Fluorescence microscopy with diffraction resolution

barrier broken by stimulated emission

Thomas A. Klar, Stefan Jakobs, Marcus Dyba, Alexander Egner, and Stefan W. Hell†
Max-Planck-Institute for Biophysical Chemistry, High Resolution Optical Microscopy Group, 37070 Göttingen, Germany

Edited by Daniel S. Chemla, E. O. Lawrence Berkeley National Laboratory, Berkeley, CA, and approved May 12, 2000 (received for review March 10, 2000)

The diffraction barrier responsible for a finite focal spot size and lin) with an intracavity frequency doubler. This system partly
limited resolution in far-field fluorescence microscopy has been converted the Ti:Sapphire pulses into visible ones. The pulse
fundamentally broken. This is accomplished by quenching ex- trains were temporally adjusted by an optical delay stage and
cited organic molecules at the rim of the focal spot through coupled into the setup by dichroic mirrors (Fig. 1a).
stimulated emission. Along the optic axis, the spot size was The duration of the visible pulses was 0.2 ps to ensure
reduced by up to 6 times beyond the diffraction barrier. The temporally defined excitation of the fluorophore. The near-
simultaneous 2-fold improvement in the radial direction ren- infrared STED pulses were stretched by a grating to ␶ ⫽ 40 ps.
dered a nearly spherical fluorescence spot with a diameter of This allowed us to extend STED over a time period much longer
90 –110 nm. The spot volume of down to 0.67 attoliters is 18 than the relaxation time (⬇0.2 ps) of the vibrational substate of
times smaller than that of confocal microscopy, thus making our the electronic ground state into which the molecule is quenched.
results also relevant to three-dimensional photochemistry and This is important because it allows the quenched molecules to
single molecule spectroscopy. Images of live cells reveal greater escape re-excitation by the same beam through vibrational
details. relaxation. Because we elected to use dyes with fluorescence
wavelengths ␭fl from 650 to 800 nm, we adjusted the excitation
and stimulation wavelengths to ␭exc ⬇ 560 nm and ␭STED ⬇ 765
I t is generally assumed that the extent of the focal spot of a
microscope is not reducible beyond the wavelength of light ␭,
because diffraction demands an extent of at least ␭兾3 in the
nm, respectively. The broad spectral range of the fluorescence
(80–120 nm) and the comparatively sharp spectrum of the laser
lateral and ␭ in the axial direction. In consequence, for more light (⬍7 nm) enabled fluorescence separation from the laser by
than a century, focused light was abandoned whenever higher suitable emission filters, as in any fluorescence microscope. The
spatial definitions were required. The diffraction limit also fluorescence was collected in the backpropagating mode as the
stimulated the invention of electron, scanning probe, and near- stimulating light beam passed straight through the sample.
field optical microscopy, but these techniques are largely sur- To start off from the smallest possible ‘‘classical’’ spot, we used
face-bound and not able to image the intact cellular interior. a 1.4 numerical aperture oil immersion lens (Leica 100⫻,
Therefore, far-field light microscopy, encompassing the conven- Planapo, Wetzlar, Germany), which is a standard objective lens
tional and confocal fluorescence microscope, remained the most featuring the highest available semiaperture angle (67.3°). Fig. 1b
widely applied microscopy in biological research (1); however, displays an axial section of the focal spatial distribution of the
the limited spot size hinders the visualization of fine subcellular excitation light featuring a pronounced maximum with a radial
structures. and axial extent of 220 and 560 nm, respectively. It was measured
We now report the generation of fluorescence focal spots of by registering the back-scattered light of a 100-nm-diameter gold
substantially reduced extent and their application to three- bead that was scanned with a piezo stage operating at 5-nm
dimensional imaging. For the first time, the axial and lateral precision (Melles Griot, Cambridge, U.K.). Behaving as a bright,
resolution of a lens is found to be in the same range (90–110 nm). point-like object, the gold bead efficiently probed the intensity
Confocal sectioning is improved up to 5-fold. The improvement in the focus and hence the spatial distribution of excitation,
is attained by quenching through stimulated emission the excited which we aimed to reduce in size (Fig. 1b).
fluorophores at the rim of the excitation focal spot with a beam The reduction of the extent of the f luorescence spot re-
whose wavelength is in the red edge of the fluorophore emission quired a focal intensity distribution of the STED-beam that
spectrum. Stimulated emission forces excited molecules to an was intense around the focal point, but dark within it. This
upper vibrational level of the ground state, whose ultrafast modification was accomplished by the use of an optical phase
vibrational decay (2) prevents re-excitation by the same beam. plate consisting of a planar glass substrate with a 1-␮m-thin
As a result, fluorescence can be entirely stopped (3–6). ‘‘Engi- layer of MgF2, evaporated on a central circular area. By
neering’’ of the fluorescence spot, or point-spread-function introducing a delay by ␭STED兾2, this layer reverts the sign of the
(PSF) by stimulated emission depletion, was predicted to im- wave amplitude with respect to the remaining ring-shaped
prove the resolution in the transverse direction (3, 5), and initial area. Because we chose half of the total amplitude in the
experiments with nanocrystals indeed confirmed an improve- entrance pupil to be phase-reverted, the focused wave front
ment by a factor of 1.3 (6). However, this moderate improvement produced destructive interference at the focal point and
was achieved along a single direction only, and it remained rendered a central minimum, as shown in the measurement in
unclear whether it would be effective in biological imaging. Fig. 1c. Whereas the excitation PSF featured the usual three-
dimensional (3D) pattern, the STED-beam-PSF featured high
Physical Principles and Setup
intensity above and below the focal plane. In the transverse
Our setup used two synchronized trains of laser pulses: a train
of visible pulses was used for excitation and a near-infrared This paper was submitted directly (Track II) to the PNAS office.
counterpart for stimulated emission depletion (STED). Each Abbreviations: PSF, point-spread-function; STED, stimulated emission depletion; 3D, three-
excitation pulse was immediately followed by a STED pulse. The dimensional; FWHM, full-width-at-half-maximum.
pulses originally stemmed from a mode-locked Ti:Sapphire laser †To whom reprint requests should be addressed. E-mail: shell@gwdg.de.
(Coherent, Santa Clara, CA) emitting at a repetition rate of The publication costs of this article were defrayed in part by page charge payment. This
f ⫽ 76 MHz in the near-infrared. The pulses entered an optic article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
parametric oscillator (Angewandte Physik und Elektronik, Ber- §1734 solely to indicate this fact.

8206 – 8210 兩 PNAS 兩 July 18, 2000 兩 vol. 97 兩 no. 15

 level of the ground state with N*0, the temporal change of the
population is given by:
dN 1/dt ⫽ ⫺N 1␴ I STED/ប ␻ ⫹ N *0␴ I STED/ប ␻ ⫺ N 1k Fl [1]

and
dN *0/dt ⫽ N 1␴ I STED/ប ␻ ⫺ N *0␴ I STED/ប ␻ ⫺ N *0k vib . [2]

␴ is the molecular cross-section for the transition and ប␻ is the

STED beam photon energy; kFl and kvib are the rates for
spontaneous emission and vibrational relaxation, respectively.
The first and second term on the right hand side of Eq. 1 describe
the stimulated emission from the fluorescent state and the
re-excitation from the vibrational state by the stimulating beam,
respectively. Whereas the third term on the right hand side of Eq.
1 describes the decay of N1 by spontaneous emission, its coun-
terpart in Eq. 2 describes the vibrational decay of N*0. If the focal
intensity is low enough to render the rates of the stimulated
transitions much slower than kvib, re-excitation is negligible and
N*0 ⬇ 0. Hence, for a given duration ␶ of the STED pulse, the
population of the fluorescent state and the remaining fluores-
cence obeys the proportionality N1 ⬀ e⫺␶␴ISTED兾ប␻ in good ap-
proximation. For higher intensities, the depletion of N1 will be
governed by the fast vibrational relaxation alone (kvib ⱖ 3 ⫻ 1012
s⫺1). Altogether, this leads to a highly nonlinear relationship
between N1 and ISTED, the precise behavior of which is obtained
by numerical computation (3, 5).

Results
This nonlinear relationship is indeed found in the experiment
(Fig. 2a) in which we measure the fluorescence of the styryl dye
LDS 751 (Molecular Probes) as a function of the STED-beam
intensity for a given pulse length. The experiment was carried out
by focusing without phaseplate onto a dye nanocrystal. The
saturation of the depletion is so pronounced that, at STED-beam
T
peak intensities ISTED ⱖ ISTED ⬇ 0.75 GW兾cm2, the fluorescence
of the dye is suppressed to less than 10%. The ‘‘threshold
T
intensity’’ ISTED is arbitrarily elected for orientation. We also
note that the peak intensity was calculated as

冉 冕
ISTED共z兲 ⬵ PSTED ⫻ S共z, 0兲/ 2␲f␶ ⫻
⬁

0
冊
S共z, r兲r dr , [3]

Fig. 1. Microscope. (a) Excitation pulses are followed by stimulated emission where S(z, r) is the measured focal intensity distribution and
depletion pulses for fluorescence inhibition. After passing dichroic mirrors r the radial coordinate. PSTED is the focused time-averaged
and emission filters, fluorescence is detected through a confocal pinhole by a power. The axial coordinate z is taken at the location of the

APPLIED PHYSICAL
counting photodiode. (b) Measured excitation PSF. (c) Measured STED-beam- maximum. Fig. 2a also allows us to predict the effect of the

SCIENCES
PSF featuring local minimum at the center and intense maxima above and
STED-beam-PSF of Fig. 1 when the focal intensities in the
below the focal plane. Z denotes optic axis. The measurements of b and c are T
carried out with the pinhole removed.
region of the maxima are ⱖISTED : The f luorescence is inhibited
in the whole focal region except for the innermost part of the
spot. With increasing intensity, the area of inhibition grows
direction, it also displayed a weak but non-negligible, dough- and confines the f luorescence to an ever smaller volume. The
nut-shaped first maximum that is expected to reduce the result is a f luorescent spot with subdiffraction dimensions as
f luorescence spot also in the lateral direction. the one shown in Fig. 2d.
The spot in Fig. 2d was probed by a 48-nm bead stained with
Fig. 1 b and c indicates that the subtraction of the latter from
the fluorophore LDS 751 with an intensity of ISTED ⬇ 2.8
the former should result in a narrower f luorescent spot.
GW兾cm2, calculated as the average of the intensity of the two
However, it is also apparent that the mere subtraction would axial maxima. Because we placed a confocal pinhole in front of
increase the resolution only marginally, because the local the detector, switching on and off the STED-beam automatically
minimum of the STED-beam-PSF is of about the same extent contrasted the narrowed focal spot with that of a confocal
as the maximum of the excitation PSF. To really break the microscope (Fig. 2b). The difference in focal extent becomes
diffraction barrier, the minimum of the STED-beam-PSF must obvious when comparing their XZ-sections shown in the inset.
be narrower. This requirement is fulfilled by a nonlinear Because of the strong local maxima along the optic axis, the most
relationship between the intensity ISTED of the STED beam pronounced difference is found in the axial direction. Whereas
and the residual population of the f luorescent state, which is the confocal spot featured an axial full-width-at-half-maximum
in fact theoretically expected (3, 5). If we denote the popula- (FWHM) of 490 nm, that of the STED-fluorescence spot was
tion of the f luorescent state with N1 and that of the vibrational only 97 nm.

Klar et al. PNAS 兩 July 18, 2000 兩 vol. 97 兩 no. 15 兩 8207

 T
Fig. 2. (a) Fluorescence is a nonlinear function of stimulating intensity; 10% remaining fluorescence is obtained for ISTED corresponding to PSTED of 2.2 mW in
the focus. (b) Surface plot of XZ-section (Inset) of confocal fluorescence spot for 1.4 oil immersion lens. (d) Same as b but with STED-beam PSF switched on. (c)
Corresponding axial intensity profiles demonstrate 5.1-fold reduction of the axial width (FWHM) from 490 nm down to 97 nm.

We note that the STED-beam was switched on in the forward we compare axial images of randomly dispersed 100-nm-diameter
scan and off in the backward scan, line by line. Along with other beads. The fluorescent beads were custom-labeled (Polysciences)
spectroscopic evidence (6), this measure ruled out photobleach- with the dye Pyridine 2 (Lambdachrome, Göttingen, Germany). A
ing as a potential cause of the observed spot size reduction. In
the lateral direction, we measured a FWHM of 104 nm and 127
nm in x and y directions, respectively, meaning that the lateral
resolution was doubled. The breaking of the resolution limit in
the radial direction is a direct consequence of the doughnut-
shaped first maximum of the STED-beam PSF. Because of the
lower intensity in the ring, the lateral improvement was less
pronounced than its axial counterpart. It could be enforced,
however, by a differently optimized phase plate that should lead
to a similar factor of spot size reduction also in the lateral
direction.
Nevertheless, the nearly spherical spot size obtained with the
present arrangement is particularly attractive because it reduces
spot shape artifacts. Altogether, the 3D volume defined by the
FWHM of the confocal spot was narrowed down by a factor of
18, so that we obtained a focal volume of 670 zeptoliter, which
is for the first time below the 1 attoliter barrier. When compared
with the FWHM volume of the main maximum of the conven-
tional PSF in Fig. 1b, this reduction amounts to a factor of 28.
To compare the signal strength between the STED-
f luorescence and the confocal spot, we display the non-
normalized axial profiles in Fig. 2c, revealing a decrease of the
peak height by 40% in the STED case. This decrease is com-
Fig. 3. XZ-images of 100-nm-diameter fluorescent beads (a and b) and of
paratively low and is likely reduced by further optimization. 100-nm-diameter negatively stained glass beads agglomerations (c and d) as
Therefore the STED-beam indeed primarily suppresses the observed in the confocal (a and c) and the STED-fluorescence (b and d)
outer parts of the focus and leaves the center mostly unaffected. microscope. Note the artifacts indicated by arrows induced by the elongated
The improved axial resolution is also evident in Fig. 3, in which spot in the confocal image and their reduction in the STED counterpart.

8208 兩 www.pnas.org Klar et al.

similar comparison is made for ‘‘negatively stained’’ beads: i.e.,
100-nm unstained silica beads immersed in fluorescent solution.
Negative staining was performed by dispersing 100-nm fused silica
beads in Pyridine 2 dissolved in a Mowiol mounting medium [100
mM Tris䡠HCl, pH 8.5兾9% Mowiol 4-88 (Hoechst)兾25% glycerol].
In the regular confocal image (Fig. 3 a and c), the beads emerge as
axially elongated entities. Importantly, this diffraction-induced
misrepresentation is immanent to any focusing light microscope
using a single lens. In the STED-fluorescence image (Fig. 3 b and d),
however, the beads are largely spherical and better distinguished.
The peak intensities for efficient stimulated emission ISTED are
higher than those required for 1-photon excitation because the
rate of stimulation needs to be faster than the spontaneous
emission. However, they are, by about two orders of magnitude,
lower than the peak intensities of 200–500 GW兾cm2 successfully
used in live cell multiphoton microscopy (7). This is attributable
to the fact that the cross sections for 1-photon absorption and
stimulated emission are similar. On the other hand, because of
the longer pulse width, the pulse energies are of the same order.
As potential optical limiting effects usually scale nonlinearly with Fig. 4. Resolution improvement in live cells. XZ-images of a S. cerevisiae
yeast cell with labeled vacuolar membranes with standard confocal resolution
the intensity (8–10), our method is live cell compatible.
(a) and with axial resolution improved by STED (b). Whereas the confocal
Next we demonstrate that the STED method is indeed appli- mode fails in resolving the membrane of small vacuoles, the STED microscopy
cable to live cells. For this purpose, we labeled the vacuolar better reveals their spherical structure. XZ-images of membrane-labeled E.
membrane of live yeast cells with the dye RH-414 (Molecular coli show a 3-fold improvement of axial resolution by STED in d as compared
Probes). Saccharomyces cerevisiae strain RH 488 (MATa, his4, with their simultaneously recorded confocal counterparts in c.
leu2, ura3, lys2, bar1-1) were grown at 27°C in complete medium
(1% yeast extract兾2% peptone兾2% glucose兾20 mg/ml uracil and
adenine) to an absorbance at 600 nm of approximately 0.8. The We noticed that after repeated imaging the dye dispersed in
cell culture was diluted with one volume of 130 mM NaCl, 45 mM the cell, thus indicating that the STED beams induced photo-
NaH2 PO4, and 0.55 M sorbitol (pH 7.3). Vacuolar staining was stress to the yeast cell. This was not observed when imaging the
accomplished by adding RH 414 to a final concentration of 70 E. coli. In the STED-fluorescence E. coli image (Fig. 4d), the
␮M from a 25 mg兾ml EtOH stock solution. The uptake of RH bacterial membrane is better outlined than in its confocal
414 followed similar kinetics to the well characterized vital stain counterpart taken at the same time (c). To quantify the im-
FM 4-64. Because the uptake of FM4-64 has been shown to be provement of axial resolution in the sample, we precipitated a
strictly time-, temperature-, and energy-dependent, we conclude fine layer of fluorophore on the coverslip, which is easily noticed
that the labeled cells were alive (11). For microscopy analysis, the as a horizontal line in the images. We found that in the STED
cells were embedded in 1% low melting agarose. Images were fluorescence image the axial resolution is improved by a factor
recorded 1 h after adding the dye. We also imaged live Esche- of 3. We recorded several stacks of STED-images as the stained
richia coli bacteria labeled with the dye Pyridine 4 (Lamb- E. coli did not show notable alterations induced by imaging.
dachrome). For labeling the E. coli membranes, one volume of Of the dark red fluorescent dyes scrutinized, we found the
saturated ethanolic Pyridine 4 solution was diluted with 100 following 17 compounds to display a fluorescence suppression of
volumes of bacterial culture. Staining was performed for 1 h at ⬎90%: (i) styryls, such as Styryl 6, 7, and 8, LDS751, Pyridine
room temperature. After a washing step, the cells were embed- 1 and 2, and RH 414; (ii) oxazines, such as Oxazine 170, Nile Red,
ded in LB medium with 15 g兾liter agar and were placed on a and 7-AAD; (iii) carbocyanines such as DODCT, DTDCI, Cy 3,
coverslip. Cy 3.5, Cy 5, and (iv) the fluoresceines C-562 and C-563. (The
To reduce potential photostress, the yeast cells and the E. coli dyes are tradenames of Lambdachrome, Amersham Pharmacia,
were imaged with calculated ISTED ⫽ 1.07 GW兾cm2 and 2.44 or Molecular Probes. The fluorescence depletion was measured

APPLIED PHYSICAL
GW兾cm2, respectively. Because we focused into a watery me- using uniform layers of saturated solutions in EtOH or DMSO

SCIENCES
dium, these intensities are present only at the interface between mixed with Mowiol mounting medium in equal ratio. We note
the glass coverslip and the watery medium. With increasing that the Stokes shift and cross-section may slightly vary with the
focusing depth, the intensities drop off as a result of spherical solvent or mounting medium. The investigation of visible wave-
aberration induced by the concomitant refractive index mis- length dyes for STED is a next step in our studies. We expect that
T
match. Although both intensities are greater than ISTED and a number of green, yellow, and red dyes will qualify for strong
therefore in the nonlinear range of the depletion curve of Fig. 2a, stimulated emission depletion.
ISTED at the sample is expected to be significantly lower than in
the case with the bead. Hence, we expect the resolution im- Discussion and Outlook
provement by STED to be lower in the live cell sample. We have demonstrated the breaking of the diffraction barrier in
Fig. 4 compares confocal axial images of the live yeast (a) and fluorescence light microscopy up to a factor of 2 in the lateral
E. coli (c) cells with their STED-fluorescence counterparts in b and 6 in the axial direction. The improvement of resolution along
and d, respectively. In Fig 4a, the axial images of the yeast the optic axis was larger because the regions of strongest
vacuoles are observed as oval-shaped structures. Again, the STED-beam intensity were located immediately above and
diffraction-limited spot in the confocal microscope overempha- below the focal plane. By positioning similarly intense beams in
sizes the vertical parts of the vacuolar membrane. In contrast, the lateral direction, a further substantial reduction of the
because of the axially narrowed focal spot, the STED- fluorescent volume in this direction is anticipated.
fluorescence image in Fig. 4b gives a more faithful picture of the In the STED-images of the live bakers yeast and E. coli cells,
spherical shape of the vacuoles of the live yeast cell (see arrow). the gain in resolution is somewhat lower than what is found with
In particular, they reveal the spherical shape of smaller vacuoles the PSF measurement. This can be largely attributed to the fact
that cannot be recognized as such by the confocal microscope. that the cells were maintained in a watery environment whose

Klar et al. PNAS 兩 July 18, 2000 兩 vol. 97 兩 no. 15 兩 8209

refractive index (n ⫽ 1.34) was significantly different from that interference effects removal. In contrast, the STED-concept is
of the glass coverslip (n ⫽ 1.51). Spherical aberration induced by solely based on physical phenomena and can be synergistically
the mismatch in refractive index reduces the local STED-beam combined with 4Pi microscopy to further improve the resolution
intensity (12) and to a lesser extent the steepness of the (5). To the best of our knowledge, there is no reported alter-
STED-beam edges in the sample. This can be solved, however, native to the concept of STED that fundamentally improves the
by replacing the oil immersion lens with a water immersion lens axial resolution in a single lens setting and without data pro-
and adapting the phase plate diameter to the water lens entrance cessing. Computational image restoration can in addition im-
pupil. prove the spatial resolution to a certain extent (18). The up to
Besides the improvement of spatial resolution, STED also has 2-times and 5- to 6-times larger optical bandwidth of our
other important applications. The instantaneous control of the microscope, in radial and axial direction, respectively, and the
fluorescence process under microscopy conditions opens up fact that the central part of the fluorescence spot is not signif-
interesting perspectives in lifetime microscopy (4, 13) and nano- icantly suppressed are good preconditions to obtain a resolution
spectroscopy from the nano- to the subpicosecond scale. STED by deconvolution of 40–50 nm in all directions, with a single lens.
should also open up new perspectives in the field of single The concept of STED-microscopy defeats the diffraction
molecule spectroscopy (14), such as the control of the excited resolution barrier in a fundamental way. The demonstrated
state of individual molecules with tens of femtoseconds temporal breaking of the diffraction barrier by a factor of 6 and the 18-fold
resolution. reduction of the focal volume are not principle limits, because a
We will also address in the future whether repeated stimulated further increase of the STED-intensity would decrease the focal
emission influences the photostability of the molecule. Whereas volume even further. The precondition for further increasing the
fast quenching to the ground state prevents intersystem crossing, spatial resolution is to keep the STED-PSF at the center at zero
the STED beam might also transiently excite the molecule into level and to increase its intensity in the surrounding area. It is an
a higher singlet state. This would not have been directly observed important theoretical insight that, despite using diffraction
in our experiment because the radiationless internal conversion limited beams, the concept of STED-fluorescence microscopy
is shorter by an order of magnitude than the duration of our could reach far-field spatial resolution at the molecular scale.
STED pulse (40 ps). Putative transiently excited molecules In practice, however, the continuous increase in intensity will
would have also been quenched. STED pulses of several tens of be challenged by concurrent photostress inflicted on the dye and
picoseconds are preferred also because of the reduced proba- the sample that may ultimately lead to increased bleaching and
bility of nonresonant multiphoton excitation from the ground live cell incompatibility. At higher intensities and for some
state. wavelengths, stimulated emission might be accompanied by
At much lower intensities and with a broader central mini- molecular multiphoton excitation. It will be interesting to ex-
mum, the STED-beam could still be used as a ‘‘photophysical’’ plore whether these challenges can be met by selecting fluoro-
3D pinhole that, by surrounding the fluorescence spot, would phores with higher stimulated emission cross-sections or other
relax the requirement of a tiny pinhole in confocal microscopy. suitable conditions. Improving the capabilities of STED-
Such a ‘‘soft’’ 3D pinhole could enable improved confocal fluorescence microscopy so to achieve resolution at the nano-
imaging of deep regions of scattering specimens or eliminate meter scale is a challenging but tempting scientific endeavor.
focal aberrations of the excitation light. These applications We also note that the depletion by stimulated emission is not
should be facilitated by the fact that STED could be readily restricted to fluorescence, but also applicable to nonfluorescent
implemented in standard confocal beam scanners. STED can organic molecules with sufficient excited state lifetimes and
also be combined with total internal reflection microscopy to significant stimulated emission cross-sections. In fact, our mi-
create spatially controlled excitation profiles (15). croscope demonstrates the control of the spatial and temporal
The far-field lateral resolution demonstrated in Fig. 2 is distribution of molecules in their excited state. As the excited
equivalent to that obtained with most near-field optical micro- state is a first step in chemical transitions, our concept should
scopes; however, in the STED microscope, the resolution is enable restriction of chemical reactions, such as those occurring
entirely based on focused beams, does not imply delicate han- in photo induced three-dimensional data storage, to a spatial
dling of a low-throughput scanning tip, and is able to image volume hitherto inconceivable with focused light.
inside a 3D volume. Axial resolution can also be substantially
improved by the coherent use of two opposing lenses, as is the We thank Dr. S. Schröder-Köhne for supplying us with the yeast strain
case in 4Pi microscopy (16, 17). However, besides requiring RH 488 and W. Sauermann for manufacturing the phase plate. We
external control of the phase of the counter propagating waves, acknowledge financial support by the Bundesministerium für Bildung,
this method also relies on subsequent data processing for Wissenschaft, Forschung und Technologie (Bonn).

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8210 兩 www.pnas.org Klar et al.