Christiane Ho ¨ppener1* and Lukas Novotny2
1Institute of Physics, University of Mu ¨nster, 48149 Mu ¨nster, Germany
2Institute of Optics and Department of Biomedical Engineering, University of Rochester, Rochester,
NY 14627, USA
fields is the primary reason for their application in biosensing and imaging. Local field
enhancement boosts the signal-to-noise ratio in measurements and provides the possibility of
imaging with resolutions significantly better than the diffraction limit. In fluorescence imaging,
local field enhancement leads to improved brightness of molecular emission and to higher
detection sensitivity and better discrimination. We review the principles of plasmonic
fluorescence enhancement and discuss applications ranging from biosensing to bioimaging.
The ability of metal surfaces and nanostructures to localize and enhance optical
2. Principles of fluorescence
2.1. Excitation and emission of a quantum emitter
2.2. Influence of the environment
2.3. Theoretical description
3. Fluorescence emission near planar interfaces216
4. Interaction of light with metals
4.1. Optical properties of metals
4.2. Coupling to surface waves
4.3. Coupling to waveguide modes
4.4. Conversion of non-propagating modes to far-field radiation
5. Structured metal surfaces
5.1. Light transmission through sub-wavelength holes
6. Localized surface plasmons
6.1. Optical properties of spherical nanoparticles
6.2. Spontaneous emission near metal nanoparticles
6.3. Fluorescence-based biosensing and bioimaging
6.4. Modification of the radiative decay rate/quantum yield
6.4.1. NIR dyes234
6.4.2. Naturally fluorescent proteins
* Author for correspondence: Christiane Ho ¨ppener, Institute of Physics, University of Mu ¨nster, 48149
Mu ¨nster, Germany. Email: email@example.com
Quarterly Reviews of Biophysics 45, 2 (2012), pp. 209–255.
f Cambridge University Press 2012
Printed in the United States of America
6.5. Biosensing based on fluorescence quenching
6.6. Plasmon rulers240
6.7. Nanoscale imaging
9. References 246
Our knowledge of biological systems is strongly fueled by the progress in optical microscopy
over the last few decades, and in particular, by the development of optical probes such as organic
dye molecules (Zhang et al. 2002; Waggoner, 2006), metal–ligand complexes (Mason, 1999;
Lakowicz, 2006), lanthanide chelates (Hemmila & Laitala, 2005), semiconductor quantum dots
(Alivisatos, 1996; Weller, 1998), fluorescent proteins (Shaner et al. 2005), or nitrogen-vacancies
in diamond, etc. (Kurtsiefer et al. 2000; Jelezko & Wachtrup, 2006; Chang et al. 2008). The
discovery of naturally fluorescent materials, e.g. minerals such as fluorite marked the early stages
of the utilization of fluorescence contrast. Although, intrinsic fluorescence of proteins can arise
from weakly fluorescent aminoacids such as tryptophan and tyrosine, only few cellular building
blocks provide strong fluorescence properties, e.g. cholophyll, rhodopsine, etc., and thus, are
directly optically accessible. However, the majority of proteins, lipids, and nucleic acids, do not
exhibit strong spectroscopic responses upon excitation by light, e.g. photoluminescence,
Rayleigh-scattering, absorption or Raman-scattering. Thus, optical spectroscopy or microscopy
cannot directly address these components on a molecular level due to lack in sensitivity. In
order to make these entities accessible to biophysical studies as well as medical diagnostics,
secondary labeling techniques have been developed, which take advantage of the strong optical
properties of chemically designed quantum emitters, genetically encoded probes, and also of
naturally fluorescent materials. The progress in fluorescence-related methods over the last few
decades has accomplished a set of modern labeling strategies, which nowadays allows for
multiple staining of various cellular organelles. Selective detection of these labels enables life
cell imaging on a level of single molecule detection. Thus, fluorescence markers are of great
importance in various fields, such as for the identification of nucleotides, aminoacids, drugs,
pollutants, the determination of ion concentrations and the pH in cellular organelles, and also
in diagnostics of diseases. Several sophisticated techniques based on the use of fluorescence
signatures such as brightness, lifetime, anisotropy or their spectrum have been developed,
including time-resolved measurements such as fluorescence correlation spectroscopy (FCS)
(Schwille et al. 1999), fluorescence anisotropy (Ja ¨hnig, 1979), fluorescence lifetime measurements
(Verveer et al. 2000), Fo ¨rster resonant energy transfer (FRET; Fo ¨rster, 1948), and also multi-
photon fluorescence excitation (Xu et al. 1996), etc. These techniques can provide far more
information on a biological system than its spatial and chemical organization and its molecular
environment. They make use, e.g., of temporal fluctuations or modifactions of the fluorescence
intensity, and can reveal molecular concentrations and molecular dynamics on the nanosecond
timescale, e.g. to investigate the transport of substrates through cellular membranes, lateral
diffusion of membrane compounds such as proteins and lipids, lipid rafts and caveolea,
conformational changes, and binding kinetics. All in common, these techniques make use of
210C. Ho ¨ppener and L. Novotny
specific photophysical properties of the quantum emitters and/or their modification in different
Although, far-field fluorescence microscopy has been in particular very successful in biological
science due to its extraordinary sensitivity, high specificity, and versatility, research in this field is
still driven by the demand for brighter and optically more stable probes with minimized toxicity
and reduced dimensions as well as by the demand for higher detection sensitivity and optical
resolution. In terms of these demands, metal enhanced fluorescence opens up new strategies by
utilizing the plasmonic nature of metallic structures on the nanometer scale. Plasmonic nano-
structures can boost the light–matter interaction, and thus have impact on processes such as
the light absorption, emission, and also light localization. Figure 1 displays possible plasmonic
structures, which are employed for the amplification of spectroscopic responses and the detec-
tion of molecules and molecular interaction.
The aim of this review article is to provide a detailed understanding of the processes involved
in metal-enhanced biological sensing, detection, and imaging. To keep the discussion in bounds,
we will restrict ourselves to fluorescence-based interactions. We will point out the principles of
3 nm H2O
angle of incidence [°]
41.5 42 42.5
Fig. 1. Plasmon-mediated spectroscopy and microscopy. (a) Surface enhanced Raman spectroscopy of
4-mercaptoencoic acid (pMBA) on gold nanoparticle aggregates for pH probing in live cells. Modified from
Kneipp et al. (2007) with the permission of the American Chemical Society. (b) Localized plasmon reson-
ance spectroscopy: induced shift in the SPP resonance by a water layer of 3 nm on a Ag surface. The inset
shows the angle of incidence dependence on the intensity enhancement near Ag and Au surfaces. (c) Sketch
of the principle of antenna-assisted microscopy by means of a finite plasmonic nanoantenna and high-
resolution fluorescence image of individual dye molecules with random orientation of their transition dipole
Exploiting the light–metal interaction for biomolecular sensing and imaging 211
plasmon-enhanced fluorescence, its impact on current limitations in the field of nanobiopho-
tonics, and we will discuss representative results. Nanoplasmonic structures provide also great
potential for label-free detection of biomolecules, such as in (localized) surface plasmon reson-
ance (L)(SPR) spectroscopy for bioaffinity reactions or surface enhanced Raman scattering
(SERS) (cf. Fig. 1), which also may pave the way for new high-resolution imaging techniques. For
detailed information on these techniques, we refer to recently published review articles (Homola
et al. 1999; Willets & Duyne, 2007; Kneipp et al. 2002).
2. Principles of fluorescence
The chemical structure of a molecule is directly connected to its optical properties, e.g. its ability
to emit fluorescence. Fluorescence occurs naturally in minerals, bacteria, and plant cells, and is
encountered also in synthetically engineered materials. These classes of intrinsic, e.g., aromatic
amino acids, neurotransmitters, porphyrins, green fluorescent protein (GFP), and extrinsic
fluorophores, such as organic dye molecules, are characterized by conjugated carbon chains or
2.1 Excitation and emission of a quantum emitter
The conjugated delocalized p electrons of chromophores lead to electronic states with transition
frequencies in the UV or visible spectral range. The electronic states are split into vibrational and
rotational sub-levels. The Jablonski diagram in Fig. 2 shows the relaxation pathway of an excited
molecule. The relaxation pathway involves internal conversion from higher vibrational states to
the lowest vibrational level of the first excited singlet state within picoseconds. This is followed
either by radiative or non-radiative decay to the electronic ground state within nanoseconds.
Radiative decay involves the emission of a fluorescence photon whose frequency is redshifted
with respect to the wavelength of the excitation frequency. On the other hand, in non-radiative
decay, the energy difference between excited and ground state is dissipated to heat via molecular
to the emission of phosphorescence. As typical triplet state lifetimes are in the millisecond range,
wavelength / nm
Fig. 2. Jablonski diagram showing the energy levels of an organic dye molecule. Spontaneous emission of
the molecule is accomplished by its excitation and subsequent internal conversion and vibrational relaxation
the ground state of the first excited state, followed by the radiative decay to the ground state of the
molecule. Competing decay routes comprise non-radiative decay via dissipation into heat and the inter-
system crossing to a triplet state with long lifetime.
212C. Ho ¨ppener and L. Novotny
intersystem crossing leads to dark periods in the fluorescence emission of a molecule (fluor-
escence blinking). Biological research often demands for quantum emitters with high quantum
efficiency, which is given by the probability of transitioning from excited to ground state by
emitting a fluorescence photon.
In the regime of weak excitation, far from saturation of the excited state, the fluorescence
emission rate cem
can be considered as a sequence of two sequential processes, namely the
excitation from ground state to excited state and the subsequent relaxation back to the ground
state via emission of a fluorescence photon, i.e.
the molecule is in free space and does not couple to the local environment. The subscript ‘i’
indicates that the quantum yield is defined by the intrinsic properties of the molecule.
As indicated before, Qi
of a fluorescence photon. In terms of the radiative decay rate cr
is the excitation rate and Qi
0is the quantum yield. The superscripts ‘0’ specify that
0is the probability of relaxing from excited to ground state by emission
0and the non-radiative decay rate
0we can express the intrinsic quantum yield as
If we change the local environment of the molecule we will affect its excitation and decay rates.
Thus, Eqs. (1) and (2) get modified as
Here, cabsaccounts for dissipation to heat in the environment and cmaccounts for coupling
to non-radiative electromagnetic modes. The total decay rate c=cr+cnrdefines the lifetime
t=1/c of the excited state. In general, the fluorescence emission is not only a function of the
molecular properties but also of external parameters accounting for the local environment of
the molecule (Lichtman & Conchello, 2005). In later sections, we will discuss factors that
influence the excitation rate enhancement (cexc/cexc
0) and quantum yield enhancement (Q/Qi
2.2 Influence of the environment
In conventional fluorescence microscopy, the design of brighter and more stable probes aims at
the minimization of the internal and environmentally conditioned non-radiative processes,
yielding a higher spontaneous-emission rate. Probably the most fascinating example for such an
optimization is given by nature itself. The GFP exhibits a chromophore consisting of three
aminoacids, which are common also for many other proteins with non-fluorescent properties
(Shimomura et al. 1962; Tsien, 1998). What makes this chromophore in GFP fluorescent is its
protected location within the protein structure; hosted in a b-barrel, the chromophore is shielded
from its environment. Only the precise arrangement and orientation of the chromophore in this
protein enable its fluorescence properties. Slight deviations of the optimized conformational
Exploiting the light–metal interaction for biomolecular sensing and imaging213
form can lead to complete loss of fluorescence. Nowadays, biochemical modifications of GFP
and other fluorescent proteins have established a spectrum of genetically encoded probes
spanningthe UV and visiblerange, withhigher quantumefficiency and photostability comparable
with their wild-type counterparts (Shaner et al. 2004). Engineering of genetically encoded fluor-
escent probes has become an extensive and highly dynamic field of research, which requires a
detailed understanding of the involved internal photochemical processes as well as of the energy
dissipation in the local chemical environment. The high sensitivity to modifications in the en-
vironment of GFP can be used for sensing of local refractive index changes or variations in
the chemical composition. For example, local refractive index changes can be extracted from
fluorescence lifetime measurements. Fluorescence lifetime imaging of GFP is being used for live
cell imaging of ligands and receptors in cellular membranes (Suhling et al. 2002).
The fact that the local environment of a fluorophore can have significant effects on its pho-
tophysical properties is known since Purcell’s studies on the spontaneous emission probability of
a free atom in a high-Q cavity in 1946 (Purcell, 1946). Since then, his theoretical considerations
have been verified in various experiments studying the modification of the fluorescence lifetime
for molecules near metal and semiconductor interfaces, in microcavities and photonic crystals,
etc. (Drexhage et al. 1966; Drexhage, 1974; Kleppner, 1981; Lodahl et al. 2004; Rigneault et al.
2000; Danz et al. 2002). This pioneering work stimulated the intent to control the emission
rate of a fluorophore by modifying its local environment and has led to intensive research on
the fundamental processes responsible for molecular fluorescence near structured surfaces.
For molecules near metal surfaces, fluorescence lifetime changes are due to modifications of
both the radiative and the non-radiative decay rates. This is in contrast to conventional fluor-
escence microscopy, where the fluorescence lifetime depends mostly on non-radiative
decay channels, such as in FRET, low quantum-yield DNA markers, or pH- and ion-sensitive
fluorophores (Fo ¨rster, 1948; Tsien et al. 2006; Slavik, 1982; Rye et al. 1992; Cohen & Salzberg,
The main reason for coupling fluorophores to metal structures is to control crand cexc
in addition to cnr. The optimization of these competing processes requires a fundamental
understanding of the influence of the material properties and the geometry on the light–metal–
molecule interaction (Novotny, 1996; Barnes, 1998; Ford & Weber, 1984; Chance et al. 1973;
Novotny, 1997; Gersten, 2005). In the following we will summarize the major processes
affecting the total ascertainable fluorescence signal. In addition to excitation and emission rate
enhancements, the fluorescence intensity can also be affected by changes of the angular emission
pattern and hence the detection efficiency.
2.3 Theoretical description
We consider a molecule characterized by its transition dipole m located at r0. For weak excitation
fields, we can describe the excitation and emission processes in terms of first-order perturbation
theory. The molecule’s fluorescence rate is then described by a two-step process according to
Eq. (3). The local electric field E has typically two contributions, namely the incident excitation
field E0and the field originating from structures in the local environment E. The excitation rate
is proportional to the absolute square of E along the direction of the absorption dipole moment
and thus, is described by
214C. Ho ¨ppener and L. Novotny
The molecule’s dipole moment is defined by the quantum wavefunctions and hence by the
molecule’s internal potential. For weak excitation fields, it is not affected by the local field E. As
the local environment changes the excitation field from E0to E, the excitation rate enhancement
Here, nmdenotes the unit vector in the direction of m. Thus, the enhancement of the local field by
metal structures leads to an increase of the molecule’s excitation rate. Note that the direction of
the local field E is not necessarily in the direction of the excitation field vector E0.
In the next step, we calculate the total decay rate from excited state to ground state, which can
be related to the local density of states (Novotny & Hecht, 2006). For a two-level system, the rate
of spontaneous decay from the excited state |im with energy Ei=? hvito a set of final states of
equal energies Ef=? hvfis given by Fermi’s golden rule:
nf j^ H HIjim
where^ H HI=xm ?b E E denotes the interaction Hamiltonian in the dipole approximation. Because of
can be easily solved because of the delta function d(vixvf). The result can be represented in
terms of the partial local density of states r as (Novotny & Hecht, 2006):
the continuous distribution of the final states, the sum in Eq. (7) reduces to an integral. The latter
m j j2r(r0,v0), (8)
where v0=vixvfand r can be expressed in terms of the system’s dyadic Green’s function G
as (Novotny & Hecht, 2006)
The Green’s function has two parts, namely G
of free space and G
scattaccounting for structures in the local environment. Thus, metal structures
in the local environment alter the local density of states r and lead to a modification of the
molecule’s decay rate c.
The same result for c can be obtained from a purely classical perspective, where the molecule
is treated as a harmonically oscillating dipole with angular frequency v0and dipole moment m
(Chance et al. 1978). In free space, the dipole oscillation satisfies
$(r0,r0;v0)} ? nm
scatt, with G
0being the Green’s function
with c0being the free-space decay rate. To account for an inhomogeneous environment, i.e.
structures with which the molecule interacts, we need to add a secondary source term to the
right-hand side, namely
Exploiting the light–metal interaction for biomolecular sensing and imaging215
Here, Escattis the dipole’s field that acts back on the dipole after it has been scattered from
the environment. Evidently, Escatt=0 in free space because there are now structures that
would cause the field to be scattered back to the dipole. Note that Escatt–ES. While ESde-
notes the scattered part of the excitation field E responsible for the excitation of the
molecule according Eq. (5), Escattis the field emitted by the molecule that acts back on itself.
In the regime of weak damping (c< <v0) the solution for the normalized decay rate becomes
(Novotny & Hecht, 2006)
m j j2
k3Im m* ? Escatt(r0)
of refraction. The ratio c/c0is identical to P/P0, which is the ratio of powers emitted by an
oscillating dipole in an homogeneous and inhomogeneous environment, respectively. It can be
shown that this classical result is identical with the result in Eq. (8), which justifies the
phenomenological classical approach originally introduced by Chance, Prock and Silbey in 1978
(Chance et al. 1978). This model has been successfully used to explain lifetime changes of
molecules and ions in inhomogeneous environments, e.g. for Eu3+ions in the vicinity of a silver
surface (Drexhage, 1970).
0is the intrinsic quantum yield introduced earlier and k=(v0/c)n, with n being the index
3. Fluorescence emission near planar interfaces
In the previous section, the alteration of fluorescence emission through its environment has been
described in an abstract way, without referring to a particular experimental situation. It has been
shown that the decay rate is dictated by the local density of states. In this section, we apply the
established theoretical framework to a molecule near a material with a planar surface, as illu-
strated in Fig. 3. The parameters of this configuration are the dielectric properties of the material
e, the distance of the molecule from the surface z0, and the orientation of the molecule’s dipole
moment m relative to the surface normal.
Fig. 3. Fluorescence emission near planar interfaces. (a) A fluorescent molecule is located at a distance of z0
above a planar interface with an orientation of its dipole moment m. The dielectric properties of the
surrounding medium and the interface are given by e0and en. (b) Possible contributions to the radiated
power for a quantum emitter near a planar metal surface: light emission in reflection or transmission, e.g.
allowed and forbidden light, and non-radiative decay routes via coupling to phonons, surface phonon
polaritons, SPPs, and generation of heat.
216 C. Ho ¨ppener and L. Novotny
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