Nikon Note 12
Authors: Karla Balaa &
Langevin Institute, ESPCI, Paris, France
Surface Plasmon enhanced
Abstract • Introduction • Technology
Total Internal Reflection Fluorescence
microscopy (TIRFM) is a powerful technique
for imaging dynamic membrane events in
However the information gained from TIRFM may be limited by
the intensity of the florescence signal and by background noise
emanating from the inner part of the cell. Here we describe how
Surface Plasmon Mediated Fluorescence Microscopy (SPMFM) can
enhance TIRFM by increasing the fluorescence signal 5-6 fold and
by reducing background noise dramatically. The imaging
configuration is that for TIRFM except that the coverglass is
coated on one side with a nanometre film of silver. For SPMFM
(as in TIRFM), a high numerical aperture (N.A.) objective is
essential for achieving required illumination angles
TIRFM is used widely to study dynamic events close to the
membrane of living cells. As with all fluorescence imaging
techniques, information may be limited by the intensity of the
fluorescence signal and /or background noise.
In TIRFM a laser beam passes through a high numerical aperture
(N.A.) objective and undergoes total internal reflection when
reflected from a high-refractive medium (e.g., glass) into a low-
refractive medium (e.g. cell/water). By using a high N.A. objective,
the laser beam can leave the front optical plane of the objective at
a supercritical angle to result in total internal reflection. This
produces an electromagnetic evanescent wave which penetrates
the cell membrane adjacent to the coverglass and excites
fluorophores within an ultra thin optical section of ~100 nm,
thereby reducing background noise from out-of-focus fluorescence.
However, because of a partial loss of light confinement due to
light scattering in the cell, excited fluorophores in the inner part of
the cell may contribute to background noise and compromise
sensitivity. Here we describe how the fluorescence signal in TIRFM
can be enhanced, and the background noise simultaneously
reduced, with a new technique known as Surface Plasmon-
Mediated Fluorescence Microscopy (SPMFM)1.
Surface plasmons are oscillations of free electrons at the surface
of a metal film, which propagate along the surface creating an
associated evanescent electromagnetic field. Surface plasmons
cannot be excited directly with incident light from the same side.
However it is possible to excite surface plasmons propagating on
the opposite side efficiently with plane polarized light. To achieve
this, light must be directed at the metal from the medium with
the higher refractive index and at a precise angle (the surface
plasmon angle). Strong coupling occurs when the phase velocity
of the plane polarised light matches that of the surface plasmon
(the Kretschmann-Raether configuration). Surface plasmon
excitation is associated with a sharp decrease in the reflected
light, a phenomenon commonly used in Surface Plasmon
Resonance (SPR) biosensors. The evanescent electromagnetic field
associated with the surface plasmon on the sample side is very
similar to that created in TIRFM. It has the same penetration depth
for the same incident angle. However, the intensity of this field can
be much greater in the presence of a metallic thin film when
excited at the correct angle. In the case of a 40 nm-thick silver thin
film excited at 532 nm, intensity is increased 13 fold. Fluorophores
near the metallic surface consequently receive much more light.
The metallic thin film also changes fluorophore emission
dramatically and, hence, the ability to detect emitted photons.
Collection efficiency varies with fluorophore-metal distance. For
very short distances (<10 nm), fluorescence is quenched. For larger
distances (>150 nm), most of the emitted fluorescence is reflected
by the metal (>90%). The detection efficiency is high for
intermediate distances only (10-50 nm) and can reach values as
high as 50 % of the whole fluorescence emission (for silver thin
film). The mechanism is similar to that for excitation; near-field
components of the fluorophore emission couple to the surface
plasmon to be converted to light on the glass side of the metal
film. The molecular detection efficiency at large distances is,
therefore, low and is useful in reducing background fluorescent
noise from the inner part of the cell. The presence of the metallic
thin film acts as a strong distance-dependent filter at the detection
level, effectively selecting fluorophores at the correct distance for
live membrane observation. Indeed, the basal membrane cell is
estimated to lie within this range. The quenching of fluorescence
at very short distances is also an advantage as it reduces noise from
fluorescent molecules adhering to the glass surface.
When comparing the overall yield in suface plasmon-enhanced
TIRFM compared with standard TIRFM, SPMFM provides a 5-6 fold
signal at a fluorophore surface distance of 20 nm in the case of a
45 nm thick silver thin film (for isotropically distributed flurophores
with a typical fluorescence yield).
Figure 1: SPMFM on a standard objective-based TIRF microscope.
The excitation laser beam passes through a beam expander (lenses L1 and
L2). Light is then directed (mirror M1) and focused (lens L3) on the back
focal plane (BFP) of a Nikon Eclipse Ti inverted microscope. The mirror and
focusing lens are mounted on a computer controlled micro-translation
stage to tune the excitation beam incident angle. A sensitive EMCCD
camera is used for fluorescence detection.
The incident p-polarized light reaches the sample plane with an adjustable
angle (insert image). When this angle is equal to the surface plasmon
angle, an intense evanescent field is created on the sample side.
Applications • Conclusion • Acknowledgements
Copyright © Nikon Corporation. All Rights Reserved.
Figure 2: HEK cells transfected
with mCherry on silver thin
film (upper) and on a
standard glass slide (lower) at
a subcritical excitation angle
(left) and at a supercritical
angle corresponding to
surface plasmon excitation
of the silver (right).
SPMFM can be used to enhance the sensitivity of TIRFM applications
(such as the observation of endo- and exocytosis, protein dynamics,
cell-substrate interactions and signalling events). Figure 2 shows
images of live Human Embryonic Kidney (HEK) cells transfected with
mCherry (excitation at 530 nm; emission 580 nm). The images
compare silver-coated glass (upper) and a standard glass slide (lower)
using epi-fluorescence imaging at a subcritical incident angle (left)
and a supercritical angle for surface plasmon excitation (right).
All images were captured under identical experimental conditions
(exposure time, gain, laser power etc.) and no brightness or
contrast correction was applied. The epi-fluorescence image with a
silver thin film (top left) is darker than glass alone (lower left)
because of low light transmission through the film. The SPMFM
image appears very bright when the surface plasmon angle is
achieved. This provides a straightforward way of adjusting and
optimising the excitation light beam (difficult in standard TIRFM).
The SPMFM image has clearly reduced background noise with
high lateral resolution.
SPMFM is a powerful technique with which to obtain highly
sensitive, real time images of membrane events in living cells.
The fluorescence signal is enhanced and background noise reduced
dramatically in SPMFM compared with standard TIRFM.
The authors would like to deeply acknowledge Yannick Goulam,
Alexandre Roland, Viviane Devauges and Sandrine Lévêque-Fort
for their decisive contribution in the results presented in this note.
The authors welcome email enquiries for metal coated slides for
anyone wishing to try this new imaging approach.
References • Special Features Download full-text
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The sensitivity of TIRFM can be enhanced by
using surface plasmon properties of silver
Surface plasmons efficiently transmit excitation
and fluorescence light through the metallic
thin film resulting in an enhanced signal and
a drastic reduction in background noise
High 1.49 N.A. TIRF objectives are essential for
SPMFM in enabling excitation of the surface
plasmon above the critical angle
No specific additional equipment is required
for SPMFM except a coverslip coated with a
metallic thin film (preferably silver).
Emmanuel Fort was appointed Associate Professor at the Paris Diderot
University in 2000. He joined the Langevin Institute (City of Paris
Industrial Physics and Chemistry Institution – ESPCI) in 2009. He is a
founder and leader of the Applied Plasmonic Imaging Centre
dedicated to applied plasmonic research in live science, spectroscopy
and integrated nanophotonics for telecommunication and computing.
Professor Fort’s interests include surface nano-fabrication, plasmonic
properties and plasmon-fluorophore coupling, fluorescence imaging,
nanoplasmonic sensors for biomedical applications and in vivo
imaging. He is also involved in studies on classical systems with particle-
wave duality and quantum-like behavior.
Karla Balaa received her Ph.D. degree from University of Nantes,
France, in 2007, her Master’s degree from the Lebanese University,
Lebanon in 2003. She’s currently a research engineer at the Applied
Plasmonic Imaging Centre in Langevin Institute, ESPCI-ParisTech.
Her research fields are plasmonics for biomedical applications and
technological development in fluorescence imaging.
1. Balaa K., Devauges V., Goulam Y., Studer V., Lévêque-Fort, Fort E. Live cell
imaging with Surface Plasmon-Mediated Florescence Microscopy. Proc. SPIE
(2009) In press.
2. Le Moal E., Fort E., Lévêque-Fort S., Cordelières F. P., Fontaine-Aupart M.P.,
Ricolleau C. Enhanced fluorescence cell imaging with metal-coated slides.
Biophys J. 2007;92(6):2150-61.
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Emmanuel Fort Karla Balaa