REVIEW OF SCIENTIFIC INSTRUMENTS
`
`VOLUME 71, NUMBER 4
`
`APRIL 2000
`
`A sample-scanning confocal optical microscope for cryogenic operation
`J.-M. Segura,a) A. Renn, and B. Hechtb)
`Physical Chemistry Laboratory, Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092 Zu¨rich,
`Switzerland
`~Received 20 September 1999; accepted for publication 9 December 1999!
`
`A sample-scanning confocal optical microscope for single-molecule imaging and spectroscopy
`working at superfluid helium temperature, liquid nitrogen, and room temperature is described. An
`optical resolution of 800 nm full width at half maximum as well as a detection efficiency of ’3.5%
`are achieved. The sample scanner features an exceptionally large scan range of 23 mm at 1.8 K. A
`position sensor allows for continuous observation of the scanner motion and for a correction of
`piezoelectric hysteresis and creep at 77 K and at room temperature. Coarse positioning of the sample
`in x-y is achieved by an inertia drive with high reproducibility and nanometer precision. We
`demonstrate combined high–resolution confocal imaging and spectroscopy of single molecules at
`1.8 K. © 2000 American Institute of Physics. @S0034-6748~00!00704-8#
`
`I. INTRODUCTION
`
`Optical detection and spectroscopy of single molecules
`recently generated considerable interest in many fields of
`research.1,2 Experiments with single chromophores circum-
`vent
`the averaging effect
`inherent
`in conventional spec-
`troscopies. In contrast to mean values, distributions of pa-
`rameters can be accessed. Single molecules have also been
`used as ultimate local probes or markers to study changes in
`their local environments or to trace dynamics of labeled en-
`tities.
`At superfluid helium temperature, spectroscopic infor-
`mation greatly enhances the sensitivity of single-molecule
`experiments.3,4 At 1.8 K the absorption line of certain types
`of impurity molecules in solid matrices, the zero phonon line
`~ZPL!, becomes extremely narrow ~some tens of megahertz!.
`Due to inhomogeneities in the solid these ZPLs are spectrally
`distributed over a range of several gigahertz in frequency and
`can thus be selectively excited by a narrow-band laser. The
`sharpness of the ZPL and its resulting high sensitivity to-
`wards external perturbations combined with a strong en-
`hancement of the photostability represent the main motiva-
`tions for performing single-molecule experiments at
`low
`temperature. Spectral dynamics and line broadening of the
`ZPL can provide detailed information on the immediate na-
`noenvironment. This opens the possibility for many experi-
`ments with single molecules that would not be possible at
`room temperature.
`low-temperature experiments,3,4 only
`In the classical
`spectral separation is used to isolate single molecules thereby
`losing all information about spatial positions. To address this
`problem, wide-field optical microscopy techniques5–7 have
`been introduced, which are capable of imaging the spatial
`positions of large numbers of single molecules distributed
`over large sample areas. While such experiments offer the
`great advantage of studying the properties of many mol-
`
`a!Electronic mail: segura@phys.chem.ethz.ch
`b!Electronic mail: hecht@phys.chem.ethz.ch
`
`0034-6748/2000/71(4)/1706/6/$17.00
`
`1706
`
`ecules in parallel, it would sometimes be more desirable to
`use scanning confocal optical microscopy ~SCOM!8,9 to spa-
`tially isolate a single molecule and correlate position with
`spectral and temporal behavior. SCOM provides diffraction-
`limited three-dimensional optical resolution and high back-
`ground rejection. It has been successfully applied for imag-
`ing and spectroscopy of
`single molecules
`at
`room
`temperature.2
`SCOM can be combined with other scanning probe tech-
`niques like scanning near-field optical microscopy ~SNOM!
`and atomic force microscopy ~AFM!. This would open the
`way to a new class of quantum optical experiments that re-
`quire single molecules to be precisely positioned relative to
`nanostructures.10,11 We envisage studying quantitatively the
`interaction of single molecules with sharply pointed nano-
`structures such as tips of scanning probe microscopes, in
`order to develop new high-resolution optical microscopies
`based on high-resolution spectroscopy.
`We have designed and realized a SCOM working at
`1.8 K with a sample scan range of 23 mm. Such a large scan
`range is a necessary condition for successful operation of the
`microscope because the field of view has to be reasonably
`large in relation to the optical resolution which is ’800 nm
`in the current setup. The sample scanning design has been
`chosen as opposed to a beam scanning configuration12 in
`order to maintain compatibility to a future scanning probe
`microscope extension. Unlike scanning tunneling micros-
`copy, AFM, or SNOM,13–15 where low-temperature designs
`have already been published, no sample scanning SCOM
`having a scan range of more than a few microns can be found
`in the literature.16,17 The main reason is the difficulty to de-
`sign a scanner based on piezoelectric materials which has the
`desired properties.
`We present single-molecule images and frequency scans
`at selected positions recorded at superfluid helium tempera-
`ture in order to demonstrate the capabilities of the instru-
`ment.
`3SHAPE EXHIBIT 1128
`© 2000 American Institute of Physics
`3Shape v. Align
`IPR2019-00157
`
`

`

`Rev. Sci. Instrum., Vol. 71, No. 4, April 2000
`
`Confocal optical microscope
`
`1707
`
`FIG. 1. Scheme of the optical path and
`elements involved in the SCOM: ~L!
`laser system, ~W! wave meter, and
`spectral analyzer, ~P! power stabilizer,
`~SP! spatial filter, ~FL! flippable lens,
`~H! holographic notch filter, ~C! cry-
`ostat, ~MO! microscope objective, ~S!
`sample, ~F! cutoff filter and notch fil-
`ter for 830 nm, ~FM! flippable mirror,
`~O! ocular,
`~SPAD!
`single-photon
`counting avalanche photo diode.
`
`II. INSTRUMENTATION
`
`A. Optical setup
`
`The optical setup is similar to what is used in a room-
`temperature SCOM. However, some particularities of low-
`temperature single-molecule spectroscopy have to be taken
`into account. The optical path is sketched in Fig. 1. The
`excitation light is provided by a single-mode tunable con-
`tinuous wave ~cw! ring dye laser ~Coherent, 699! pumped by
`a cw Ar1-ion laser ~Spectra Physics, Model 2045!. The use
`of a tunable narrow-band laser (Dn51 MHz! is essential in
`order to perform spectroscopy of the narrow ZPL of the
`single molecules (Dn’40 MHz!. A beam splitter deflects
`10% of the light to a wave meter ~Burleigh, Model WA-210!
`and a spectrum analyzer ~Tropel, Model 240! for monitoring
`of the wavelength and the linewidth. After power stabiliza-
`tion ~CRI, LS-100!, the laser light is coupled into a short
`piece of a single mode fiber ~40-692.11 from Cabloptic, core
`diam 3 mm! for spatial filtering and then collimated using a
`50.2 mm lens to a 10 mm diam beam in order to completely
`fill the microscope objective entrance aperture ~5 mm diam!.
`The mode field diameter at the fiber exit defines the size of
`the ‘‘pinhole’’ in the excitation path. A slightly ~less than
`10°! tilted holographic notch filter ~Kaiser! is then used as a
`dichroic beamsplitter with extremely sharp frequency band-
`width. It attenuates the excitation light with an optical den-
`sity .5 and transmits ’80% of the ’10 nm redshifted fluo-
`rescence emission. The beam enters a helium bath cryostat
`via bottom windows and is focused to a diffraction-limited
`spot on the sample using a microscope objective ~Newport,
`0.85 numerical aperture ~NA!, 603, focal length52.9 mm!.
`The microscope objective is located inside the cryostat to be
`very close to the sample in order to achieve a high numerical
`aperture and is used with a conjugated plane at infinity in
`order to simplify the detection optics. This, however, does
`not significantly diminish the optical performance. Thermal
`deformations during the cooling and especially the difference
`in refractive index between air (n51)and superfluid helium
`(n51.028), that fills the space between the lenses of the
`objective, have a much larger effect on the final optical reso-
`lution @800 nm full width at half maximum ~FWHM!#. The
`same objective collects the fluorescence and the remaining
`Rayleigh-scattered excitation light, which is filtered out by
`the holographic beamsplitter and a subsequent cutoff filter.
`Another holographic notch filter centered at 830 nm removes
`
`stray light introduced by the optical scanner motion detection
`system ~see Sec. II C!. A flippable mirror provides the pos-
`sibility to either direct the collimated transmitted fluores-
`cence light to an ocular for direct visual observation or focus
`the beam onto a single-photon counting avalanche photo di-
`ode ~SPAD! ~Radio Corporation of America, SPCM-100,
`200 mm diam active area!. The small active area of the
`SPAD acts as the detection pinhole. The focusing lens
`~300 mm focal length! produces a 90 mm spot ~min. to min.
`diameter! optimized for high detection efficiency9 and suffi-
`cient background rejection nedded for single molecule spec-
`troscopy. An additional lens can be flipped in the excitation
`path in order to establish wide field illumination conditions
`necessary for visual investigation of larger sample areas with
`the ocular. The SPAD is connected to a counter, which is
`read out by a computer.
`Considering the elements in the optical path, the detec-
`tion efficiency of the setup can be estimated. Assuming iso-
`tropic emission, the microscope objective with NA50.85 can
`collect 24% of the emitted light. Since molecules radiate like
`dipoles, the collection efficiency will be more or less favor-
`able depending on the orientation.18,19 The overall losses of
`the three windows of the cryostat, of the lenses and filters
`add up to 75%. Finally, the SPAD has a quantum efficiency
`of ’60% at 600 nm. All together, the total detection effi-
`ciency can be estimated to be 3.5%, which is large compared
`to other low-temperature single-molecule detection setups
`described previously.3 This enhancement is mainly due to the
`higher quantum efficiency of a SPAD as compared to a pho-
`tomultiplier.
`
`B. Mechanical design
`
`The helium bath cryostat has been homebuilt in order to
`meet the requirements of the setup. The inner diameter of
`100 mm is sufficient to contain the rather large experimenta-
`tion stage ~ES!, that is sketched in Fig. 2. The cryostat may
`be optically accessed via bottom windows allowing for a
`design where the different modules and extensions are as-
`sembled in a stack-like fashion. The ES rests on a kinematic
`mount on the bottom of the cryostat centered above the win-
`dows, compensating for the thermal dilatation. In order to
`adjust the focus, the objective can be moved in 500 nm steps
`along the optical axis using a stepper motor geared down by
`a factor of 1000. Movement is achieved by turning the ob-
`
`

`

`1708
`
`Rev. Sci. Instrum., Vol. 71, No. 4, April 2000
`
`Segura, Renn, and Hecht
`
`FIG. 2. Experimentation stage: ~KM! kinematic mount, ~M! stepper motor,
`~G! gear box, ~MO! microscope objective, ~BS! x-y bimorph scanner ~for
`clarity the bimorph piezos are not shown!, ~ZS! z scanner, ~SH! sample
`holder, ~F! optical fiber of the motion detector, ~PD! four-segment photodi-
`ode, and ~CS! shear piezo coarse-positioning scanner.
`
`jective in a fine thread ~0.5 mm pitch!. The stepper motor
`~Princeton Research Instruments, size A! is designed for
`high-vacuum applications and prepared for cryogenic opera-
`tion.
`
`C. x-y-z scanner
`
`Fine tuning of the focus is achieved by means of a z
`piezoelectric element, which is part of an x-y-z sample scan-
`ner @see Fig. 3~a!#. The z piezo consists of a bimorph disk
`~Piezomechanik GmbH! with a concentric circular opening.
`The bimorph disk is fixed to the scanner only at its outer rim.
`The sample is held by a tubelike piece of aluminum fixed to
`the inner rim of the piezoelectric disk @see Fig. 2~SH!#. By
`applying a voltage to the electrodes of the piezos, a fine
`motion of the sample in the z direction is obtained. At super-
`fluid helium temperature the maximal scan range of 6 mm
`corresponds to several motor steps. The z-motion capability
`is not only useful to precisely adjust the focus, but allows
`also for scanning and imaging of the sample in the z direc-
`tion. In combination with the x-y scanner, three-dimensional
`imaging is possible.
`The main task in designing an x-yscanner working at 1.8
`K arises from the smaller piezoelectric constant, which is
`reduced by a factor of 10 at superfluid helium temperature as
`compared to room temperature.20 We have extended a previ-
`ously used design16,21 to obtain the necessary performance.
`Figure 3~b! sketches the principle of the scanner. Four bi-
`morph piezos ~Piezomechanik GmbH! are connected by flex-
`ible metal sheets to form a square. Two facing piezos are
`
`FIG. 3. Scanner: photograph of the SCOM from the top ~a! and sketch of
`the bimorph scanner principle ~b!. ~BS! x-y bimorph scanner, ~ZS! z scan-
`ner, ~SH! sample holder, ~MO! microscope objective, and ~D! motion de-
`tector.
`
`FIG. 4. Motion detector: sketch of the x-y position detection principle. The
`numbers indicate the four quadrants of the photo diode. The formulas relate
`the signals of the quadrants to the actual x and y positions.
`
`fixed at their middle to the body of the experimentation
`stage, whereas the other pair supports the disk piezo and the
`sample. Applying a voltage to the electrodes of the bimorph
`piezos leads to a bending. The piezos that are fixed to the
`body predominantly move at the ends whereas the piezos
`that support the sample holder primarily move in the middle.
`This way, x-y scanning is achieved. A possible bending state
`of the scanner is sketched in Fig. 3~b!. For a given type of
`bimorph, the scan range is limited only by the length of the
`piezos. By using enough long piezos ~51 mm!, movements as
`large as 23 mm at 1.8 K could be achieved, which represents
`a significant improvement compared to previous scanners.
`Due to its large physical dimension the scanner has a
`low resonance frequency of 55 Hz and is therefore quite sen-
`sitive to vibrations. In order to reduce noise, the cryostat
`rests on a vibration-damped optical table and is additionaly
`isolated from the table by rubber plates. This way the sample
`position is stable within 610 nm, which is much more accu-
`rate than the optical resolution. The resonance frequency
`limits the maximal scanning frequency to ’1 Hz. However,
`scans are anyway slow in practice in order to guarantee
`enough integration time.
`We use commercial electronics for the control of the
`scan and the data acquisition ~ECS SPM Control System!.
`With this system two scanners can be controlled simulta-
`neously, allowing a further extension by a scanning probe
`microscope. Two high voltage amplifiers ~Ergonomics! gen-
`erate the scanning voltages ranging from 230 to 300 V.
`A very practical feature of the setup is the ability to
`monitor directly the actual position of the scanner. This can
`be used to linearize the scanner motion by means of a posi-
`tion feedback loop as explained later. Furthermore, problems
`with the scanner can be easily recognized, which is impor-
`tant when working in a cryostat at liquid helium temperature,
`where direct visual inspection is not possible. The position
`detector ~see Fig. 4! consists of a single mode infrared ~IR!
`fiber attached to the scanner and a four-segment photodiode
`integrated in the body of the experimentation stage. IR light
`at 830 nm emitted by a diode laser ~VOMAG, 57PNL008! is
`coupled into the far end of the fiber. The light emerging from
`the fiber end is projected onto the four segments of the pho-
`todiode. The distribution of the resulting spot over the dif-
`ferent segments depends on the actual position of the scan-
`ner. The diameter of the spot is chosen large enough to
`guarantee linearity of the detector. By measuring the relative
`IR intensity on the four segments, it is possible to monitor
`
`

`

`Rev. Sci. Instrum., Vol. 71, No. 4, April 2000
`
`Confocal optical microscope
`
`1709
`
`FIG. 5. Interferometric measurement of the slip-stick movement at room
`temperature: between the maximum and the minimum of the curve, the
`sample travels a distance of 207 nm. Each single step corresponds to a
`distance of 10 nm.
`
`the scanner motion with high accuracy.22 The resolution we
`achieved is ’50 nm and is mainly limited by intensity fluc-
`tuations and mode hops of the diode laser. In order to mini-
`mize these effects, the laser is enclosed in a 5 kg copper
`block, which acts as a temperature stabilizer. The position
`detector provides the opportunity to linearize the scanner
`motion and thus to correct for two disturbing effects inherent
`to the bimorph piezos: ~i! the piezoelectric hysteresis and ~ii!
`the piezoelectric creep. Hysteresis introduces a nonlinear
`motion of the scanner and thus causes distortions of the im-
`age. Creep manifests itself in the fact that static and dynamic
`voltage versus position relations of the bimorph piezos are
`different. At the end of a scan or a motion, the position of the
`scanner creeps to a stable point. As a result of this effect, an
`interesting molecule found during a scan at a certain location
`cannot be moved anymore into a static focus. Fortunately,
`these two effects are not present at superfluid helium tem-
`
`FIG. 7. Crystal of p-terphenyl doped with pentacene at 1.8 K. n¯
`516880.11 cm21, I exc540 W/cm2, integration time/pixel514 ms.
`
`perature making the use of the position feedback loop unnec-
`essary at this temperature.23 However, the position feedback
`is indispensable for experiments at 77 K or at room tempera-
`ture.
`
`D. Sample coarse positioning
`
`Coarse positioning of the sample is a necessary capabil-
`ity of the microscope in any real experiment. Due to thermal
`drifts, it is impossible to choose the right location on the
`sample for a given experiment before the cooling. It may
`happen that the final sample position is unattractive, which in
`the worst case necessitates a new cooling cycle if no coarse
`positioning is available. Moreover, coarse positioning can be
`very useful by allowing one to prepare several samples on a
`single coverslip and to switch between them at low tempera-
`ture.
`We have designed a coarse-positioning system based on
`a previously described inertia drive.24,25 A sapphire ball is
`glued on a set of two perpendiculary oriented shear piezos
`~Staveley, EBL2! fixed on the bimorph scanner @see Fig. 2
`~CS!#. Sapphire slides glued to the holder of the disk piezo
`sit on the top of the ball. In order to move the holder of the
`disk piezo relatively to the bimorph scanner, a sawtooth-like
`
`FIG. 6. Example of slip-stick motion. Each image has successively been
`taken after a displacement of some micrometers in each directions. Three
`molecules are surrounded by circles to facilitate comparison. The slightly
`different appearance of molecules in the images is due to laser frequency
`drifts during the acquisition time of the four pictures.
`
`FIG. 8. Line cut through a small spot as indicated at the bottom of Fig. 7.
`n¯ 516880.11 cm21, I exc540 W/cm2, integration time/point514 ms.
`
`

`

`1710
`
`Rev. Sci. Instrum., Vol. 71, No. 4, April 2000
`
`Segura, Renn, and Hecht
`
`voltage is applied to the shear piezos. During the slow in-
`crease, the load moves together with the shear piezos. How-
`ever, when the voltage is abruptly switched off, the sample
`holder slips on the sapphire balls. A macroscopic motion is
`created by inducing many of these slip-stick events. Due to
`the previously mentioned reduction of the piezoelectric elon-
`gation at liquid helium temperature, it was found to be nec-
`essary to apply high voltages to the shear piezos in order to
`overcome the contact friction. We typically use a sawtooth
`function with an amplitude of at least 600 V and a frequency
`of 1600 Hz. The speed can be tuned by changing the voltage
`or the number of steps per second. The coarse-positioning
`device is isotropic and has a high reproducibility from room
`temperature to 1.8 K. Figure 5 shows an interferometric de-
`tection of the macroscopic slip-stick motion at room tem-
`perature. Taking into account the intrinsic nonlinearity of the
`interferometer, a smooth motion by single steps of ’10 nm
`can be recognized.
`In Fig. 6, the four images of a crystal of p-terphenyl
`doped with pentacene molecules illustrate the capability of
`the slip-stick motion assembly at 1.8 K. Each picture has
`successively been taken after a displacement of some mi-
`crometers. To facilitate comparison between images, some
`molecules are surrounded by circles.
`
`III. RESULTS AND DISCUSSION
`
`Figure 7 is an illustration of typical images obtained
`with the SCOM at 1.8 K. The picture shows single molecules
`of pentacene embedded in a p-terphenyl crystal. An intrigu-
`ing feature is the distribution of spot sizes ranging from 800
`nm to some microns. As explained later, this is a typical
`effect of low temperature SCOM of single molecules. Figure
`8 displays a line cut through a small spot as indicated at the
`bottom of Fig. 7. Considering that the shape is not absolutely
`symmetric, the optical resolution can this way be estimated
`to be at least 800 nm FWHM.
`The broad features in Fig. 7 do not originate from optical
`aberrations, but rather are examples of the coupling between
`spectral properties and spatial images of single molecules at
`superfluid helium temperature. Due to the narrow ZPL, the
`absorption cross section is extremely high, and the optical
`transition can be easily saturated. If the excitation intensity is
`well above saturation in the center of the spot, the confocal
`volume needs to be notably displaced in order to produce a
`change in the emitted fluorescence. Thus, the molecules ap-
`
`FIG. 9. Frequency scan at the position indicated by the arrow in Fig. 7. The
`scan range is 1 GHz, n¯ 516880.11 cm21, I exc540 W/cm2, integration time
`585 ms.
`
`pear much broader than the actual confocal volume. On the
`other hand, the small spots represents molecules that are sig-
`nificantly detuned from resonance and therefore well below
`saturation. Figure 9 shows a local frequency scan at the po-
`sition marked by an arrow in Fig. 7. Compared to the
`lifetime-limited linewidth ~11.763.8 MHz obtained from a
`saturation study!, the line at zero detuning is strongly power
`broadened @58.9~3.5! MHz#. This proves that the optical tran-
`sition is saturated and explains the appreciable size of the
`single-molecule spot in the SCOM image. Accordingly, due
`to different excitation intensities, the observed linewidth var-
`ies depending on the position in the spot ~at the maximum or
`on the side! where the frequency scan is taken. Such local
`frequency scans are very useful for interpretation purposes
`and are a strong advantage of low temperature confocal mi-
`croscopy. Due to these broadening effects, the separation be-
`tween molecules requires a large scan range even if the op-
`tical resolution is close to the diffraction limit.
`The combination of spectral and spatial information is
`illustrated in Figs. 10~a! and 10~b! with two images of a
`crystal of p-terphenyl doped with terrylene at 1.8K. Both
`images are recorded at the same position, but with a 1 GHz
`shifted excitation frequency. Due to the narrow ZPL, differ-
`ent molecules are in resonance in the two images. It is inter-
`esting to notice that some spatially overlapping molecules
`can be separated in the spectral dimension. As an example, a
`frequency scan was performed at the position of the central
`
`FIG. 10. Single molecules of terrylene in p-terphenyl at 1.8 K. The excitation frequency is shifted by 1 GHz between images ~a! and ~b!. l5578.49 nm,
`I exc5460 W/cm2, integration time/pixel538 ms. ~c!: Frequency scan at the position of the molecule in the center of image ~b!. The scan range is 1 GHz,
`l5578.49 nm, I exc5750 W/cm2, integration time585 ms.
`
`

`

`Rev. Sci. Instrum., Vol. 71, No. 4, April 2000
`
`Confocal optical microscope
`
`1711
`
`singer, and Professor U. P. Wild for continual help and sup-
`port. This project has been founded by the Swiss Priority
`Program NFP 36 and the ETH Zu¨rich.
`
`1 Single-Molecule Optical Detection, Imaging and Spectroscopy, edited by
`T. Basche´, W. E. Moerner, M. Orrit, and U. P. Wild ~VCH Verlagsgesell-
`schaft, Weinheim, 1997!.
`2 X. S. Xie and J. K. Trautman, Annu. Rev. Phys. Chem. 49, 441 ~1998!.
`3 T. Plakhotnik, E. A. Donley, and U. P. Wild, Annu. Rev. Phys. Chem. 48,
`181 ~1997!.
`4 W. E. Moerner and M. Orrit, Science 283, 1670 ~1999!.
`5 F. Gu¨ttler, T. Irngartinger, T. Plakhotnik, A. Renn, and U. P. Wild, Chem.
`Phys. Lett. 217, 393 ~1994!.
`6 J. Jasny, J. Sepiol, T. Irngartinger, M. Traber, A. Renn, and U. P. Wild,
`Rev. Sci. Instrum. 67, 1425 ~1996!.
`7 M. Croci, T. Irngartinger, A. Renn, and U. P. Wild, Exp. Tech. Phys.
`~Lemgo, Ger.! 41, 249 ~1995!.
`8 Handbook of Biological Confocal Microscopy, 2nd ed., edited by J.B.
`Pawley ~Plenum, New York, 1995!.
`9 R. H. Webb, Rep. Prog. Phys. 59, 427 ~1996!.
`10 D. J. Norris, M. Kuwata-Gonokami, and W. E. Moerner, Appl. Phys. Lett.
`71, 297 ~1997!.
`11 W. E. Moerner, T. Plakhotnik, T. Irngartinger, U. P. Wild, D. W. Pohl,
`and B. Hecht, Phys. Rev. Lett. 73, 2764 ~1994!.
`12 M. Va´cha, H. Yokoyama, T. Tokizaki, M. Furuki, and T. Tani, Rev. Sci.
`Instrum. 70, 2041 ~1999!.
`13 R. D. Grober, T. D. Harris, J. K. Trautman, and E. Betzig, Rev. Sci.
`Instrum. 65, 626 ~1994!.
`14 G. Behme, A. Richter, M. Su¨ptitz, and C. Lienau, Rev. Sci. Instrum. 68,
`3458 ~1997!.
`15 Y. Durand, J. C. Woehl, B. Viellerobe, W. Go¨hde, and M. Orrit, Rev. Sci.
`Instrum. 70, 1318 ~1999!.
`16 W. Go¨hde, J. Tittel, T. Basche´, C. Bra¨uchle, U. C. Fischer, and H. Fuchs,
`Rev. Sci. Instrum. 68, 2466 ~1997!.
`17 S. Smith, H. M. Cheong, B. D. Fluegel, J. F. Geisz, J. M. Olson, L. L.
`Kazmerski, and A. Mascarenhas, Appl. Phys. Lett. 74, 706 ~1999!.
`18 W. Lukosz, J. Opt. Soc. Am. 71, 744 ~1981!.
`19 T. Plakhotnik, W. E. Moerner, V. Palm, and U. P. Wild, Opt. Commun.
`114, 83 ~1995!.
`20 T. M. Tritt, D. J. Gillespie, G. N. Kamm, and A. C. Ehrlich, Rev. Sci.
`Instrum. 58, 780 ~1987!.
`21 P. Muralt, D. W. Pohl, and W. Denk, IBM J. Res. Dev. 30, 443 ~1986!.
`22 U. Du¨rig, D. W. Pohl, and F. Rohner, J. Appl. Phys. 59, 3318 ~1986!.
`23 S. Vieira, IBM J. Res. Dev. 30, 553 ~1986!.
`24 D. W. Pohl, Rev. Sci. Instrum. 58, 54 ~1987!.
`25 J. W. Lyding, S. Skala, J. S. Hubacek, R. Brockenbrough, and G. Gam-
`mie, Rev. Sci. Instrum. 59, 1897 ~1988!.
`
`at 1.8 K.
`FIG. 11. Crystal of p-terphenyl doped with terrylene
`l5578.51 nm, I exc55.3 kW/cm2, integration time/pixel528 ms. The cut-
`off filter has been removed in order to see the crystal structure by means of
`Rayleigh-scattered excitation light.
`
`spot in image ~b! @see Fig. 10~c!#. It reveals the presence of
`two molecules that are separated in frequency by only about
`100 MHz. This methodology can be used to correlate data
`obtained in the spectral and spatial dimensions. Another type
`of information is obtained in Fig. 11 recorded at the same
`location as in Fig. 10, but with the cutoff filter behind the
`notch removed. As a result some of the Rayleigh-scattered
`laser light can reach the detector. Different crystal domains
`are clearly recognizable as well as some molecules of ter-
`rylene, which seem to accumulate close to the grain joints.
`This kind of image is very useful in order to correlate the
`single-molecule properties with the macroscopic environ-
`ment, which is quite difficult in classical single-molecule de-
`tection schemes.
`
`ACKNOWLEDGMENTS
`
`The authors want to especially thank A. Hunkeler and B.
`Lambillotte for excellent mechanical support. The authors
`are grateful to E. Donley, P. Nyffeler, B. Sick, W. Trabe-
`
`
`
`View publication statsView publication stats
`
`