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www.rsc.org/pccp | Physical Chemistry Chemical Physics

Manipulation of the local density of photonic states to elucidate

fluorescent protein emission rates

Yanina Cesa, a Christian Blum, a Johanna M. van den Broek, b Allard P. Mosk, b

Willem L. Vos* bc and Vinod Subramaniam* a

Received 13th October 2008, Accepted 12th January 2009

First published as an Advance Article on the web 11th February 2009

DOI: 10.1039/b817902f

We present experiments to determine the quantum efficiency and emission oscillator strength of

exclusively the emitting states of the widely used enhanced green fluorescent protein (EGFP). We

positioned the emitters at precisely defined distances from a mirror to control the local density of

optical states, resulting in characteristic changes in the fluorescence decay rate that we monitored by

fluorescence lifetime microscopy. To the best of our knowledge, this is the first emission lifetime control

of a biological emitter. From the oscillation of the observed emission lifetimes as a function of the

emitter to mirror distance, we determined the radiative and nonradiative decay rates of the

fluorophore. Since only the emitting species contribute to the change in emission lifetimes, the rates

determined characterize specifically the quantum efficiency and oscillator strength of the on-states of

the emitter, in contrast to other methods that do not differentiate between emitting and dark states.

The method reported is especially interesting for photophysically complex systems like fluorescent

proteins, where a range of emitting and dark forms has been observed. We have validated the analysis

method using Rhodamine 6G dye, obtaining results in very good agreement with the literature. For

EGFP we determine the quantum efficiency of the on-states to be 72%. As expected for this complex

system, our value is higher than that determined by methods that average over on- and off-states.

1. Introduction

The discovery, development, and use of genetically-encodable

visible fluorescent proteins (VFPs) has provided revolutionary

new capabilities to visualize molecular and cellular biological

processes using fluorescence microscopy. 1–3 Despite the

widespread use of VFPs as reporters and sensors in cellular

environments the versatile photophysics of fluorescent proteins

is still subject to intense research. Understanding the

photophysics of fluorescent proteins is essential for accurate

interpretation of the biological and biochemical processes

illuminated by the fluorescent proteins as well as for the

development of biosensors based on fluorescent proteins.

A multitude of studies has addressed the spectral complexity

of fluorescent proteins. Different chromophores can form

within one protein, 4,5 intrinsic and photoinduced spectral shifts

have been observed, 6–8 and it has been demonstrated that

VFPs exhibit rapid blinking as a result of transitions between

emitting and non-emitting states. 9–11 This photophysical

complexity influences the determination of fundamental

emission properties such as the radiative and nonradiative

a Biophysical Engineering Group, Faculty of Science and Technology

and MESA+ Institute for Nanotechnology, University of Twente,

P.O. Box 217, 7500 AE Enschede, The Netherlands.

E-mail: v.subramaniam@tnw.utwente.nl

b Complex Photonic Systems (COPS), Faculty of Science and

Technology and MESA+ Institute for Nanotechnology, University

of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands.

E-mail: w.l.vos@tnw.utwente.nl

c Photonic Bandgaps Group, Center for Nanophotonics, FOM Institute

AMOLF, Kruislaan 407, 1098 SJ Amsterdam, The Netherlands

decay rates, fluorescence quantum efficiency and oscillator

strength, since most approaches average over the different

subensembles exhibiting different photophysical properties in

the analyzed sample.

The most widely-used approach determines the quantum

efficiency of an unknown fluorophore by comparing the

wavelength integrated emission intensity of the unknown

sample to a known reference having the same absorbance at

the chosen excitation wavelength. 12 Using this value for

quantum efficiency and the measured fluorescence lifetime,

the radiative and non-radiative decay rates and emission

oscillator strengths can be calculated. However, this approach

is rigorously correct only for systems of identical emitters, and

averages over all spectral species. It does not discriminate

between (a) molecules in emitting states and those in (b)

absorbing, but non-emitting states or (c) in photoinduced dark

states created by the measurement itself. These dark states

bias the determined quantum efficiency and hence affect the

calculated values for the decay rates and oscillator strength.

Fluorescent proteins exhibit blinking attributed to spontaneous

as well as photoinduced transitions between emitting to nonemitting

states, 9–11 and exhibit intrinsic spectral fluctuations

due to chemical or environmental influences upon the

chromophore. 6,13 Consequently, the values for fluorescent

protein quantum efficiencies derived from different studies

vary considerably. A typical example is the red emitting

protein DsRed, for which values for the quantum efficiency

in the literature range from the first pioneering results reporting

23% 14 to the much higher consensus values currently, but

which still range from 68% 15,16 to 79%. 17 Another example is

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the green emitting protein Azami green, for which quantum

efficiencies of 67% 18 and 90% 19 were reported. It is therefore

evident that a method is needed for the determination of

quantum efficiencies that is independent of relative measurements

and the presence of non-emitting states.

We present here the first such experiments on fluorescent

proteins using a method to determine radiative and nonradiative

decay rates of an emitter, and thus quantum efficiency and

emission oscillator strength, that characterize the emitting

states of the chromophore and is not dependent on spectral

integration. This method has been applied in other

research fields to erbium ions, 20 semiconductor colloidal

nanocrystals, 21,22 silicon nanocrystals 23 and self-assembled

quantum dots. 24 We control the local density of optical states

by positioning the fluorescent protein at precisely defined

distances from a metallic mirror. From a classical point of view,

if an emitter is placed in front of a metallic mirror the field

emitted directly by the emitter interferes with the field from the

emitter’s mirror image. If the distance from the mirror is such

that the interference is constructive, the local density of states,

and hence the emission rate, will be increased. If on the other

hand, the distance is such that the interference is destructive, the

local density of states, and hence the emission rate, will be

reduced. In this way control over the distance from the emitters

to the mirror corresponds to control of the local density

of states. A classical model taking full account of material

properties of the mirror and dielectric environment was

developed by Chance et al. 25

The distance-dependent modification of the local density of

states results in a characteristic oscillation in the fluorescence

decay rate 25–28 that we monitored by fluorescence lifetime

microscopy. The results were modeled using the single mirror

model 25 yielding the radiative and non-radiative decay rates of

the emitting states, and by extension, a set of relevant

photophysical parameters of the protein including quantum

efficiency and emission oscillator strength. This approach has

the clear advantage that it is based on measurements of

emission properties reflecting purely the characteristics of the

emission transition, rather than those of the absorption as is

the case for other standard techniques to determine these

parameters. In the present approach, no prior knowledge of

absorption characteristics is required, representing a direct

determination of the emission band properties.

We first studied the well-characterized laser dye Rhodamine

6G (Fig. 1a) to validate our sample preparation and

measurement method and to measure the complete set of

photophysical parameters of this important dye. We then

demonstrated that this approach of controlling the local

density of states is suitable for the analysis of photophysically

complex systems like fluorescent proteins. We analyzed the

emission from the widely-used fluorescent protein EGFP

(Fig. 1b) to sensitively measure the decay rates, the quantum

efficiency of the on-states, and the emission oscillator strength.

2. Methods

To control the local density of states of the emitters, we

chose to work in a single mirror configuration. The emitters

are embedded in an isotropic medium and localized at a

Fig. 1 Structure of (a) Rhodamine 6G and (b) the green fluorescent

protein EGFP studied here. Rhodamine 6G is a common and

well-characterized laser dye with relatively simple photophysics that

was used to validate our method. EGFP is a typical representative of

the class of genetically-encodable fluorescent proteins. The emitting

chromophore forms inside the protein barrel and is completely

shielded by the protein scaffold from the external environment.

Fluorescent proteins show very complex photophysics, which makes

the determination of photophysical parameters by conventional

methods difficult.

well-defined distance from the metallic mirror. To realize these

conditions we used the sample configuration shown in Fig. 2a.

Mirror and spacer layer fabrication

The mirrors used in this work were fabricated by multilayer

e-gun deposition on a silicon wafer. First a layer of 50 nm of

chromium was deposited to improve the adhesion of the silver

layer that acts as the mirror. The thickness of the silver layer

deposited over the chromium was 200 nm. Finally the SiO 2

layer that acts as spacer was deposited with a controlled

thickness to obtain the selected distance between the emitters

and the silver mirror. The whole deposition process was

carried out in a Balzers BAK 600 e-gun evaporation machine.

The multideposition can be done keeping the vacuum in

the chamber between different material depositions while

optimizing the conditions of the surfaces for the following

deposition. This approach helps to improve the adherence,

especially between the easy to oxidize silver layer and the SiO 2 .

The mirrors and spacer layers were characterized by

scanning electron microscopy (SEM), imaging different

positions of a cross section of each mirror-spacer. A typical

cross section SEM image of the mirror and covering SiO 2 layer

is shown in Fig. 2b. The thickness of the silver mirror and of

the SiO 2 spacer layer was found to be homogeneous over the

analyzed cross sections. The refractive index of the SiO 2

layer was measured with ellipsometry, giving a value of

n = 1.46 0.05 for the different SiO 2 spacer thicknesses.

Sample film and cover

To form a thin layer of the fluorescent molecules over the SiO 2

spacer, the emitting molecules (Rhodamine 6G from Lambda

Physics; EGFP obtained as reported in ref. 6) were embedded

in a polymer matrix to obtain a nearly isotropic orientation of

the molecules in the layer. 1% PVA was selected for this

purpose. A solution of PVA and the corresponding molecule

at low concentrations to exclude dye–dye interactions was spin

coated over the surface. From the original wafer 2 cm 2cm

samples were cut and 100 ml of the PVA solution was spin

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etween the emitters and the mirrors, used in the analysis, is

defined by the nominal spacer thickness plus half the PVA

layer thickness, which is held constant for all samples.

Finally a thick layer of PMMA (n = 1.489) was deposited

onto the fluorescent layer to match the refractive index of the

spacer and reproduce the conditions of a single mirror. 25 A

drop of B2% PMMA in toluene solution was deposited and

dried over the sample to form a layer 41 mm.

To determine the lifetime of the respective emitter in a

homogeneous medium with the same refractive index of the

spacer used in the mirror samples, we followed the same

sample film and cover production procedure but used a quartz

coverslip as substrate (replacing the mirror) which has a

refractive index (n = 1.46).

Emission lifetime determination

Fig. 2 (a) Schematic of the sample design used to modulate the local

density of states. A thin Cr layer deposited on a silicon substrate

improves the adherence of the silver layer that acts as a mirror. A SiO 2

layer of well-defined thickness in the range 50 nm to 300 nm is

deposited on the mirror and acts as a spacer. The thin o20 nm

PVA layer containing the emitters is spincoated on top of the spacer.

Finally a thick layer of PMMA with a matching refractive index to

SiO 2 is used to create an optically isotropic environment for the

emitters. (b) Scanning electron micrograph of a crossection of the

mirror and spacer layer. The three layers (from bottom up: Cr, Ag and

SiO 2 ) are clearly distinguishable. The thicknesses of silver and SiO 2

layers are determined from these images. (c) AFM image of the spin

coated PVA layer spanning a region containing a scratch made on the

PVA layer. Height image (top) clearly shows the scratch, where PVA

was removed. From the averaged height profile (over the white area

indicated in the image) the thickness of the PVA layer can be

determined as 17 3 nm. The ‘‘hills’’ at the side of the scratch

originate from PVA being piled up by the scratching of the layer.

coated onto the sample at 6000 rpm for 10 s. The thickness of

the resulting PVA layer was characterized by atomic force

microscopy spanning a region containing a scratch made on

the layer with a Teflon tweezers, yielding a homogeneous layer

of 17 3 nm. A typical AFM image with the corresponding

averaged height profile is presented in Fig. 2c. The distance

The fluorescence decay curves were measured with a time

correlated single photon counting (TCSPC) system. The

sample was illuminated with a pulsed diode laser with a pulse

duration of 50 ps (FWHM) and a repetition rate 20 MHz

(BDL475, 469 nm, B&H, Germany). Epi illumination

(40, NA 0.6) was used, collecting the emission through the

same objective. A 15 nm band pass emission filter centered at

556 nm was used for Rhodamine 6G and a 10 nm band pass

emission filter centered at 510 nm was used for EGFP in the

detection path to limit the detection wavelength to the peak of

the emission spectrum. To eliminate any influence by scattered

or back-reflected excitation light, an additional long pass filter

(razor edge 473.0 nm, Semrock, USA) was inserted into the

detection path. The emission is focused to the active area of a

single photon avalanche diode (PDM series, MPD, Italy, peak

quantum efficiency of B50% at 500 nm) connected to the

TCSPC module (SPC-830, Becker & Hickl, Germany) to

perform time resolved fluorescence measurements. Integration

times per decay curve were chosen to yield a total of

approximately 20 000 counts per decay curve to assure

accurate determination of the characteristic lifetime (t) 29 by

the TCSPC card software (SPCimage, B&H, Germany).

3. Results and discussion

Rhodamine 6G

We first tested our approach by measuring a series of

Rhodamine 6G samples. A typical fluorescence decay curve

and fit for a Rhodamine 6G sample on a mirror with 150 nm

emitter–mirror distance is presented in Fig. 3. The data

obtained were fitted by a monoexponential decay function.

The quality of the fit was judged by the w 2 parameter, where a

w 2 = 1 is characteristic of an accurate fit. The residuals plotted

at the bottom of Fig. 3 show that the fitting is good in the

whole range of the decay.

Since small variations in the thickness of the layers defining

the distance to the mirror, as well as variations in the fits of the

data will give rise to changes in the recorded lifetimes, it is

necessary to collect a statistically relevant number of decay

curves for each sample. To achieve this, we collected five

separate fluorescence lifetime images at different positions of

each sample representing one emitter–mirror distance, thus

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to the lifetime of the emitter. The small FWHM shows the

good quality of our sample preparation and measurement.

The most frequent lifetime of 3.33 ns is the peak of the

Gaussian distribution for a Rhodamine 6G–mirror distance

of 150 nm. This value is used to calculate the decay rate for the

respective distance. For all the distances measured, the lifetime

presents a similar Gaussian distribution, with a FWHM of

0.090 0.009 ns. The peak positions vary as expected with the

emitter–mirror distance, corresponding to the expected

modulation of the decay rate.

Using the same measurement procedure described above,

we obtained an emission lifetime of 3.3 ns, corresponding to a

decay rate of 0.30 ns 1 , for Rhodamine 6G in a homogeneous

medium of refractive index of n = 1.46. This rate was then

used as a normalization factor for the analysis. The normalized

decay rates for Rhodamine 6G are plotted as a function of the

distance to the mirror in Fig. 4a. The error bars on the abscissa

(the distance to the mirror) are defined as the sum

of the uncertainties in SiO 2 and PVA layer thicknesses

(Dz = 6 nm), while the error bars for the decay rate were

calculated using the half-width of the lifetime (Dt = 0.045 ns).

In Fig. 4a we present the normalized decay rate of Rhodamine

6G versus the distance to the mirror. The results are modelled

by the single mirror configuration model. 25 Two main effects

Fig. 3 (a) Typical decay curve measured for Rhodamine 6G at a

distance of 150 nm to the mirror. The data (dark grey dots) was fitted

with a mono exponential decay (black line). The light grey curve

corresponds to the instrument response function (IRF) of the system.

The residuals are plotted at the bottom and show the very good quality

of the fit in the whole decay range. (b) Typical lifetime image, showing

the homogeneity of the sample. Each pixel represents the fitted lifetime

of a curve like the one plotted in (a). Several images were taken into

account to determine the lifetime distribution for each distance to the

mirror. (c) Lifetime distribution histogram. The histogram was built

from the 5120 constituent single pixel decay curves from 5 lifetime

images like the one plotted in (b). The distribution is very narrow and

shows a Gaussian shape with a FWHM of 0.087 ns.

accounting for any possible inhomogeneities in the emitter–

mirror distance within one sample. Each image contained

32 32 decay curves measured on an area of 16 16 mm,

see Fig. 3b. The decay times extracted from the monoexponential

fitting of the 5120 constituent single pixel decay

curves were collected in a single histogram that represents the

measured lifetime distribution for each distance emitter–

mirror, see Fig. 3c. From the distribution we can ascertain

the most frequent lifetime as well as the width of the

distribution as a measure of the quality of the lifetime

determination for each dye-mirror distance.

Fig. 3c shows a narrow distribution of measured lifetimes

that can be fitted by a Gaussian distribution. The width of the

distribution is 0.087 ns FWHM, which is very small compared

Fig. 4 (a) Normalized decay rate vs. the distance to the mirror for

Rhodamine 6G at l = 555 nm. The data are compared to theory 25 for

different orientations of the emitter dipoles with respect to the mirror

surface (n Ag (l = 555 nm) = 0.129 + i3.25; Q = 95%): parallel

(long dashed), perpendicular (short dashed) and isotropic distribution

(solid line). The experimental points are followed by the predicted

behaviour for isotropic dipole orientation for distances to the mirrors

Z 110 nm. Below 110 nm Rhodamine 6G–mirror distance the

measured decay rates are below the predicted values (grey points),

for unknown reasons. (b) Total decay rate as function of the

normalized LDOS. A linear relation between the measured decay rate

g(o,z) for Rhodamine 6G and the calculated normalized LDOS can be

observed as expected according to Fermi’s golden rule. The solid line

represents a linear fit with a nonradiative decay g nrad =0.015 0.02 ns

and a radiative decay rate g hom

rad = 0.29 0.02 ns. The resulting

quantum efficiency is Q = 95% 5%.

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are expected. First, the decay rates oscillate with the increase

of the distance to the mirror, since the phase of the reflected

field changes with this distance. Second, the oscillation

amplitude decreases with increasing distance, since the

radiation field of the dipole decreases with the distance

between the emitter and its mirror image. Similar behaviour

is predicted for different transition dipole orientations with

respect to the mirror. Indeed a damped oscillation of the

normalized decay rate as a function of the distance to the

mirror is observed in Fig. 4a, consistent with the prediction by

theory for a single mirror configuration model.

For our preparation method of embedding emitters in a

polymer film we expect an isotropic distribution of the

emission dipole orientations with respect to the mirror.

Fig. 4 shows that for emitter–mirror distances Z 110 nm,

the oscillation in measured data indeed follows the values

predicted for isotropically distributed emitters (solid line).

Fig. 4 also depicts the normalized decay rates calculated for

the parallel (long dashed) and perpendicular (short dashed)

orientations. The calculation was performed for the

wavelength corresponding to the Rhodamine 6G emission

peak (555 nm), the refractive indexes for silver at the

corresponding wavelength (n Ag (l = 555 nm) = 0.129+i3.25 30 )

and a quantum efficiency Q = 95% (value obtained from

analysis below)). For emitter–mirror distances less than

110 nm the measured decay rates are below the predicted

values (grey data points in Fig. 4a), even considering a

deviation from the isotropic distribution orientation and all

transition moments being oriented parallel to the mirror. For

these emitter–mirror distances, we repeated the experiment

and reproduced the same decay rates. The observed deviation

from the model was only found for Rhodamine6G, and

not for the fluorescent protein EGFP (see below). While

no explanation has been found for this discrepancy yet,

these two data points were tentatively excluded for the

following analysis.

The measured decay rate g(w,z) is the sum of two contributions,

the non-radiative decay rate g nrad (o) and the radiative decay

rate g rad (w,z). The radiative decay rate, according to Fermi’s

golden rule, 31 is proportional to the projected local density of

optical states r(w,z) (LDOS) and depends explicitly on the

distance to the mirror z, and the transition frequency o.

Therefore the total decay rate can be expressed as

gðo; zÞ ¼g nrad ðoÞþg hom rðo; zÞ

rad ðoÞ ð1Þ

r hom ðoÞ

where g hom

rad (o)andr hom (o) are the decay rate and corresponding

LDOS respectively in a homogeneous medium of the

corresponding refractive index. The total decay rate is plotted

as a function of the normalized LDOS in Fig. 4b. The error

bars for the decay rate are based on an uncertainty in

the lifetime of half-width of the lifetime distribution

(Dt = 0.045 ns) and for the normalized LDOS are based on

the interval corresponding to the uncertainty Dz in the distance

to the mirror.

A linear relation was found between the measured decay

rate g(w,z) for Rhodamine 6G and the calculated normalized

LDOS as expected from Fermi’s golden rule. The data

were fitted by a linear function (Fig. 4b), yielding for the

non-radiative decay rate a value of g nrad = 0.015 0.02 ns 1

and for the decay rate in a homogeneous medium

g hom

rad = 0.29 0.02 ns 1 . This results in a quantum efficiency

of Q = 95% 5%. Although Rhodamine 6G was embedded

in polyvinylalcohol in our study, the determined quantum

efficiency can be compared with values determined for

Rhodamine 6G in ethanol, since ethanol and polyvinylalcohol

provide a chemically very similar environment to the emitter.

Indeed our value and the values reported in the literature for

the quantum efficiency of Rhodamine 6G in ethanol

determined with different methods, ranging from 0.94 to

0.96, 32–36 agree very well.

Inasmuch as our method only addresses emitting or ‘‘on’’

states of the fluorophore, the correspondence of our result for

Q with the literature reports reflects the relatively non-complex

photophysics of Rhodamine 6G. Although dark states have

been observed for Rhodamine 6G, these states have been

found to be linked to the triplet state and a dark state formed

through the triplet state. 37 Although the intersystem crossing

rates to the triplet state were found to vary depending on the

solvent used, 38 the probability for inter-system crossing to the

triplet state for Rhodamine 6G is generally low enough to

exclude a noticeable buildup of molecules in the triplet state,

and thus a formation of dark states, under the applied

conditions.

From the homogeneous decay rate g hom

rad , the emission

oscillator strength f emiss of the transition can be calculated 39

f emiss ðoÞ ¼ 2m ee 0 pc 3

q 2 no 2 g hom

rad ðoÞ

where m e and q are the electron mass and charge respectively,

e 0 is the vacuum permittivity, c is the speed of light and n is the

refractive index of the surrounding media. Here we use the

frequency corresponding to the peak of the emission (555 nm),

in the center of the detection band to calculate the effective

emission oscillator strength of the transition.

The effective emission oscillator strength for Rhodamine 6G

is thus determined to be f emiss = 0.92 0.03. To the best of our

knowledge, no value for the emission oscillator strength has

been reported for Rhodamine 6G. The absorption oscillator

strength has been reported before to be f abs =0.69. 36

Enhanced green fluorescent protein (EGFP)

We show the decay curves from the fluorescent protein EGFP

for two sample–mirror distances in Fig. 5a. The data

correspond to distances to the mirror of 170 nm and 60 nm.

The decay of EGFP emission at 60 nm distance to the mirror is

decidedly faster than the EGFP decay recorded for a

EGFP—mirror distance of 170 nm. Both decay curves were

well-fitted with a monoexponential function (t = 2.26 ns,

w 2 = 1.02 for 170 nm and t = 1.36 ns, w 2 = 1.6 for 60 nm).

Clearly, placing the EGFP molecules at different distances

from the silver mirror induced a change in the emission

lifetimes, as expected from theory. To accurately determine

the characteristic lifetime of EGFP for each distance to

the mirror, fluorescence lifetime imaging was used to

determine the most frequent lifetime and the width of

the lifetime distribution for each distance. The width of the

ð2Þ

This journal is c the Owner Societies 2009 Phys.Chem.Chem.Phys., 2009, 11, 2525–2531 | 2529


Fig. 5 (a) Typical decay curve measured for EGFP for distances to

the mirror of 60 nm (circles) and 170 nm (triangles). Curves are

normalized to unity to clearly visualize the difference in lifetime. The

black lines correspond to mono-exponential fits. At shorter distances

to the mirror a shorter lifetime is observed (t = 2.26 ns, w 2 = 1.02 for

170 nm and t = 1.36 ns, w 2 = 1.6 for 60 nm), as predicted by the

theory. The grey circles correspond to the IRF of the system. (b)

Normalized decay rate vs. the distance to the interface for EGFP. The

measured values (circles) follow the predicted behaviour for Q = 72%

and isotropic dipole orientation (solid line), which is clearly different

from the expected modulation for Q = 100% (dashed line). (c) Total

decay rate as a function of the normalized LDOS. The linear fit (solid

line) yields values of the nonradiative decay g nrad = 0.14 0.03 ns 1

and the radiative decay rate g hom

nrad = 0.36 0.03 ns 1 . From these

decay rates the quantum efficiency is calculated to be 72% 6%.

lifetime distributions was found to be narrow (0.08 0.01 ns

FWHM) and could be fitted by Gaussian functions.

In Fig. 5b we present the observed decay rate, derived from

the most frequent lifetime observed, as a function of the

sample–mirror distance. For EGFP we expect to see the

influence of its moderate quantum efficiency of 60% reported

in the literature 40 on the modulation depth of the decay rate as

a function of distance to the mirror. A reduced quantum

efficiency results in a lower modulation depth of the decay

rate. 41 Indeed, we observe this behaviour. The normalized

decay rates show less modulation than expected for unity

quantum efficiency, but agree very well with calculations

considering a quantum efficiency of 72% (derived from

analysis below). The calculated normalized decay rate for

unity quantum efficiency is also shown in Fig. 5b to illustrate

the change in modulation depth, clearly noticeable for shorter

distances. The calculations were performed using the

wavelength corresponding to the EGFP emission peak

(512 nm), and the refractive index for silver at this wavelength

(n Ag (512 nm) = 0.129 + i3.02 30 ).

The total decay rate is plotted as function of the normalized

LDOS (Fig. 5c) and fitted with a linear function from which

the non-radiative decay rate of g nrad = 0.14 0.03 ns 1 and

the decay rate in a homogeneous medium g hom

rad = 0.36

0.03 ns 1 are derived. Using these values we calculate a

quantum efficiency of Q = 72% 6% and effective emission

oscillator strength (eqn (2)) of f emiss = 0.97 0.06.

By modulating the local density of states and observing the

change in emission lifetime, one obtains the photophysical

properties characteristic of the emitting states of the

fluorophore. Non-emitting states do not contribute to the

observed emission lifetime and are thus not accounted for.

Since conventional measurements are adversely affected by

absorbing and non-emitting as well as non-absorbing states

photoinduced during the measurement process, we expected to

find an increased value for the on state quantum efficiency.

Indeed the value for the quantum efficiency we obtain (72%) is

markedly higher than the value of Q = 60% reported

before, 16 consistent with reports that a significant fraction of

green emitting proteins can reside in dark states even at very

low excitation intensities. 42,43 The reported values for the

radiative and nonradiative decay rate for a green fluorescent

protein mutant closely related to the EGFP analyzed in this

study were smaller than the values we determine, which is a

consequence of the lower quantum efficiency used to calculate

the rates in this report. 44 The effective emission oscillator

strength of EGFP we determine is f = 0.97. To the best of

our knowledge no emission or absorbance oscillator strength

has been experimentally determined for any fluorescent

protein to date. The anionic chromophore of the green

fluorescent protein, which is also the chromophore of

EGFP studied here, has been analyzed by computational

methods. 45,46 These studies have shown that the

HOMO–LUMO coupling has the largest absorbance

oscillator strength and thus is the source of the absorption.

The calculated strong HOMO–LUMO coupling agrees well

with the effective emission oscillator strength that we

determined.

4. Conclusions

We have presented for the first time the determination of

fundamental photophysical parameters of the on states of a

fluorescent protein by systematically changing the local

density of optical states. The local density of states is changed

by controlling the spacing between the emitters and a metallic

mirror, which results in characteristic changes of the emission

lifetime. Analyzing the change of emission lifetime as a

function of the emitter distance to the mirror gives access to

the decay rates, quantum efficiency and emission oscillator

strength. Since only emitting states contribute to the emission

lifetime, only the properties of the emitting states are

determined, in contrast to other methods that average over

2530 | Phys. Chem. Chem. Phys., 2009, 11, 2525–2531 This journal is c the Owner Societies 2009


emitting and non-emitting states. The method presented does

not require measurements relative to a reference molecule. We

show that changing the local density of optical states is an

especially potent method to analyze complex photophysical

systems like fluorescent proteins, a class of molecules which

are known to exhibit significant residence times in intrinsic or

photoinduced dark states. We validated our approach with the

photophysically simple dye Rhodamine 6G, and recovered a

quantum efficiency in excellent agreement with literature. In

contrast, for the complex biological fluorescent protein EGFP,

we determined a clearly higher value for the quantum

efficiency of the emitting states exclusively (Q = 72%) than

reported values averaged over on- and off-states (Q = 60%).

The increased value agrees well with reports that a significant

fraction of green emitting proteins can reside in dark states.

Theoretical studies predict a strong HOMO–LUMO coupling,

which agrees well with our measurement.

The consistency between our measurements and previously

reported values from both experimental as well as theoretical

determinations, serve to validate the method as a new and

potentially very powerful manner to obtain fundamental

insights into the complex fluorescent protein emission

properties. We even envision the individual characterisation

of different emitting forms coexisting in many fluorescent

proteins. In this case, the decay characteristics are

multiexponential, and the influence of a modified local density

of photonic states on each individual decay component can be

analyzed.

Acknowledgements

We thank Niels Zijlstra and Merel Leistikow for their

contribution to the LDOS calculations and Willem Tjerkstra

for useful discussions. This work is part of the research

program of the ‘‘Stichting voor Fundamenteel Onderzoek

der Materie’’ (FOM), which is supported by the ‘‘Nederlandse

Organisatie voor Wetenschappelijk Onderzoek’’ (NWO).

W.L.V. also thanks NWO-Vici and STW/NanoNed. C.B.,

Y.C. and V.S. also acknowledge support from the MESA +

Institute for Nanotechnology.

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