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Detection and Imaging of Single Molecules by Optical Near-Field Microscopy.

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tion of benzene consisted in his recognition that the second half of the double
bond. unlike the single bond. I S a nomadic entity which cannot always be
assigncd a unique position in the molecule. ... The electronic theory of valencq ... shed a flood of light on structural chemistry and revealed the fundamental
distinction between 1oc:tlized and non-localized electrons. a ) C . A. Russell, Tlrc
H ~ ~ / ~ JI bw
l e io
i r /I., Humanities. Neu York. 1971; b) F. G. Ariidt. in a letter see:
E C'anipaigne. .I C h m . E h c . 1959.36. 336; c) H . C. Longuet-Higgens. Proc.
C ' / i ( w i . Soc., London. 1957. 157.
1. H . b m ' t Hot'f, Dir AniiPcs (/mi.\ I'llitroire rlioie T / i k ) r t c . P M . Bazebdi.ik.
Oxford. 1891
Rotterdam. 1887: CIiciiii.s/ri (17 S p a c ~ Clarendon.
J. E Marsh, P/ii/u.\. M O R . 1888. 76. 426.
See. L. Pauling. 3Ltifin.e of i h ('iieii7icoi
h i i d , 3rd cd.. Cornell University Press.
Ithnca. NY. 1964. chap. 14.
G. Reddelien. J. Pro!,/ Chrrri. 1915, 213.
a ) A . A. Lwine. A . G. Cole, J A m . Clion. Sor. 1932.54. 3%: b) similar results
w i - e 1;iter published. see: J. P. Wibnut. P. W. Haayman. Siirnce 1941. 94. 49.
a ) L Pauliiip. J .Am. C h o i i . Soc. 1931. 1367; b) L. Pauling. ;bid. 1932. 54. 988:
c) L Pauling. Q. W. Wheland. J. Clirwi. P/IJ.\. 1933. I . 362.
L. E. Sutton. L. Pauling, T,.nii.\. I.irrar/u? Sol. 1935. 31, 939.
Thl\ di\tinctioii of single versus double minima is clearly stated by Sutton and
Patiling. " I t appe;irs very definitely that benzene is a single molecular species
and ... ueciiii say that it IS ii hybrid ofthe two Kekule structures ... and that the
angles between pairs of external valencies are all 60 . ... Our procedure here IS
different from that of Mills and Nixon. for they assumed that the niolecule had
either structure I or structure 11, whereas we consider that initially i t has the
symmetrical resonating structure and then calculate how much it 15 distorted
when a ring is attached."
[I 71 N. Frank. J. S. Siegel. unpublished.
1181 R. Boese. D. Blhser. W. E. Billups, M. M. Haley. A. H. MaulitL, D. L. Mohler.
K. P. C. Vollhardt. A n x m ~Clirni.
1994, 106. 321 -325. Ati,ccu.. C h m Irir. Ed.
Eiigl. 1994. 33. 313-17.
[I91 A. Stanger, J. A m Clicni. Soc. 1991. I I 3 . 8277.
[ X I K. K. Baldridge, J. S. Siegel. J. A i t i . Chmi. Snc 1992. 114. 95x3.
[21] W. Natakul. R . P. Thummel. A. D. Txggart. .lAi7i. C ' / i c i i i . Sot. 1979. I O i .
[I21 a) R. Neidlein. D. Christen, V. Poignee. R. Boese. D. Bliser. A Gieren, c'.
Ruiz-Perez, T. Hubner. A i i g w . Chwii. 1988. 100. 292-293, Aiigrw. C/I~WI.
Ed. Eiigl. 1988. 27, 294-295: h) R. Boese, D. Bliser. ihid. 1988. /Of). 293 -295
and 1988. 27. 304-305.
[23] I n this regard. perfluoro-1 serves as a good model for these strained iiionocqclic
annelated bemenes in general. see: R. P. Thummel, J. D. Korp. I . Bernal. R. L
Harlow, R. L. Soulen. J. Am. C h i ? .Soc. 1977, YY. 6916.
[24] M. Saunders. M. R. Kates. J A m Clion. Sue. 1980. 102, 6867.
[25] H. C. Longuet-Higgins. C. A. Coulson. Trmi.\. Frtrurlri?, So<.. 1946. 41. 746.
Detection and Imaging of Single Molecules by Optical Near-Field
Thomas BaschC
,.. . . we
or niolrcwle.
never experiment with .just one electron or aton?
In tliocight esperinients u'e sometiiiies tissunie we do:
this invariably entails ridiculous consequences. ''
Erwin Schrodinger, 1952
The successful storage and spectroscopic characterization of
individual atoms in electromagnetic traps and the development
of microscopic techniques with atomic resolution such as scanning tunneling microscopy (STM) and atomic force microscopy
(AFM) are two examples that make us realize that
Schrodinger's notion has been outpaced by reality. One should.
however. bear in mind that the observation of single particles, as
commonplace as it may seem today, has only become possible
within the past 10-15 years, regardless of the technique. This
Highlight deals with new developments in the field of optical
detection of single molecules: other methods of detection are
not covered.
The first optical detection of a single molecule in the condensed phase was achieved in 1989 with the spectroscopic characterization of an individual pentacene molecule in an organic
mixed crystal at low temperatures ( T = 1.5 K)."' Not much
later. and independent of the research on solids. efforts to detect
individual dye molecules in a liquid were successful.[21
A single molecule in a solid at low temperature is a very
sensitive spectroscopic probe for the properties of its local envi[*I
Dr. T Basche
lnstitut fur Physikalische Chemie der Universitit
Sophimhtrasc 11. D-X0333 Munchen ( F R G )
Telel'ax. Int. code + (89)590-2602
ronment, owing to its extremely narrow optical linewidth. In
contrast, for single-molecule detection (SMD) in liquids or,
more general, for all techniques that can be performed at room
temperature, analytical aspects are as important as the measurement of spectroscopic details. S M D represents the ultimate limit
in chemical trace analysis and has been proposed as a realistic
method for sequencing the DNA of the human genome within
a reasonable period of time. In molecular biology the detection
of single fluorescence-labeled species (proteins, viruses) is a foreseeable and increasingly important development.
Optical spectroscopy of single molecules has already given
rise to a host of new experimental results. including the development of techniques for the optical characterization of individual
molecules with ever increasing sensitivity. Ultrasensitive fluorescence detection is ideally suited for the optical observation of
single molecules. The experimental challenge here lies in the
discrimination of the weak fluorescence of a single molecule
from the matrix background light (Rayleigh and Raman scattering, matrix luminescence). Since the signal of the molecule is
proportional to the intensity of the incident radiation (poweri
area), and since the background light and the concomitant
background noise level increase with the power, enhanced sensitivity is achieved by decreasing the area of illumination. Consequently, tight focusing of the exciting laser light is essential.
Focusing with lenses, however. invariably involves the fundamental Abbe diffraction limit to resolution. which states that
foci of dimensions less than the wavelength of the light used are
not possible. Fortunately. the method of near-field scanning
optical microscopy (NSOM) developed during the last few years
circumvents this limit and is therefore ideally suited for fluorescence detection of single molecules. Recently, using this new
microscopic technique. Betzig et aLi3' and Ambrose et al.i4]independently succeeded in imaging individual dye molecules on
the surface of a substrate at room temperature. Before these
experiments are described, let us first look at the principles and
potential applications of near-field optical microscopy.
Conventional optical microscopy has a limited resolution of
about i.!2 of the optical wavelength employed. This theoretical
resolution limit is a consequence of diffraction effects due to the
wave characteristics of light; for green light under ideal conditions this limit is roughly 0.3 pm, and with good optical microscopes it can in fact be reached. (However, even in confocal
microscopy resolution better than 2 / 2 can be achieved using
special "tricks".) When light is passed through a small aperture.
whose diameter is less than the wavelength. the dimensions of
the light beam at a distance close to the aperture are nearly
unchanged. This area is called the optical near field. Such an
aperture can be scanned raster fashion across the surface of a
close-lying object. Many recorded points can then be combined
to reconstruct an image of the surface. The resolution of this
microscopy is not limited by the wavelength of light, but only by
the size of the aperture and its distance from the object.
Near-field microscopy in the visible regime was first demonstrated independently by Pohl et al.[5' and Lewis et al.'" Since
then, several optical near-field probes have been developed. In
most cases the light is directed from the light source to the
aperture by a some sort of wave guide. To collect images quickly
and with a good signal-to-noise ratio. it is of special importance
to pass as much light as possible through the miniscule aperture.
To this end, the tips of tapered optical fibers and micropipettes
with dimensions on the order of 10 nanometers turned out to be
ideal apertures. Other aperture geometries have also been proposed and were recently successfully employed, for example, a
partially metal-coated tetrahedral tip.''] The aperture can also
be used to collect light from the object and to scatter evanescent
waves from the near field into propagating modes of the optical
We will next describe a schematic setup of a near-field microscope in the so-called illumination (or transmission) mode
(Fig. 1 ) . Here the tip of a tapered optical fiber is used as an
aperture and scanned raster fashion across the object of interest.
The light is coupled into the fiber through an objective. The
fiber tapers from the original fiber diameter to a tip having a flat
face perpendicular to the fiber axis. The tip is produced by
simultaneously heating the fiber with a CO, laser and pulling
the ends apart. This process leads to apertures from 20 to
500 nm with high reproducibility. Finally, the tip is vapor-coated with a metal film (Al) to prevent the light from leaking out
at the sides. The sample is placed within about 10 nm of the fiber
tip and is moved in the X,J direction with a piezoelectric transducer. The signal is detected at each point, and the image is
generated pixel by pixel. During the scan the distance between
the fiber tip and the object must be kept constant by using an
active feedback mechanism. An elegant approach to this is shear
force feedback detection (not included in Fig. I), in which the
fiber tip is excited at one of its a high-quality mechanical resonance frequencies. The damping of the vibration as the object
approaches the tip is then recorded as a distance-dependent
I/ piezelectric
Al coating
Fig. 1. Schematic setup o f a scanning near-field microscope The inset (middle left)
shows the fiher tip with the ncar-tield and far-field regions For details Fee text
signal. A lateral resolution (s,~)of about 12 nm (or 4 4 3 ) could
be demonstrated with a setup similar to that shown in Figure
The best resolution currently possible in NSOM is roughly
two to three orders of magnitude less than that of other scanning
microscopic techniques like STM and AFM. Yet NSOM offers
some highly interesting possibilites: 1) All contrast mechanisms
known in conventional optical (far-field) microscopy can be
employed. e.g. polarization. refractive index. and fluorescence.
2) NSOM is possible under ambient conditions, and light has
only a weak disturbing effect on samples an important prerequisite for the study of biomolecules. 3) Specific interactions
in the optical near field (evanescent modes) can, for example,
lead to local amplifications of the fluorescence. 4) NSOM can
be combined with other optical spectroscopies without loss of its
high local resolution. A good review of the latest, in part spectacular research in near-field microscopy, frequently conducted
on biological systems. is presented in the conference proceedings'"' of the Second Near-Field Optics Conference. Raleigh.
NC, USA, 1993. In addition to NSOM images of blood cells or
DNA, images of, for example, quantum layers are shown. First
investigations on magnetooptical data storage with NSOM
have yielded storage densities of 7 Gbitcm-2 at a local resolution of 30-60 nm.('O1
Figure 2 shows an NSOM image (Betzig et al.) of lipophilic
carbocyanine molecules spread with high dilution (about 23
molecules per pm2) over a thin poly(methy1 methacrylate)
(PMMA) film.['] High dilution ensures a mean distance between
the dye molecules which is larger than the diameter of the aperture- this condition is necessary for the distinction of individual
molecules. The setup of the near-field microscope was as shown
in Figure 1 . The excited, fluorescing single molecules, which
appeared as microscopic points of light. were detected with a
lateral resolution of 50 nm and a vertical resolution of 5 nm.
The view that the emissions indeed arise from individual molecules is supported by a number of arguments. Most convincing
is the fact that photoinduced bleaching, which happens to some
of the molecules after longer illumination. does not lead to the
gradual decrease of the signals, but to the discrete and total
extinction of individual emissions. The very good signal-to-
basically the same NSOM setup.[41An interesting result of their
investigation is the observation of reversible photobleaching
of individual molecular chromophores. The authors attribute
this effect to hopping motions that change the molecular orientation and consequently the excitation probability of the
molecules. In a sense. this implies that a photoinduced reaction of a single molecule ;it room temperature has been obscrvcd.
We can expect that near-field microscopy will lead to many
tkscinating experiments with individual molecules in the near
futurc. These include investigations on the conditions for lightinduced photobleaching of individual chromophores. an unwanted effect that limits all sensitive fluorescence measurements
at room temperature. Time-resolved measurements after excitation with picosecond pulses or by analysis of the fluorescence
autocorrelation function are also just being tackled.l"] New
perspectives of special interest are offered by investigations of
single molecules at low temperatures with a combination of
NSOM and optical spectroscopy. Here. an extremely high spectral resolution can be used additional to the high local resolution
in NSOM.
Gcrm'iii \ersioii:
I'ig 2 Ncar-lield inicroscopic pic1 tirc of Iluorescsnt \inple carhocq.iniiie d > c inoleculer on the \urf;ice o f a P M M . 4 suhstrate (Trom rel: [ 3 ] )
noise ratio in Figure 2 is attributed to the low background signal
in the NSOM technique; a s already discussed. only ii very small
sample volume is illuminated with high intensity. It is not yet
clear if fliioresceiice amplification by evanescent fields in the
optical near field contributes to the signal. At closer inspection.
different geometric shapes of the structures in Figure 2 are apparent. They result from different orientations of the molecular
transition dipoles. From an analysis of these shapes, the authors
could determine the arrangement of the individual molecular
transition dipoles, in other words of the individual molecules. on
the surface. Moreover, by rcverse reasoning, Betzig et al. were
also able to use the molecular point dipole to map the optical
near field of their fiber tip.
Ambrose et al. succeeded in imaging the fluorescence of
single rhodamine 6G molecules on a silica surface using
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