Journal of Microscopy, 2009
doi: 10.1111/j.1365-2818.2009.03188.x
Received 2 April 2009; accepted 7 May 2009
A compact STED microscope providing 3D nanoscale resolution
D. WILDANGER, R. MEDDA, L. KASTRUP & S.W. HELL
Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics,
Am Fassberg 11, 37077 Göttingen, Germany
Key words. Fluorescence nanoscopy, resolution, STED microscopy,
supercontinuum.
Summary
The advent of supercontinuum laser sources has enabled the
implementation of compact and tunable stimulated emission
depletion fluorescence microscopes for imaging far below the
diffraction barrier. Here we report on an enhanced version
of this approach displaying an all-physics based resolution
down to (19 ± 3) nm in the focal plane. Alternatively, this
single objective lens system can be configured for 3D
imaging with resolution down to 45 × 45 × 108 nm in
a cell. The obtained results can be further improved by
mathematical restoration algorithms. The far-field optical
nanoscale resolution is attained in a variety of biological
samples featuring strong variations in the local density of
features.
Introduction
Despite its limitations regarding the spatial resolution, farfield fluorescence microscopy enjoys ongoing popularity in
the life sciences. Simple sample preparations under ambient
conditions and the ability to perform imaging in 3D are
compelling arguments in its favour. The introduction of
stimulated emission depletion (STED) microscopy (Hell &
Wichmann, 1994; Hell, 1997; Klar & Hell, 1999; Klar
et al., 2000) has conceptually and practically overcome the
diffraction barrier in far-field fluorescence microscopy and
thus opened the door for far-field imaging with resolution in
the nanometre range (Donnert et al., 2006). The basic idea
behind STED and related optical microscopy concepts (Hell &
Kroug, 1995; Hell, 1997, 2003, 2007; Hell et al., 2003) is
to switch off or inactivate fluorescent molecules in the focal
region using a dedicated beam of light (STED beam) featuring
a zero at a moving coordinate in space. The result is that only
molecules at sub-diffraction proximities to the zero are active
and hence contribute to the signal at the given coordinate.
Correspondence to: Stefan W. Hell. Tel: +49 (0)551 201-2500; fax: +49 (0)551
201-2505; e-mail: [email protected]
C 2009 The Authors
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Journal compilation More recent approaches in far-field fluorescence nanoscopy
do not switch the molecules with spatially structured beams
but individually and stochastically in space (Betzig et al., 2006;
Hess et al., 2006; Rust et al., 2006). Complementing the earlier
approaches, they have substantially broadened the range of
applicability of emerging far-field optical nanoscopy.
The central zero of the STED beam is produced by phase
modifying the wavefront of a laser beam, which is then
spatially overlaid with the focal spot of the excitation
beam. The wavelength of the STED laser is chosen such
that if molecules become excited, they are immediately
quenched to the ground state by stimulated emission.
Thus, molecules subject to the STED beam are essentially
deprived of their ability to fluoresce or simply switched off,
whereas those located within a small range at the zero
maintain their fluorescence capability. Consequently, the
effective point-spread function (PSF) of the STED microscope
is narrowed down to sub-diffraction dimensions, which upon
scanning translates into an enhanced spatial resolution,
because features closer than the diffraction limit are recorded
sequentially in time. The full-width half-maximum (FWHM)
of the region from which the fluorescence can be emitted is to
first approximation given by (Hell, 2003, 2004; Westphal &
Hell, 2005)
r (I ) ≈
λ
√
2n sin α 1 + I /Is
(1)
where λ is the STED wavelength, n is the refractive index of
the medium, α is the semiaperture angle of the objective lens
and I is the intensity maximum engulfing the zero. Is is the
characteristic intensity required for reducing the fluorescence
ability by a factor of two for the dye being used; it typically
amounts to 10–30 MW cm−2 when implementing STED
microscopy with pulses on the order of 100 ps. It should be
noted that STED microscopy does not improve the resolution
by narrowing the employed light beams, which remain
diffraction limited of course; rather the resolution increase
rests on the specific employment of distinct molecular states
(a fluorescent and a non-fluorescent one) for the separation of
adjacent features.
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The use of pulsed lasers is not strictly necessary (Willig et al.,
2007), but due to the electronic and vibrational kinetics of the
fluorophore, STED microscopy is most efficiently implemented
using pulsed lasers with pulse durations smaller than the
fluorescence lifetime of a few nanoseconds. In addition,
the use of pulse repetition rates low enough to allow a
relaxation of the triplet state reduces photobleaching (Donnert
et al., 2007a). For this reason, STED microscopy has been
notoriously associated with complex setups and expensive,
high-maintenance laser systems.
Recent developments in laser technology have allowed
revisiting the light source issue in STED microscopy. Initially,
pulsed supercontinuum sources were used as a source of
fluorescence excitation only (Auksorius et al., 2008) but,
eventually allowed a replacement of the STED laser as well.
Concretely, a STED microscope realized at a fraction of the
cost and complexity of previous setups was introduced, which
used a single commercial all fibre-based supercontinuum
source and a commercial vortex phase plate for preparing
the focal doughnut (Wildanger et al., 2008). The system
delivered a lateral spatial resolution in the 30–50 nm range
in various colours. Although it required little alignment
and afforded long-term stability, the reported compact STED
microscope did not yet fully exploit the potential of the
supercontinuum laser source. This stems from the fact that
the vortex phase plate requires circular polarization. However,
because the supercontinuum beam is unpolarized, half of
the power was dumped. By adding a second beam path
with orthogonal circular polarization, we have now extended
the performance of our supercontinuum STED microscope,
achieving an all-optics based spatial resolution down to 20 nm.
Alternatively, we used the second beam to realize 3D subdiffraction resolution in a compact single objective lens based
STED system.
Materials and methods
STED microscope
Because the basic design of the supercontinuum STED
microscope has been detailed elsewhere (Wildanger et al.,
2008), the subsequent discussion concentrates on the relevant
changes in the setup (Fig. 1). In order to take advantage of
the full spectral power density of the supercontinuum laser
(SC-450-PP-HE, Fianium Ltd., Southampton, UK), a total
of three beams are derived from the source: an excitation
beam and two STED beams. The laser output is first filtered
to remove the infrared components (λ > 950 nm) of the
spectrum and is then split using a polarizing beamsplitter
cube to provide two orthogonally polarized but otherwise
equal beams. Both beams are individually directed to prismbased monochromators selecting a ∼20-nm-wide spectral
band at 700 nm for STED. The STED beams are then coupled
into two polarization-preserving single mode optical fibres.
Fig. 1. Experimental setup: the supercontinuum beam is split into two orthogonally polarized beams (polarizing beamsplitter cube), which are spatially
filtered through single-mode optical fibres after the STED wavelength has been extracted (WS). After collimation, the beams pass through separate phase
plates (PM 1 and 2) and are recombined at a polarizing beam splitter. A dichroic mirror (DC3) couples the STED light into an objective lens. The excitation
beam is extracted from the s-polarized supercontinuum beam by employing a dichroic mirror (DC1). After the desired wavelength has been extracted
with an interference filter (EF) the beam is spatially filtered (SMF) and coupled into the microscope by a further dichroic mirror (DC2). A quarter-wave
plate in front of the objective lens ensures circular polarization for all beams. The fluorescence light passes through the dichroic mirrors and is focused
into a multi-mode fibre, which acts as confocal pinhole and is finally detected with an avalanche photodiode; object scanning is performed with a piezo
scan stage. Inset: combinations of phase plates and resulting STED PSFs used to achieve either ultimate lateral resolution (HR) or 3D superresolution
(3D).
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Journal compilation STED MICROSCOPY WITH A SUPERCONTINUUM SOURCE
The excitation laser beam is derived from the s-polarized
beam using a shortpass dichroic beamsplitter (Z670SPRDC,
AHF Analysentechnik GmbH, Tübingen, Germany) positioned
prior to the monochromator. It is then directed through a
bandpass filter (Z570/10×, AHF Analysentechnik GmbH) and
coupled into a polarization-preserving single mode fibre.
At the fibre outputs, the two STED laser beams are separately
collimated, recombined with a polarizing beamsplitter cube
and coupled into the objective lens with a dichroic beamsplitter
(Z680SPRDC, AHF Analysentechnik GmbH). Likewise, the
excitation light leaving the optical fibre is collimated and
is coupled into the objective lens with a second dichroic
mirror (Z568RDC, AHF Analysentechnik GmbH). Careful
adjustment of the orientation and tilt of a superachromatic
quarterwave plate (RSU 1.4.15, B. Halle Nachfl. GmbH, Berlin,
Germany) placed in front of the objective lens renders circular
polarization for both STED beams.
For the purpose of 3D nanoscopy, two different phase
plates were placed into the STED beams to achieve a lateral
(xy) and an axial (z) resolution enhancement, respectively
(Fig. 1, inset). In this configuration, a vortex phase plate (RPC
Photonics, Rochester, NY, U.S.A.) is used in conjunction with
a custom-made phase plate introducing a π phase shift in a
central disk that covers half of the beam’s photon flux. For
the experiments targeted at the highest possible lateral (xy)
resolution, two vortex phase plates were placed into the STED
beams. To ensure the appropriate circular polarization for the
s- and the p-polarized beams the helical phase ramps have
to be placed in opposite senses of direction. The pulse timing
was adjusted by equalizing the optical path lengths of all three
beams.
To account for the improved resolution of the new
microscope the piezo scan stage was replaced with a model
which allows higher scan speeds and provides superior
scanning precision (P-733.3DD and controller E-710.2CD
with dynamic scan linearization, Physik Instrumente GmbH,
Karlsruhe, Germany). The coarse positioning was carried
out with two crossed linear stages (M426, Newport SpectraPhysics GmbH, Darmstadt, Germany) mounted on a custombuilt elevation (z) stage. Typically, images of 10 × 10 μm up to
20 × 20 μm were acquired with a 10–20 nm pixel size and pixel
dwell times of 0.2–1.0 ms. The STED and confocal reference
images were recorded sequentially. Under these conditions,
the total acquisition time for an image of 10 μm × 10 μm,
10 nm pixel size, 0.2 ms dwell time is 4 min.
Cell culture
The SH-SY5Y neuroblastoma cell line was grown as described
previously (Encinas et al., 2000). Cells were seeded on standard
glass cover slips to a confluence of about 80%. A total of 10 μM
all-trans-retinoic acid (EMD Biosciences Inc., San Diego, CA,
U.S.A.) was added the day after plating. After 5 days in
the presence of retinoic acid, the cells were washed three
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Journal compilation 3
times and incubated with 50 ng mL−1 human brain derived
neurotrophic factor (Alomone Laboratories, Jerusalem, Israel)
in serum-free medium for >7 days.
Fluorescence staining
Immunostaining of neurofilaments and vimentin was
performed with anti-O-glycosylated NF-M [NL6] mouse
IgG (Sigma, Munich, Germany) and anti-vimentin [V9]
mouse IgG (Sigma), respectively, as primary antibodies and
with ATTO 590 sheep anti-mouse IgG (ATTO-TEC GmbH,
Siegen, Germany/Dianova GmbH, Hamburg, Germany) as
a secondary antibody, respectively. After several washes
the cover slip was embedded in Mowiol mounting medium.
Immunolabelling of microtubules in PtK2 cells was carried
out according to the same protocol but with anti-beta-tubulin
mouse IgG (Invitrogen GmbH, Karlsruhe, Germany) as the
primary antibody.
Results and discussion
In order to characterize the resolution improvement by the use
of a second STED beam, we imaged fluorescent nanoparticles
[FluoSpheres® , red fluorescent (580/605), Invitrogen GmbH]
dispersed on a microscope cover slide. Unless otherwise noted,
44 nm beads were used for the single-beam experiments,
whereas 24 nm particles were used in the dual-beam
measurements. Figure 2 shows the comparison between the
confocal reference and the STED recordings with a single
(A, B) and with two beams (C, D) corresponding to timeaveraged STED powers of 2.5 and 5 mW which give rise to
pulse energies (peak intensities) of 2.5 nJ (9.2 GW cm−2 )
and 5.0 nJ (18.3 GW cm−2 ), respectively. The beads in the
STED images exhibit FWHM of (45 ± 7) nm and (35 ±
4) nm, respectively. The example shown in Fig. 2(C) also
demonstrates that two beads spaced only 36 nm apart are
separated by a pronounced dip in brightness of 30%. When the
bead diameters are taken into account, the optical resolution
of the microscope can be inferred to be ∼42 nm and ∼31 nm,
respectively. This corresponds to a near 1.4-fold increase in
resolution, which is in perfect agreement with Eq. (1), which
√
predicts an enhancement by a factor of ∼ I2 /I1 ≈ 1.4.
Figures 3 and 4 show exemplary recordings of
immunolabelled neurofilaments and vimentin fibres. In
each case, the confocal reference is contrasted with the
respective STED image, showing a near 10-fold resolution
improvement taking advantage of physical phenomena only.
The images of these fibrous structures demonstrate that
the improved resolution discerns rich detail about the fibre
bundles, including the organization of individual filaments.
By measuring the width of single filaments, features down
to (19 ± 3) nm in size are found, which is about the size of
the antibody construct. Hence, in the immunofluorescence
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D. WILDANGER ET AL.
Fig. 2. Red fluorescent beads (Ø = 44 nm) imaged confocally (A) and with one STED beam (B). Red fluorescent beads (Ø = 24 nm) imaged confocally
(C, deconvolved) and with two STED beams (D, deconvolved). Scale bar: 500 nm. (E) Line profile along the path indicated in (D): two beads separated by
36 nm are clearly discernible. (F) Histograms of the measured FWHM of 24 nm beads imaged with a single (green, I STED = 9.2 GW cm−2 ) and with two
STED beams (orange, I STED = 18.3 GW cm−2 ).
imaging, the resolution is superior to that of the imaging of
beads. We ascribe this fact to the superior properties of the dye
ATTO 590 at the used wavelengths.
The fluorescent marker also showed good photostability. It
was possible to image the same regions in the vimentin or
neurofilament labelled cells with adequate contrast multiple
times. Specifically, the signal dropped to about half of its initial
value only after four nanoscale recordings. To investigate
whether the observed photostability was a unique feature of
the ATTO 590 dye, we prepared a series of neurofilament
samples using the same immunofluorescence protocol but
with different dyes attached to the secondary antibody: ATTO
590, ATTO 594 (both from ATTO-TEC GmbH), DyLight
594 (Thermo Scientific/Perbio Science, Bonn, Germany)
and Alexa 594 (Invitrogen GmbH). A small region of each
sample was repeatedly imaged with the same time-averaged
excitation power (0.27 μW) and STED power (3.8 mW),
corresponding to pulse energies (peak intensities) of 0.3 nJ
(4.2 MW cm−2 ) and 3.8 nJ (13.9 GW cm−2 ), respectively.
All other experimental parameters were kept constant, in
particular the excitation and STED wavelengths, pulse lengths
and pixel dwell times. In each image, an approximately
3 × 3 μm2 region containing representative fluorescent
features was selected.
The integrated fluorescence from these regions normalized
to the first frame is plotted as a function of frame number
in Fig. 5(A). Among the investigated dyes, ATTO 590
displayed the best photostability. The bleaching curves follow
monoexponential decays, and fitting these decays provide
the following fractional loss of fluorescence per image: 17%
(ATTO 590), 19% (ATTO 590), 20% (DyLight 594), and 27%
(Alexa 594). These numbers indicate that the variations in
photostability are relatively small among these dyes and that
several commercially available fluorescent dyes are suited for
STED microscopy with STED wavelengths around 700 nm.
A further series of bleaching measurements was performed
with the same dye throughout (ATTO 590) but with increasing
STED intensities. The other experimental conditions were
maintained, but now the STED power was varied between
0 and 3.8 mW to afford pulse energies in the range of 0–
3.8 nJ (Fig. 5B). The experiments show that fading is caused
by both the excitation and the STED beam. The photobleaching
caused by STED is acceptable but, in total terms, it accounts
for most of the observed loss in fluorescence. Although the
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Fig. 3. Fluorescence imaging of immunostained neurofilaments. Bundled neurofilaments imaged with confocal (left) and with STED microscopy (right,
data deconvolved). Insets (i) and (ii) show intensity profiles along the paths indicated by the pink and orange arrows, respectively. Although the fibre
bundles are blurred in the confocal images individual strands are well resolved in the STED images. Also the organization of the filaments can be clearly
discerned. Measurement of point objects indicate a resolving power of (19 ± 3) nm focal plane resolution. Scale bars: 1 μm.
exact mechanisms underlying the STED photobleaching have
not been investigated yet in full, they surely involve photon
absorption of excited singlet (S 1 ) (Dyba & Hell, 2003) and
triplet state (T 1 ) molecules (Donnert et al., 2007a). Our
observation is that the importance of each state in the
bleaching pathway of a particular dye strongly depends on the
dye and to some extent also on the molecular environment.
Nonetheless, the observed photostabilities are remarkable
compared to earlier experiments in which >50% loss of
C 2009 The Authors
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Journal compilation fluorescence after a single recording was not uncommon
(Dyba & Hell, 2003). We assume that an important
ingredient to the observed photostability is the relatively
low repetition rate of 1 MHz, which allows an efficient
relaxation of the triplet population between successive pulses,
thus eliminating the photobleaching pathways via the triplet
state (T-Rex effect) (Donnert et al., 2007a). Furthermore, the
temporal and spectral structure of the laser pulses obtained
from the supercontinuum spectrum may differ from the
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Fig. 4. STED imaging of densely packed immunostained neurofilaments: confocal (left) and with STED microscopy (right, data deconvolved). Insets
highlight densely packed fibre bundles that are entirely blurred in the confocal images whereas individual strands are well resolved in the STED images.
Scale bars: 1 μm.
conventional laser sources used previously and may augment
photostability; this hypothesis merits further investigation.
Although the doughnut employed in our configuration
improved the lateral resolution by ∼10-fold over that of
the confocal microscope, the axial (z) resolution remained
limited to ∼600 nm. Because the objects shown in Figs 3
and 4 are rather sparse, the signal-to-background ratio
and the spatial resolution are not compromised by the
fluorescent background. However, if dense structures rather
than isolated filaments or clusters are imaged, both the
contrast and the resolution can suffer substantially. One
approach to overcoming the background issues is to embed
the sample in a polymer resin and cut it into 50–100-nmthick slices (Punge et al., 2008). This procedure also minimizes
photobleaching and allows 3D reconstruction. However,
the low photobleaching rates observed in our recordings,
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Fig. 5. Photobleaching experiments. Neurofilaments were immunolabelled with various dyes (DyLight 594, ATTO 594, ATTO 950, Alexa 594) and
were repeatedly imaged. The integrated and normalized fluorescence intensity is plotted as a function of the frame number (A). To study the influence of
the STED pulse energy the measurement was repeated with a ATTO 590 stained sample using increasing STED laser powers (B). The STED pulse energies
and the corresponding lateral resolutions are given in parentheses. All images were acquired with the same pixel size (20 nm) and dwell time (0.2 ms).
The excitation intensity was kept constant.
prompted us to pursue a purely optical axial superresolution
approach by STED. In contrast to the slicing technique, it is
non-invasive and leads directly to a 3D stack which needs no
further alignment. To this end, we replaced the vortex phase
plate in the s-polarized beam with a phase plate that introduces
a constant π phase shift in a circular region covering half the
area of the back aperture of the objective lens. The resulting
STED focal spot features a central zero along with two main
maxima, one above and one below the focal plane. To confine
the effective fluorescence excitation both laterally and axially,
the z-doughnut is incoherently overlaid with the doughnutshaped STED spot produced by the p-polarized beam (Fig. 1,
inset). By varying the intensities of the two STED beams, the
aspect ratio of the resulting effective PSF can be adjusted over
a wide range, including a nearly isotropic PSF.
The resolution provided by this configuration was again
tested on 44 nm fluorescent nanoparticles. An xz-section
(10 × 1 μm2 , pixel size 20 × 33 nm2 ) containing a single
fluorescent bead was acquired, followed by analysis of two
line profiles placed along the optic axis (z) and within the
focal plane (x), see Fig. 6(A). By fitting Lorentzian functions
to these profiles and taking the 44 nm diameter of the
fluorescent particles into account, the FWHM of the effective
PSF were estimated to be 45 nm laterally and 108 nm
axially corresponding to a ∼175-fold decrease in focal volume
compared to the confocal PSF. Three-dimensional imaging
was further pursued on immunostained microtubules. A
C 2009 The Authors
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Journal compilation region of 15.3 × 15.3 × 2 μm3 with a voxel size of
20 × 20 × 66 nm3 was scanned. After deconvolution and
background subtraction, a 3D reconstruction of the data was
created with the visualization software package AMIRA (Visage
Imaging, Berlin, Germany). An isosurface rendering of the
reconstruction along with a maximum projection is shown on
the top of Fig. 6(B) whereas in the lower panel the rendering
shows an xz section of the same dataset. The intensity line
profile shown in the inset discloses features which extend less
than 100 nm in the z-direction. As a result, structures which
are 200 nm apart are clearly resolved. Finally, Fig. 6(C) shows
five subsequent slices from a different image stack.
Conclusion
We have demonstrated a compact supercontinuum laser
source providing a lateral resolution of 20–25 nm in STED
microscopy. The beams can also be prepared to render a 3D
STED microscopy resolution up to 45 × 45 × 108 nm in a
single-lens setup. The gain in resolution provides object details
that are completely indiscernible by conventional or confocal
microscopy. In combination with the supercontinuum laser
source, various dyes proved remarkably photostable and
allowed 3D superresolution imaging by purely optical
sectioning. It should be noted that, unlike in fluorescence
nanoscopy concepts utilizing single molecule switching, the
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D. WILDANGER ET AL.
Fig. 6. STED microscopy with superresolution in 3D. (A) Resolution measurement on fluorescent nanoparticles (44 nm) reveals 52 nm and 110 nm in
the lateral (x-) and axial (z-) directions, respectively. (B) 3D imaging of immunolabelled microtubules: isosurface rendering (top) and maximum intensity
projection along the y-axis (bottom) of a stack of images comprising 30 slices. The inset shows an intensity profile at the indicated position. (C) Slices from
a different image stack acquired at a 100 nm pitch in the z-direction.
practically obtained spatial resolution does not depend on
the local concentration of fluorophores or on the density of
the features to be imaged; the resolution is just a function
of the dye and the employed aperture and wavelength. This
is particularly obvious in Fig. 4, where neurofilaments are
clearly discerned in a single image despite the strong variations
in local packing density.
The current laser system operates at a repetition rate of only
1–2 MHz which, by limiting the attainable scan rate, accounts
for the relatively long recording times. In the near future,
supercontinuum sources operating at 5–10 MHz repetition
rate are expected to become available. These systems should
accelerate image acquisition by a similar factor and enable
comparable or superior resolution with a single STED beam.
Another modification will extend the microscope to twocolour operation as has already been demonstrated in more
complex setups (Donnert et al., 2007b). In conclusion, STED
microscopy using supercontinuum sources has a significant
potential for future STED microscopy implementations that
will prove invaluable in many applications.
Acknowledgements
The research leading to these results has received funding from
the European Community’s Seventh Framework Programme
FP7/2007–2011 under grant agreement no. 201837. D.W.
acknowledges a doctoral fellowship by the German National
Academic Foundation.
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