. Rodrı ́guez graduated in Biochemistry in 2001 at the
National University of Patagonia San Juan Bosco in Comodoro
Rivadavia. He obtained his Ph.D. in Chemical Sciences at the
University of Buenos Aires (UBA) in 2009. He belongs to the staff of
the National Scientific and Technological Research Council of
Argentina (CONICET) at the Research Institute of Theoretical and
Applied Physical Chemistry in La Plata. Present research interests are
the development of heterogeneous photosensitizers, the photophysics
and photochemistry of dyes in nanostructured heterogeneous media
and the study of molecular interactions and energy and charge transfer
processes.
Martin Mirenda graduated in Chemical Sciences in 2003 at UBA. He
obtained his Ph.D. in Chemical Sciences at UBA in 2009. He is
presently Professor at the School of Science and Technology,
National University of San Martin and belongs to the research staff
of CONICET and the National Atomic Energy Commission. Present
117
Article
research interests are photophysics in highly absorbing media and
innovation in liquid materials for efficient management of excitation
energy, either by light or ionizing radiation.
M. Gabriela Lagorio obtained her B.S. (Chemical Sciences) in 1982
at UBA. She worked in the private industry from 1983 to 1987.
Subsequently, she started her research activities, obtaining her Ph.D.
in Chemical Sciences (UBA) in 1991. She is presently Professor at
UBA and researcher of CONICET. Her research field is reflectance
and fluorescence of plant material and hybrid systems, and her work is
focused on the modeling and analysis of the interaction of light with
biological material.
Enrique San Román obtained his Ph.D. in Chemistry at UBA in
1977. He is presently retired and holds a Consulting Full Professor
position at UBA and a contract from CONICET at the Institute of
Physical Chemistry of Materials, Environment and Energy (UBA-
CONICET). Present research interests include the photochemistry of
heterogeneous materials and the development of instrumental
methods and theoretical tools for their study.
■
ACKNOWLEDGMENTS
The authors dedicate this work to Silvia E. Braslavsky for her
standing support and encouragement. This work has been
supported by UBACyT 20020130100166BA, CONICET
PIP11220110100467 and PIP11220130100795, and ANPCyT
PICT2014-2153.
■
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118 DOI: 10.1021/acs.accounts.8b00271

Acc. Chem. Res. 2019, 52, 110−118