Download Parallelized STED fluorescence nanoscopy

Parallelized STED fluorescence nanoscopy
Pit Bingen,1,2 Matthias Reuss,1,2 Johann Engelhardt,1,2 and Stefan W. Hell1,2,3
1
German Cancer Research Center (DKFZ), Optical Nanoscopy Division, Im Neuenheimer Feld 280, 69120
Heidelberg, Germany
2
Bioquant Center, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany
3
Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077
Göttingen, Germany
*[email protected]
Abstract: We introduce a parallelized STED microscope featuring m = 4
pairs of scanning excitation and STED beams, providing m-fold increased
imaging speed of a given sample area, while maintaining basically all of the
advantages of single beam scanning. Requiring only a single laser source
and fiber input, the setup is inherently aligned both spatially and temporally.
Given enough laser power, the design is readily scalable to higher degrees
of parallelization m.
©2011 Optical Society of America
OCIS codes: (350.5730) Resolution; (180.2520) Fluorescence microscopy; (000.2170)
Equipment and techniques; (080.4865) Optical vortices.
References and links
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20. M. A. Schwentker, H. Bock, M. Hofmann, S. Jakobs, J. Bewersdorf, C. Eggeling, and S. W. Hell, “Wide-field
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relaxation,” Nat. Methods 4(1), 81–86 (2007).
1. Introduction
Since the beginning of the 20th century, far-field optical microscopy has been essentially
limited to >λ/2 > 200 nm in resolution, with λ denoting the wavelength of light. While
electron and scanning probe microscopy have provided much finer details since then, they
lack the possibility of non-invasively imaging (living) cells and tissue as well as other
transparent materials in three dimensions (3D). In the 1990s it was discovered that the
diffraction resolution barrier in far-field fluorescence microscopy can be effectively overcome
by employing basic transitions between the states of the fluorophores in use [1]. Specifically,
transitions ensuring that fluorescent molecules remain transiently non-fluorescent allowed the
separation of nearby features by causing them to emit sequentially. This simple strategy forms
the basis of Stimulated Emission Depletion (STED) microscopy [1, 2] as well as of more
recent far-field fluorescence nanoscopy techniques [3–11]. While they maintain most of the
advantages of far-field imaging, these concepts procure resolution down to the nanoscale.
To separate features by sequential detection, STED microscopy forces a part of the
fluorophores to dwell almost exclusively in the ground state [1]. The nearly permanent
occupation of the ground state is enforced by the so-called STED beam, whose wavelength
and intensity ISTED are tuned such that it is capable of stimulating excited fluorophores
instantaneously back down to the ground state. The focal pattern of the STED beam features
(at least) a (single) point [12] or line [13] of vanishing intensity at which the occupation of the
fluorescent state is inherently allowed. By precisely positioning the zero point(s) or line(s) in
the sample and adjusting ISTED one can define the coordinate in space where the fluorophores
can still assume the fluorescent state, i.e. are not 'switched off' by the enforced occupation of
the ground state [9, 10].
The most common STED microscopy configuration is to overlay a diffraction-limited
focused excitation beam with a doughnut-shaped STED beam. Although both beams are
limited by diffraction, resolution beyond the diffraction limit is possible, because above a
certain threshold intensity IS, molecules will almost exclusively remain non-fluorescent; the
time they spend in the fluorescent state becomes negligible. The resolution can be increased to
subdiffraction dimensions by making sure that the doughnut intensity surpasses the threshold
IS at subdiffraction distances to the zero- intensity point, so that virtually all fluorophores are
kept non-fluorescent but those residing at the closest proximity to the doughnut zero. The
resolution increases in proportion with the square-root of ISTED,m/IS, with ISTED,m denoting the
intensity at the (doughnut) maximum engulfing the zero [10, 14]. In fact, any reversible
transition between two states can be used to create subdiffraction resolution images [15],
which is why the STED principle has been generalized to any reversible saturable optical
linear (fluorescence) transitions (RESOLFT) [10]. Hence, any reference to STED in this paper
will equally apply to the more general RESOLFT concept.
It should also be noted that the principles of STED or RESOLFT do not require the
imaging of the fluorescence onto a point detector, which is the hallmark of confocal
microscopy. Therefore, STED and RESOLFT superresolution need not be implemented as
point-scanning or confocal systems. In essence any pattern that uses an intensity minimum
(zero) to confine a signaling or a non-signaling state to subdiffraction dimensions can be used
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to create superresolution images. However, because it facilitates the suppression of
background and elegantly provides three-dimensional (3D) imaging, confocal arrangements
have been preferred in most STED implementations. Furthermore, since the diffraction
resolution problem applies only to features that are closer than λ/2, it is conceptually
straightforward to create patterns of multiple local zero-points or zero-lines separated by
distances > λ/2. Such patterns automatically parallelize STED or RESOLFT nanoscopy.
Nonetheless, to date most STED microscopes are still based on single point-scanning, even
though the image field is far greater than λ/2. This is a drawback for imaging large field of
views, since an n-fold higher resolution in the x- and the y -direction in the focal plane
requires an n × n-fold denser pixelation and hence a recording time longer by this factor.
Effective parallelization has been accomplished by spinning disc arrangements with
multiple pinholes, which have permitted to increase the recording speeds in confocal
microscopy [16]. Other methods have used cascaded beam splitters to create 64 parallelized
beams (LaVision BioTec, Germany). Another way of parallelizing the scanning process is to
apply arrays of zero-lines and valleys, which is usually called structured illumination [17].
Within the super-resolution techniques, saturated structured illumination SPEM/SSIM do
provide parallelization of subdiffraction imaging, but the reported versions are laid out such
that they require data post-processing [4, 18]. Moreover, they are not confocally arranged,
meaning that 3D imaging is more difficult to implement. The stochastic super-resolution
methods [6–8], which ensure that molecules capable of fluorescent emission are further away
than λ/2, are inherently parallelized. Nevertheless, the stochastic nature of single molecule
detection and the fact that at most a single emitting molecule is allowed within a λ/2 spatial
range, compromises their imaging speed. Providing images based on the nearly simultaneous
emission of an arbitrary number of fluorophores from predetermined positions [9, 10], STED
and RESOLFT are advantaged with regard to time resolution. Yet, while theoretical studies
have been given [19] and initial steps with RESOLFT parallelized with arrays of line-shaped
zeros have been shown [20], parallelized imaging with these concepts still requires
development. In particular, the widely applied STED microscopy has so far not been
parallelized.
STED microscopy can be parallelized using arrays of multiple doughnuts [19] or with
arrays of lines [9, 14] ('structured illumination'). The latter is presently less attractive simply
because of the large power requirement for the STED beam, which stems from the fact that
the maximum intensity ISTED,m has to exceed IS. A challenge in parallelizing STED by
implementing multiple doughnuts is to retain the zero intensity at the doughnut center while
guaranteeing the alignment with the excitation spots. We now demonstrate a parallelized
STED microscopy using m=4 doughnuts realized by beam multiplication, which is scalable to
a higher number m of spots. Additional properties are the inherent alignment of both
excitation and STED beams by launching them from a single fiber. Both parallelization and
the creation of multiple doughnuts are achieved just by polarization optics [21]. The
fluorescence originating from the individual excitation spots is spatially separated and
collected by m=4 single photon-counting modules. The photons collected in these
independent detection channels stem from different positions in the focal plane as determined
by the spatial arrangement of the doughnut pattern. Different scanning schemes are
demonstrated that combine the individual channels in order to reduce the required recording
time by a factor of four. Furthermore a new type of beam-scanning device is presented.
2. Design of the parallelized setup
Parallelization of STED by focused beam multiplication requires a device generating multiple
beam pairs (excitation/STED) and the co-alignment of these beams. Furthermore, their
wavefronts need to be controlled such that the generated focal intensity spots are nearly
diffraction-limited and doughnut-shaped with central zeros for the STED light. Last but not
least, in pulsed STED setups such as the one implemented here, one should be able to tune
individual laser intensities and the relative delay between the laser sources.
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Fig. 1. Parallelized STED microscope: A stack of Wollaston prisms (W1,W2) are used for
generating multiple foci and a segmented waveplate (SWP) for shaping the beams to either
excitation spots or doughnut-shaped STED beams (Inset: Image Plane). Unpolarized excitation
(635 nm) and STED (745 nm) originating from outputs pumped by a common laser are
combined using a dichroic mirror (DC) and coupled into a common single-mode-fiber after
adjusting timing by inserting an optical delay (not shown). Both unpolarized output beams are
split into four polarized beamlets in the conjugated backfocal plane (Inset: Conjugated
Backfocal Plane; arrows indicate linear polarization orientation of the individual beamlets).
Depending on the available laser power, this configuration is scalable to any power of 2 by
adding further Wollaston prisms. All beamlets are then reflected by a bandpass filter (BP) and
focused into a scanning device (Quadscanner), consisting of four motorized mirrors, placed in
the conjugated image plane (Inset: Conjugated Image Plane). The first pair of of mirrors
induces a lateral displacement in one direction (X-Scan) while the second pair displaces the
beams in the perpendicular direction (Y-Scan). All beamlets are circularized using a quarterwave plate (QWP) to avoid a bias on STED induced by molecular orientation before passing
through the SWP which acts as beamshaping device (Inset: Separating element). The
fluorescence passes back through the scanner and bandpass filter before being focused onto a
mirrored pyramid which splits the fluorescence originating from the four focal volumes. Each
fluorescent signal is focused onto a single-photon counting module (APD).
Optical devices, such as Wollaston prisms, can separate light beams into two orthogonally
polarized sub-beams that exit the device at a specific angle determined by the devices’
geometry. Both laser wavelengths required for STED can originate from a single fiber to cross
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the Wollaston prism while remaining perfectly aligned. For large scanning angles or
differences in wavelength between STED and excitation wavelength one should account for
birefringence dispersion by using achromatic Wollaston prisms [22]. The axial alignment of
the beams depends of the micro objectives used. For APO type objectives used in the present
system they are usually shifted by 100-200 nm which is well within the 400-500 nm axial
resolution and can hence be neglected. Stacking these prisms doubles the number of beams
for each prism while maintaining their alignment. The number of beams is therefore scalable
depending on the available laser power. Moreover the intensities can be evenly and accurately
distributed among the individual beams by rotating the input polarization and individual
prisms. We therefore decided to use this type of beamsplitting device in order to minimize
losses of the limited laser power and construct a stable parallelized STED setup.
Two approaches [21, 23] have been reported to simplify STED nanoscopy by making
excitation and STED beams pass through a common beam-shaping device. Both
configurations leave the wavefront of the excitation wavelength largely unaltered and cause
the STED beam to interfere destructively at the focal point, thereby creating the desired
doughnut shape. Consisting of a chromatic segmented wave plate (SWP) that alters the
polarization of individual beam segments in the aperture, the (easySTED) type proposed by
Reuss et al. [21], is polarization-independent with respect to keeping the central intensity
minimum of the doughnut as low as possible. Non-circular components will basically lead to
asymmetry effects in connection with the random orientation of the molecules. Such effects
can be avoided by providing a uniform distribution of polarization directions, e.g. circular
polarization. The handedness of the circular polarization is unimportant. In addition, they do
not need to be perfectly circular because non-circular components do not fill up the central
intensity minimum of the SWP induced doughnut. For all these reasons, we preferred the
SWP over the helical vortex phase plate which has been commonly used so far [24].
The polarization-independence of the beam-shaping device is also important for
implementing a single-fiber as a common source for the STED and the excitation light using
Wollaston prisms, because a stack of N Wollaston prisms generates 2N beams where 2N-1
beams have left-handed and the other 2N-1 beams right-handed circular polarization after
passing a quarter wave plate. In comparison the more commonly used vortex phase plate only
works with one handedness. In order to produce the desired auto-aligned spots of light, the
SWP is simply placed centrally close to a plane that is optically conjugate to the back focal
plane. It can be placed right after the objective lens, because fluorescence can pass without
major alterations, which in turn makes the preceding pupil plane accessible for the
beamsplitting elements. More versatile optical devices such as spatial light modulators (SLM)
obviously exist, that could combine beamsplitting and shaping tasks, but these devices would
waste a large part of the available laser intensity for controlling the wavefront amplitude and
phase. Moreover, SLMs would not allow for simultaneous and individual control of the
different wavelengths in an easySTED configuration.
Scanning was accomplished with a home-built beamscanner, here referred to as
Quadscanner, consisting of four galvanometric mirrors oriented in a configuration that
essentially creates a parallel displacement of the focused beams in the conjugated image plane
as disclosed in the published patent application WO 2010/069987. This in turn displaces the
excitation pattern in the sample by an amount set by the magnification factor objective/tube
lens combination. The Quadscanner manipulates the beams close to their beam waist (as
opposed to regular beamscanning which rotates the beams in a plane conjugate to the pupil
plane) thereby inducing fewer aberrations by the limited flatness of light-weight
galvanometric mirrors. The two degrees of freedom per scan axis offer a flexible way to
control both the lateral position as well as the plane of rotation of the beamscanning. This
ensures a standing beam in the SWP plane and thereby guarantees that the beam remains in
the center of the SWP. The fluorescence has to be separated from the excitation beam path
before passing back through the Wollaston prism to avoid splitting up the fluorescence, which
would reduce the signal and lead to crosstalk between the detection channels. For this reason
and to make sure that the beams rotate in the SWP plane under the presence of the Wollaston
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prisms, these are inserted near the conjugated backfocal plane (cBFP), preceding the scan lens
and the bandpass filter (Fig. 1). Compared to conventional commercial beam scanning
devices, which operate in the conjugated backfocal plane, the Quadscanner reduces the
required number of optical images, thereby simplifying the setup and increasing its stability.
Each of the excitation foci overlaid with a doughnut-shaped STED PSF leads to
independent fluorescent sources at well-defined positions in the focal plane. The signal from
each of these channels must be isolated by focusing the fluorescence onto point detectors,
after being separated using a beam separating element, or onto a CCD Camera. We decided to
use avalanche photodiode (APD) point detection which enables faster recording times. For
larger degrees of parallelization, CCD cameras or APD arrays could represent an easier
solution. Different imaging approaches can now be taken regarding scanning and
recombination of the different channels to obtain faster acquisition due to parallelization. The
most straightforward approach is to scan four non-overlapping areas and to recombine the
individual frames by stitching them together in a mosaic-like pattern. For larger field-of-views
(FOV) one has to create an array of scanning positions in order to avoid multiple scanning of
the same positions. A second approach consists of scanning four large area images
simultaneously at a four-times shorter pixel dwell time and adding the overlapping regions of
the full-scale frames. Besides reducing the recording time, the advantage of such an approach
could be a bleaching-reducing effect by reducing deposited energy dose imposed by each of
the individual beams and by allowing the relaxation of the molecules from long-lived dark
states back into the ground state [25]. Depending on the FOV however, the non-overlapping
region can represent a major proportion, which would gradually reduce the useable area. Still
another option would be to use a comb like scanning pattern using different prism rotations to
produce a line of spots which is not further pursued here.
3. Experiment
We constructed a four channel multispot STED setup using Wollaston prisms for multispot
generation, a chromatic segmented waveplate (SWP) to shape the focal spots and four single
photon detection units for detection (Perkin-Elmer, USA). To further simplify our setup we
used a custom laser system (SC450-20-2, Fianium, United Kingdom) with a single laser pump
operating at a repetition rate of 18.45 MHz and generating two unpolarized outputs: a
supercontinuum spectrum ranging from 520 nm to 720 nm and a high power output used for
STED at 745±5 nm. The excitation band (Z633/10 X, Chroma, USA) is selected from the
supercontinuum spectrum and recombined with the STED line using a dichroic mirror
(SP750, AHF, Germany), after an optical delay maximizing STED. Once coupled into a
single fiber, excitation and STED laser beams no longer required any spatial or temporal
alignment. The collimated unpolarized beams passed two Wollaston prisms (Jenoptik,
Germany) rotated by 45° with respect to each other to generate four uniform beams (see Fig.
1. inset) at an angle of 20 arcminutes, producing a rhombus-like pattern in the sample with a
separation of 5.75 µm at an angle of 45°. The offset between the wavelengths due to the
prisms’ birefringence dispersion of ~1% can be neglected in the present system. The beams
are reflected by a bandpass filter (z625-745 rpc, Custom-Made, AHF), also acting as
fluorescence filter in the detection path, before being focused into the homebuilt beamscanner
(Quadscanner) consisting of four galvanometric mirrors (Cambridge Technology, USA)
placed next to a commercial microscope (DMI 3000B, Leica Microsystems, Germany). Used
as beamshaping device, the SWP (B. Halle, Germany) consists of four segments of crystal
quartz cut from a single block and assembled with the fast axes oriented as described
elsewhere [21]. The thickness is adapted such that a birefringent retardation of 2.5 λ is
achieved for 750 nm and 3λ for 635 nm between the slow and fast axis of the crystal. The
SWP was placed just before the 1.46 numerical aperture 100x oil objective lens (Leica
Microsystems, Germany). The sample was clamped directly to the objective lens by a homebuild sample holder to reduce thermal drifts. Excitation was performed with a total timeaveraged optical power of 5 µW at the back aperture of the objective lens. The time-average
power of the STED beam at the back aperture of the objective lens was 70 mW,
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corresponding to a pulse energy of 3.8 nJ. This energy, which was distributed equally among
the four spots considering transmission losses, yields a time-averaged power density of 4.1
MW/cm2 per STED doughnut in the focal region. This in turn corresponds to an average pulse
intensity of 1.1 GW/cm2 of the 200 ps STED pulse in the focal region. For separating the
fluorescence in the detection path we used a custom-made silver-coated pyramid to separate
the four channels in the image plane and four lenses to focus the fluorescence onto four single
photon counting modules (SPCM-AQR-13/14, Perkin-Elmer, USA). Scanning as well as data
processing of the collected signal was controlled using a FPGA board (PCI-7833R, National
Instruments, USA) and self-built scanning software (LabVIEW, National Instruments, USA).
Additional shortpass (SP750, Semrock, USA) and bandpass (HQ690/60x, Chroma, USA)
interference filters were placed in the detection path to isolate the fluorescence from reflected
excitation and STED light.
Figure 2. shows the rhombus-like distribution of the STED and excitation light in the focal
plane. For one beam pair the overlaid spot of excitation (green) and STED (red) focal spots
were obtained by scanning an 80 nm gold bead (BBInternational, UK) across the focal region.
We obtained four-leaf-shaped doughnuts for the STED beam at a wavelength of 745 nm due
to the fourfold segmentation [21] while the excitation spots remained largely unaltered. The
central intensity of the doughnut resides below signal background which should guarantee for
good resolution improvement and fluorescence signal. The measured excitation spots were
separated by 5.75±0.07 µm in the long axis at an angle of 45°.
Fig. 2. Beamshaping by the segmented waveplate in combination with two Wollaston prisms.
Left: STED (top) and excitation (bottom) spots are arranged in the rhombus-like pattern when
focusing onto a camera in the backfocal plane (scale bar: 1 µm). Top right: Focal intensity
distributions of one of the four parallelized excitation (green) and STED (red) beam pairs. The
segmented wave plate converts the 745 nm STED beam into a doughnut and the 633 nm beam
into a regular spot (Scale bar: 500nm). Bottom right: line profiles of excitation (green) and
STED (red) focal spots along the direction indicated by the arrows on the top image.
To investigate the resolution of our system, 20 nm fluorescent beads (Invitrogen, USA)
filled with Crimson fluorophores (625/645) were imaged both by confocal and STED
microscopy in the four separate channels (Fig. 3) and recombined using either mosaic-like
stitching or large-scale addition of overlapping frames. Please note the distinct rhombusshaped stitching pattern of the first scanning scheme. In all four detection channels a 5-fold
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resolution improvement was obtained with the smallest feature imaged on the scale of 35 nm.
The second scanning scheme, which was performed with a four times smaller pixel dwell
time, provided similar resolution and signal. It is however exposed to the risk of
compromising the resolution gain due to drift or residual errors in the offsets. Crosscorrelation of the detection channels can minimize the recombination errors. No asymmetries,
which could be attributed to molecular orientation effects or polarized excitation, were
observed. All detection channels displayed similar detection count rates.
Fig. 3. Fluorescent beads measured using parallelized confocal (top) and STED (center) by
either adding overlapping frames (left) or stitching non-overlapping frames together (right).
Scale bars: 500 nm. Both scanning schemes offer a fourfold faster acquisition of
superresolving STED microscopy. Bottom: Line profiles across the beads indicated by the
white arrows above. All images represent raw data and were obtained using pixel dwell times
of 50/200 µsec (overlay/stitched) and a pixel size of 20 nm.
These observations confirm the proper alignment of our system regarding SWP position,
beam quality, polarizations and detection. Please also note that the lenses placed in the
detection path were adapted such that the detectors openings acted as confocal pinholes.
Relatively low STED beam intensities were used to avoid extensive bleaching induced by the
joint action of the excitation and the STED beams, which allowed the comparison of the same
structure in the different detection channels (Fig. 4.).
In Fig. 4 we tested the resolution improvement in all four channels over the entire field of
view (FOV), approximately 80 x 80 µm2, by taking images using STED and confocal
microscopy of tubulin in mammalian (PtK2) cells. We did not observe reductions in signal or
resolution towards the image border. The different channels show identical images and
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resolution and are shifted laterally in the rhombus-like shape as expected (Fig. 4). Four
parallel tubulin filaments can be clearly distinguished in the four STED images but cannot be
resolved using confocal microscopy. Minor variations are always expected due to the multiple
scanning and potential bleaching of each recording and the differing detection efficiencies.
To test the speed-increasing capabilities of our setup we scanned an area of 5.8 x 4 µm2 of
vimentin in PtK2 cells, immunolabeled with the organic dye KK114, using STED and
stitching the four channels together (Fig. 5). The individual frames (5.8 x 4 µm2) were then
recombined by stitching, resulting in an effective scanning area of 11.6 x 8 µm2. After the
scanning, a second single large scan was taken using only a single beam and the same region
identified. The same features were detected with similar resolution as in the stitched image.
Minor discrepancies observed were most probably due to previous bleaching. Overlapping of
individual subframes can be minimized by carefully controlling scanning angle and area, but
small errors may still occur in the border regions if stitching offsets are not carefully
calibrated. Multiple scanning in the bordering region can also increase bleaching and lead to
artifacts.
Fig. 4. Left: Confocal overview and magnified region (inset) of KK114-labelled tubulin strands
in a fixed mammalian (PtK2) cell. Right: Magnified STED images of the four parallelized
detection channels 1-4, each corresponding to the confocal region. Note that all STED channels
show identical features and that four parallel tubulin fibers (arrows) can be observed using
STED which cannot be resolved using confocal microscopy. The confocal images have been
recorderd using one of the detection channels. The other three channels provide similar images
(not shown). All images represent raw data and were obtained using a pixel dwell time of 100
µsec and pixel size of 20 nm. Scale bars: 1 µm / 10 µm (Magnifications/Overview).
Finally we tested the second scanning approach by overlaying four large frames of fixed
PtK2 cells with Atto647N-labelled tubulin after adding an offset in each of the channels at a
dwell time of 50 µsec and compared it to a single image taken at 200 µsec (Fig. 6). The
images gave comparable resolution and signal as a single frame at four times longer dwell
time. The advantage of this approach is that recombination errors are spread across the entire
image and no longer restrained to the stitching borders. However depending on the scanning
area, the non-overlapping region can represent a major proportion of the image, which
reduces the gain in recording speed. In brief, we demonstrated four times faster scanning
capabilities of STED with respect to single beam scanning and acquisition. Importantly using
this configuration, parallelization can be scaled up to more than m=4 excitation and STED
beams and the acquisition rate can be increased accordingly using the scanning schemes
demonstrated here.
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Received 13 Sep 2011; revised 29 Oct 2011; accepted 31 Oct 2011; published 7 Nov 2011
21 November 2011 / Vol. 19, No. 24 / OPTICS EXPRESS 23724
Fig. 5. Top left: Confocal overview of KK114-labelled vimentin fibers in mammalian (PtK2)
cells. Scale bar: 5 µm Right: Confocal (top) and STED (bottom) images of area indicated on
the left (white square) using a stitched scanning scheme. Note the Rhombus-like stitching
pattern. To avoid overlapping of frames, overlapped regions were considered only once. Scale
bars: 1 µm. Bottom left: Large single channel STED image following STED and confocal
stitched images for comparison. The other detection channels show similar images (not
shown). No significant disparities between stitched and single frames images can be observed
when following individual vimentin fibers along multiple frames. All images represent raw
data and were obtained using a pixel dwell time of 20 µsec and pixel size of 20 nm. Scale bar:
1 µm.
Fig. 6. Comparison of single STED image (left) at 200 µsec pixel dwell time of the first spot
passing the sample (to avoid comparing a bleached image) and by adding the four detection
channels at a pixel dwell time of 50 µsec (right) of Atto647N-labelled tubulin in PtK2 cells.
Scale bars: 1 µm. All images represent raw data and were obtained using a pixel size of 20 nm.
4. Conclusion
We have designed an alignment-free parallelized STED setup that provides m = 4 -fold
increased scanning speed with respect to regular single spot STED setups while delivering a
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(C) 2011 OSA
Received 13 Sep 2011; revised 29 Oct 2011; accepted 31 Oct 2011; published 7 Nov 2011
21 November 2011 / Vol. 19, No. 24 / OPTICS EXPRESS 23725
similar resolution. Being based on an easySTED setup and incorporating only one laser
source makes all STED and excitation beams quasi identical and intrinsically pre-aligned,
both spatially and temporally. A stack of 2 Wollaston prisms in combination with an
(easySTED) segmented waveplate (SWP) creates m=4 independent subdiffraction fluorescent
sources separated by an angle set by the Wollaston prisms. The fluorescence from these
detection channels can be recombined in different scanning modes and used to increase the
scanning speed by the number of implemented channels.
The chromatic segmented waveplate (SWP), previously used for easySTED, transforms
the STED wavelength into a doughnut-shaped spot with a nearly perfect central intensity zero
while leaving the excitation wavelength unaltered, irrespective of the input polarization. Only
this property enables the use of Wollaston prisms as beamsplitting elements for generating
multiple spots for STED nanoscopy, which generates two spatially separated output beams
with perpendicular linear polarizations. The benefit is that the relative intensities between the
beams can be matched to each other by controlling the polarization of the input. Furthermore
the angle between the beams, which directly translates to spot separation in the image plane,
can be manufactured to any desired value and remains constant thereafter, i.e. virtually
without drift.
We also introduced a novel beamscanning device based on four galvanometric mirrors
offering two degrees of freedom for each scanning axis thereby allowing to control both
position and plane of rotation of the beamscanning. This Quadscanner is placed close to the
first conjugated image plane where each mirror pair effectively displaces the focused beams
laterally, which in turn translates into point-scanning in the sample. This configuration makes
the scanning lens obsolete and opens the first conjugated back-focal plane for beam-shaping
devices, such as the Wollaston prisms in our setup, and allows for more compact and rugged
beamscanning STED microscopes.
In conclusion, this paper demonstrated the first experimental parallelization of STED
superresolution fluorescence microscopy. Scaling the parallelization further up is mainly
limited by the power of the laser sources available, However, switching the fluorescence by
transferring the fluorophores between long-lived states requires lower light intensities
[5,9,10,14], in which case enough laser power is available to scale parallelization further up
using this and related optical schemes.
Acknowledgments
We thank Ellen Rothermel for preparing the Ptk2 cells. PB was funded by an AFR PhD grant
from the National Research Fund, Luxembourg (TR-PHD BFR08-059).
#154558 - $15.00 USD
(C) 2011 OSA
Received 13 Sep 2011; revised 29 Oct 2011; accepted 31 Oct 2011; published 7 Nov 2011
21 November 2011 / Vol. 19, No. 24 / OPTICS EXPRESS 23726