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Optical measurements on the nanometer scale
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Optical measurements on the nanometer scale Weihong Tan 1
Department of Chemistry and The UF Brain Institute, University of Florida, P.O. Box 117200, Gainesville, FL 32611-7200, USA Near-¢eld optics has seen a variety of novel applications, including ultramicroscopy, microspectroscopy, photofabrication, single molecule studies and biochemical sensing. Intracellular calcium responses upon drug stimulation have been monitored in single living cells. Single molecule optical probes have been nanofabricated, and these probe's optical and spectroscopic properties have been characterized. Ultrasensitive detection schemes, using near-¢eld optical probes, have been developed for ultratrace level analysis. Biomolecule immobilization has been carried out on optical ¢ber probes for the preparation of sensitive biochemical sensors. These research activities will further enrich the ¢eld of near-¢eld optics, and it is expected that near ¢eld optics' potential will be fully explored for a variety of applications. z1998 Elsevier Science B.V. All rights reserved. Keywords: Near-¢eld optics; Single molecule detection; Biochemical sensors; Single molecule probes; Biomolecule immobilization; Imaging; Microspectroscopy; Calcium measurement; In-vivo monitoring; Glutamate; Lactate; Optrode
1. Introduction Laser technology has been the driving force for the development of many analytical and biophysical techniques. These techniques can carry out precise and accurate determinations with excellent detection limits and reliability for a variety of samples. One of the most important recent advances, scanning probe microscopy (SPM ), has also bene¢ted from laser technology. There are many different types of SPMs, among which an optical microscope, near-¢eld scanning optical microscopy (NSOM ), has been depend1
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ent on lasers. NSOM, based on near-¢eld optics (NFO ), has begun to gain recognition in several ¢elds, including microscopy, spectroscopy, photofabrication, single molecule studies and biochemical sensing [ 1,2 ].
2. Near-¢eld optics (NFO ) The wealth of optical phenomena offers many opportunities for the investigation of individual nanometer-sized structures if the relevant radiation can be con¢ned to a suf¢ciently small extension. However, in most conventional optical techniques, the standard rules of interference and diffraction lead to the Abbeé diffraction limit [ 3 ] on the resolution of optical microscopes. This limit is approximately V / 2, where V is the wavelength of light. No classical optical microscope can overcome this diffraction barrier. Electron and Xray microscopes do not overcome it either, except that their V is signi¢cantly shorter. Their better absolute resolution comes, however, at the price of their use of highly ionizing radiation, which may cause severe damage to some samples. Through the years, as technological and scienti¢c studies required ¢ner and ¢ner resolution, the bounds imposed by far-¢eld diffraction pushed the optical microscope to its fundamental limits. The search for better resolution has led to the concept of near-¢eld optics [ 4,5 ]. NFO has enabled researchers to examine optically a variety of specimens without being limited in resolution to one half the wavelength of light. NFO is generating considerable interest and has been applied in microscopy, spectroscopy, single molecule studies, biochemical analysis and photonanofabrication. NFO enables us to bypass the optical diffraction limit by utilizing a small light source which effectively focuses photons through a tiny aperture that may be as small as V / 50. The principle underlying this concept is schematically shown in Fig. 1. The near-¢eld apparatus consists of a near-¢eld light source, with the sample in the near-¢eld and a far-¢eld detector. To form a
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only in the 1980s was the principle followed by optical experiments. Major advances in SPM that provide sample and probe manipulation with subnanometer precision and for three-dimensional computer imaging from a series of line scans have facilitated the development of visible NSOM. Today's NSOM has indeed borrowed some of the electromechanical and computer control scanning techniques from STM and AFM.
3. Near-¢eld optical probes
Fig. 1. Schematic drawing of near-¢eld optics.
subwavelength optical probe, light is directed to an opaque screen containing a small aperture. The radiation emanating through the aperture and into the region beyond the screen is ¢rst highly collimated, with dimension equal to the aperture size, which is independent of the V of the light employed. The region of collimated light is known as the `near-¢eld' region. The highly collimated emissive photons only occur in the near-¢eld regime. To generate a high-resolution image, a sample has to be placed within the near¢eld region of the illuminated aperture. The aperture then acts as a subwavelength-sized light probe which can be used as a scanning tip to generate an image. That is why this optical microscopy is called near-¢eld scanning optical microscopy. As with many scienti¢c developments, the expression of the fundamental concepts behind NFO considerably predated their successful implementation. The NFO principle has been discovered and rediscovered several times [ 4^8 ]. In the 1920s, Edward Hutchinson Synge discussed an instrument very similar to today's NSOM with Albert Einstein, and published his ideas in The Philosophical Magazine. His basic vision included the design of a subwavelength aperture scanned in the near-¢eld of a sample. Synge's ideas were independently rediscovered several times in the 1950s but it was not until 1972 that we saw the ¢rst experimental demonstration of near-¢eld scanning microscopy by Ash and Nichols. They used microwaves ( wavelength 3 cm ) to obtain a subwavelength line scan. The photon `scanning probe technique' preceded all other scanning probe microscopies, such as scanning tunneling microscopy (STM ) [ 9 ] and atomic force microscopy ( AFM ) [ 10 ]. However,
The major technical challenge in NFO is that we need a small subwavelength light source with enough intensity. Another is that the sample has to be scanned closely and quickly. The latter requirement is not too dif¢cult nowadays ( magnetic memories are scanned extremely quickly at even closer distances ). There are other problems, such as a `feed-back' mechanism to avoid physical contact and damage to the source. There are also wonderful recent solutions [ 11 ], like combined NFO and force, or combined NFO and AFM operation. These further enrich the contrast mechanisms of near-¢eld optics: refraction, re£ection, polarization, luminescence, lateral force interactions, and so on. However, the ¢rst requirement of a tiny but intense and scannable light source has been much more challenging. NFO is realized by subwavelength optical probes. Light can be apertured down to sizes
Fig. 2. Scanning electron microscopy micrograph of a subwavelength optical ¢ber probe.
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much smaller than its wavelength, with no obvious theoretical limit until one reaches the size of an atom. Subwavelength probe development has been the key to the advances in NFO instrumentation and application. Originally, tiny nanofabricated ori¢ces in a thin metal sheet or ¢lm were used as light sources. However, the photon throughput was very limited. This led, among others, to the idea of a glass micropipette which has been used for intracellular measurements. Further development has led to the advent of active subwavelength light sources ( ¢ber optic tips and luminescent probes ) [ 12^14 ]. Now glass micropipette and optical ¢ber tips are both used for NFO applications, with the most frequently used being the ¢ber optic tip, as shown in Fig. 2. There are two techniques in NFO ¢ber probe fabrication: optical ¢ber micro-pulling and etching. In NFO probe nanofabrication process, the ¢rst step is the preparation of ¢ber optic tips of appropriate size and shape. For example, a ¢ber tip is produced by drawing an optical ¢ber in the puller with appropriate heating by a CO2 laser and pulling force. The second step is the metal coating of such tips. The optical ¢ber tip is coated with aluminum, by vapor deposition, to form a small aperture. Pulling such ¢bers is mostly done today with a commercial puller ( such as Sutter Instrument Corp., Novato, CA, P-97 or P-2000 ). One can now reproducibly pull robust and ef¢cient ¢ber optic tips with ori¢ces as small as 20 nm, optically streamlined and clean enough on the outside to facilitate metal coating. Fiber optic tips can also be prepared by etching with chemical solutions [ 15 ]. There
is a large difference in light throughput between the pulled and the etched optical ¢ber probes. Etched tips may provide two to four orders of magnitude higher light throughput. There are also other recent efforts in preparing better NFO probes. For example, a new technique based on photolithography has been developed to fabricate a ¢ber probe with a nanometric protruding tip. An optical ¢ber is ¢rst sharpened by chemical etching and coated with metallic ¢lm. Then, it is coated with photoresist and its apex region is selectively exposed to an evanescent wave. Finally, the metallic ¢lm at the apex region is etched away. The tip diameter of the NFO probe fabricated by this method is about 30 nm [ 16 ]. One of the major concerns troubling today's NFO technology is the quality of NFO tips. The major problems in this area are: reproducibility, limited light throughput for probes with sizes smaller than 50 nm, and leakage of light through pinholes.
4. Near-¢eld scanning optical microscopy and spectroscopy NSOM belongs to the SPM family, sometimes referred to as the `children of the STM'. However, the rich contrast mechanisms in optics and the ability to image a broad range of samples in a variety of environments make NSOM unique. The very same tip can be scanned over the same sample with an alternation of the contrast mechanism ( for example, £uorescence and shear force ), yielding images with a high degree
Fig. 3. NSOM and shear force images of a latex sphere sample. The sample was obtained from TopoMetrix, and prepared by coating latex spheres on a glass slide. The glass slide was then coated with a thin layer of aluminum. The latex spheres were washed away, and holes were left on the glass surface. Left: topographic image obtained through shear force. Right: optical image by NFO probe. All features ( such as A ) correspond well to each other in these two images. A is the metal coating site: it is higher in the topographic image ( bright ), and does not transmit light ( dark ).
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of ¢delity, as well as additional information content ( this is like adding the natural color to a three-dimensional topographic map ). One of the major advantages of NSOM is its ability to obtain simultaneous optical and topographic images. The optical image is obtained through the optical signal, while the topographic image is obtained through shear force, shown in Fig. 3. This is potentially very useful for a variety of studies where the functionality is closely linked to the sample's morphological conditions. NSOM has led to a revolution in physical science by providing optical î with rigid samples resolution at the level of 120 A [ 17 ]. Very impressive biological images with tens of nanometer resolution have been obtained for cytoskeletal actin, blood cells, DNA molecules and photosynthetic units [ 18^21 ]. Simultaneous topographic and optical images of human chromosomes have been obtained using a sharp and bent optical ¢ber probe. These images have provided useful information on native chromosome structure [ 22 ]. Near-¢eld scanning optical spectroscopy (NSOS ) is based on near-¢eld optical microscopy. It basically adds one more dimension, spectroscopy, to NSOM and can be used to obtain the spectrum of various nanostructures, such as subcellular organelles and quantum wells. NSOS inherits all the advantages of NSOM: its non-invasive nature, its ability to look at non-conducting and soft surfaces, and the addition of the spectral dimension. The ability to obtain spectroscopic information with a nanometer-sized resolution makes NSOS very promising for a wide variety of biomedical and chemical research. Examples include the detection of £uorescent labels on biological samples and isolating local nanometer-sized heterogeneity in microscopic samples. Using NSOS, tetracene and perylene doped in polymethylmethacrylate as well as microscopic crystals have been studied, which demonstrates that nanoscopic inhomogeneity can be detected in what might at ¢rst appear to be homogeneous [ 23,24 ]. Near-¢eld optical spectra have been obtained for single molecules and quantum wells [ 25,26 ]. Low-temperature near-¢eld scanning optical microscopy was used for spectroscopic studies of single, nanometer-dimension, cleaved edge overgrown quantum wires [ 27 ]. A direct experimental comparison between a two-dimensional system and a single genuinely one-dimensional quantum wire system, inaccessible to conventional far-¢eld optical spectroscopy, is enabled by the enhanced spatial resolution. The photoluminescence of a single quantum wire is easily distinguished from that of the surrounding quantum well. Near-¢eld microscopy / spectroscopy
provides a means to obtain optical information from a variety of samples on a nanometer scale.
5. Single molecule detection and localization Molecular structure and dynamics have traditionally been inferred through averaging techniques, such as X-ray crystallography, electron diffraction, and various spectroscopies. Near-¢eld optical microscopy and spectroscopy are new tools providing highly improved imaging and localization techniques for single molecule studies. Among the most recent exciting advances in NFO research are studies of single molecules [ 28^30 ]. Using NFO to localize and detect single molecules has its unique advantages. Actually, there is no need for a molecular size NFO probe for single molecule localization and detection. The size of the probe can be quite large compared to that of one molecule ( 100 nm vs. 1 nm ). Single molecule localization is not single molecule imaging. The sample is prepared with a very low surface concentration of the molecule of interest. The reason a 100 nm probe can localize one molecule ( 1 nm ) [ 28 ] is that there are no other molecules within this 100 nm area. Therefore, one molecule can be studied. Very elegant observations of single molecules together with their optical spectroscopy have been performed by a variety of techniques. Individual carbocyanine dye molecules are localized with NSOM [ 28 ]. The imaging resolution is about 50 nm, and the molecular location is resolved within about 25 nm in the horizontal plane and 5 nm in the vertical direction. About two dozen isolated dye molecules are imaged within minutes. Information has also been obtained on the orientation of these individual molecules. Along with single molecule localization, studies of single molecule spectroscopy, dynamics, photochemistry and photophysics have also been performed by NFO. The ability to observe the optical spectrum of a single molecule or, alternatively, of molecular aggregates can afford insights into the interactions that distinguish one molecular environment from another [ 26 ]. Near-¢eld spectroscopy of single molecules has been performed in air at room temperature. In this experiment, time-dependent emission spectra of a single molecule have been obtained. The spectra of individual molecules exhibit wavelength shifts from, and have typically smaller line widths than, those from the bulk material, as is expected since the spectral lines from the bulk material will be broadened by the range
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Fig. 4. A NFO probe inserted into a vascular smooth muscle cell. Measurements are done by monitoring £uorescence intensity of a calcium ion dye excited by light exiting from the NFO probe. Very localized quantitative measurements are due to the near¢eld optical excitation of the probe.
of local environments of the individual molecules. NFO has also been used to study the molecular dynamics of individual molecules dispersed on a glass surface. The £uorescence lifetimes of single molecules are measured on a nanosecond time scale, and their excited-state energy transfer to the aluminum coating of the near-¢eld probe is characterized [ 30 ]. The feasibility of £uorescence lifetime imaging with single molecule sensitivity, picosecond temporal resolution and a spatial resolving power beyond the diffraction limit has been demonstrated. NFO has opened a new avenue in single molecule studies. This may facilitate the manipulation of single molecules in the basic physical sciences as well as in medical diagnostics and in biotechnology.
6. Subcellular monitoring with NFO probes for single point monitoring NFO has been applied to study biological samples by monitoring intracellular analyte changes. There are two approaches for cellular monitoring: park the probe outside on the cell membrane and insert the probe into a cell. In the ¢rst approach, a new feedback method, photon density feedback, has been developed to mon-
itor the registration of a near-¢eld illumination probe with living cell membranes [ 31 ]. In this method, the £uorescence intensity of a uniformly distributed £uorochrome is monitored while the sample is moved in the z-axis towards the probe. Upon contact between the cell membrane and the near-¢eld probe a maximum intensity is detected. Using this method, physiological properties of neurons, astrocytes or mast cells have been monitored, indicating that this high-resolution optical detection method will permit a new approach to the study of molecular distribution and action within living specimens. Another method of intracellular monitoring is to insert the probe inside a living cell. For example, NFO probes are used to penetrate vascular smooth muscle cells (VSMC ) to monitor Ca2 £uctuations during cell stimulation ( J. Bui, T. Zelles, H. Lou, V. Gallion, I. Phillips and W., Tan, submitted ). As shown in Fig. 4, a NFO probe has entered a single VSMC, without any visible damage or leakage. Fluorescence images of these cells have con¢rmed that the cells are still alive after the penetration of the probe. The penetration of the probe into a cell is very similar to cell microinjection. The NFO ¢ber probe is controlled by a micromanipulator. The tip of the NFO probe de¢nes the spatial resolution of the localized subcellular measurements. Single cell
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responses to neurohormone stimulation which contracts VSMC by increasing intracellular Ca2 have been observed. The results indicate that this technique is useful for detecting resting Ca2 and Ca2 increases after drug administration. Different response pro¢les of intracellular Ca2 in£ux have been observed after ionomycin and bradykinin administration. Further, responsive heterogeneities due to ionomycin among different cells of the same type have been detected. NFO probes make possible real-time visualization of intracellular events during drug administration by probing nanoscopic cellular regions.
7. Laser desorption, Raman studies and ultrasensitive detection Laser desorption and Raman studies have also been carried out using NFO probes [ 15,32,33 ]. Near-¢eld Raman spectra have been obtained from single Ag colloidal nanoparticles with excitation intensities as low as 10 nW. Far-¢eld confocal studies have been carried out on spatially isolated, single Ag nanoparticles. A direct comparison of the near-¢eld and confocal results shows that near-¢eld optical excitation does not lead to signi¢cantly different selection rules in Raman spectroscopy. This work further demonstrates that single metallic nanoparticles can be attached to the tip of a near-¢eld probe for potential application in surface-enhanced optical microscopy and nanofabrication [ 33 ]. As mentioned in Section 3, etched NFO probes have much higher light throughput. Thus the rapid heating of these probes with pulsed laser radiation can be used for thermal desorption of organic crystals and for modi¢cation of polymer surfaces. A lateral resolution of 75 nm is achieved in these studies [ 15 ]. In addition, NFO has also been used for ultrasensitive detection of dye molecules in extremely small samples [ 34 ]. A NFO probe is inserted into an aqueous sample contained in a 5 Wm diameter pore for a speci¢c duration. The optical signal from the £uorophores, physically immobilized onto the probe, is monitored by an avalanche photon diode, and is related to the concentration of the £uorophores. Rhodamine 6G (R6G ) molecules and R6G-labeled DNA molecules have been tested. Excellent linearity has been observed for a wide range of concentrations. NFO probes are able to detect R6G concentrations down to 10315 M. It is expected that this technique will be extremely useful for measurements of intracellular biomolecules.
Fig. 5. Schematic drawing of near-¢eld nanofabrication: photo-nanofabrication of a nanometer biochemical sensor probe. The probe is prepared with a near-¢eld optics controlled biochemical immobilization process. Depending on the molecules or biomolecules in the solution, different types of probes can be prepared with a variety of biochemical sensitivities.
8. Photo-nanofabrication and ultrasmall biochemical sensors NFO enables a major advancement in nanofabrication techniques for nanometer devices. Microfabrication based on conventional optics has been widely used in photolithography. However, it has not been able to produce nanometer structures and devices due to the diffraction limit. Using near-¢eld optics, a novel nanofabrication technology for the preparation of nanometer structures has been developed. The nanostructures or probes can be converted to sensors for highly localized biochemical measurements by attaching the appropriate chemical indicators to the probe tips. 8.1. Photo-nanofabrication
Photo-nanofabrication is a new and controllable nanofabrication technology [ 14,35 ]. Using the near¢eld optics principle, photo-nanofabrication controls the size of the luminescent material grown at the end of an optical ¢ber probe, by photochemical reactions. These reactions are initiated and driven by an appropriate wavelength of light. The luminescent material is formed ( synthesized ) only in the presence of light and is `bonded' only to the area where light is emitted. The
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Fig. 6. Microphotograph of a NFO biochemical sensor. It is prepared by photo-nanofabrication. Bright tip area shows that sensing molecules are attached to the tip end surface.
key to photo-nanofabrication is a near-¢eld photochemical reaction, in which the electromagnetic waves of the light source are mapped by the photochemical process. Thus the size of the luminescent probe is de¢ned by the light emitting aperture and is independent of the wavelength of the light used to promote the chemical reaction. The photochemical reaction only occurs in the near-¢eld region, where the photon £ux and the absorption cross-section are the highest [ 2 ]. NFO has thus revolutionized nanofabrication techniques for ultrasmall nanometer devices and patterns. The NFO based nanofabrication process is shown in Fig. 5. To illustrate the principle of photo-nanofabrication, here we describe the near-¢eld photopolymerization process, by which submicrometer optical ¢ber pH sensors have been prepared. The metal-coated ¢ber tip is ¢rst silanized, then is used for photopolymerization which is controlled by the light emanating from the near-¢eld light source. The size of the light source and the near-¢eld evanescent photon pro¢le control the size and shape of the immobilized photoactive polymer. The pH sensors are prepared by incorporating £uoresceinamine derivative, acryloyl£uorescein, into an acrylamide-methylenebis( acrylamide ) copolymer that is attached covalently to a silanized ¢ber tip surface by photopolymerization. The rate and size of polymer formation is controlled by visually monitoring the distal tip through a microscope. This process enables the incorporation of pH sensitive dye molecules covalently bonded to the optical ¢ber sur-
face and the production of nanometer biochemical sensors [ 35 ]. The size of the polymer grown on the aperture of the optical ¢ber tip is equal to or smaller than that of the aperture. By photo-nanofabrication, one is able to produce submicrometer structures at the end of NFO probe, as shown in Fig. 6. 8.2. Miniaturized biochemical sensors
Photonanofabrication produces nanometer sized optical probes with or without a speci¢c biochemical sensitivity. Probes with a speci¢c biochemical sensitivity are nanometer biochemical sensors. The reduced size of the light sources, together with the near-¢eld enhanced molecular excitation cross-section and the good spectral and time resolution have enabled the development of rugged, ultra-small, ultra-sensitive and ultra-fast ¢ber-optic biochemical sensors. The size of the sensors ranges from 50 nm to a few micrometers, and no mechanical con¢nement is used. Thus the analytes have immediate access to the dye on the sensor tip. This gives the NFO sensors the shortest response times among any reported optical ¢ber sensors. These nanometer intracellular sensors require only attoliters ( 10318 liter ) of sample, zeptomoles ( 10321 mole ) of unknown and milliseconds or microseconds of response times. The sensing occurs in the near-¢eld regime of the optical excitation, thus highly increasing the sensitivity per photon and per sensor molecule. This has decreased the volume needed for ( non-destructive ) analysis. Since the initial pH sensor
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research in 1992, many different optical sensors have been successfully prepared for direct, real-time chemical analysis of intracellular processes and other studies. These sensors monitor pH, calcium, sodium, potassium, chloride, oxygen and glucose [ 14,35^45 ]. Even smaller probes, so called PEBBLE ( probes encapsulated by bio-listic embedding ) optochemical sensors, have been recently developed ( Clark and Kopelman, submitted ). These ¢berless probes have all their dimensions in the nanoscale range and typically are spheres with a radius of 100 nm. These probes have been applied to monitor biological samples, such as single mammalian cells. PEBBLE probes are introduced into a cell with a `gene gun', i.e., a particle delivery system usually used for the `dry' insertion into viable cells of DNA molecules.
9. Biomolecule probes for biochemical sensing In addition to the above photonanofabrication method for nanometer sensors, there have also been development in direct covalent immobilization techniques for biomolecule sensors. A variety of biomolecules ( enzymes, antibody, antigen, proteins and
DNA ) can be immobilized onto solid supports for the study of biomolecule interactions. These solid supported biomolecules have proved to be useful for many biological studies. However, these solid supports cannot be used to study biological processes at the subcellular level. The NFO probes should easily allow preparation techniques for biomolecule probes suf¢ciently small for studies in nanoscopic regions. Photo-nanofabrication is one such preparation technique for ultrasmall biochemical sensors. However, to have biomolecules covalently bonded to a NFO tip, special functional groups, such as vinyl monomer, are required. A generalized immobilization technique for most biomolecules has been developed recently by creating a procedure applicable to the synthesis of various nanometer biomolecule probes ([ 45 ]; Liu and Tan, submitted ). New immobilization procedures, including tip silanization, conjugation, and chemical binding of these compounds, are being created to coordinate the NFO tip's characteristics with the speci¢c properties of biological compounds. As shown in Fig. 7, biomolecules with free amine functional groups can be immobilized to NFO probes, thus preparing NFO biosensors. These sensors operate by detecting distinct changes in optical properties of the speci¢c biomolecules adhered to the tips.
Fig. 7. GDH immobilization on NFO ¢ber probes.
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Using this method, a glutamate dehydrogenase ( GDH ) probe has been prepared and used to detect glutamate ( Glu ). By covalent bonding, GDH enzymes can be immobilized on a ¢ber tip surface. The ¢ber is ¢rst silanized with trimethylacylpropyldiethylenetriamine to cover the tip with amine groups. As amines are found in most enzymes as well as in other biomolecules, it is then necessary to incorporate a homobifunctional crosslinker as a conjugate reagent between the enzyme molecules and the amine groups on the ¢ber tip surface. Therefore, the carboxyl group will conjugate to the amino acid side chains of the enzyme. This approach has been successful for the immobilization of GDH on optical ¢ber probes. Further miniaturization of GDH probes to approximately 100 nm will increase the probe's sensitivity and the ability to work with minute biological samples. The GDH probe has been used to detect Glu ef¢ciently, down to 0.2 WM. The GDH is highly selective in detecting Glu due to the speci¢city of the GDH enzymatic reaction. The miniaturization of Glu sensor has also resulted in fast response times.
10. Single molecular probes For better spatial resolution and higher sensitivity in biochemical analysis, smaller probes are required and are in the making. There are recent exciting new developments in optical supertips [ 2,46 ]. These probes can be developed as a single molecule sensor. Potential applications, which are unique to single molecule probes, include sequencing DNA, probing nanometer scale environments, monitoring individual molecule reactivities, studying the variability of molecular conformations, detecting disease infection at an early stage, and devising nanostructures and molecularscale imaging probes for single molecule microscopy. Existing designs for optical supertips are based on the same principle as the green plant photosynthetic system [ 47 ]. A submicrometer antenna collects the photons by absorption and transfers the excitation energy to a single active center [ 2 ]. From there the energy is either ( i ) radiated as a photon or ( ii ) transferred to the sample in an energy transfer process ( Foërster^Dexter ) [ 48 ]. In either case the result is generally affected by the nearby sample molecule. ( i ) The radiated excitation may be affected, e.g., by intermolecular spin-orbit coupling ( Kasha effect, external heavy atom effect ) [ 49 ]. ( ii ) The energy transfer results in a £uorescence or phosphorescence typical of the sample molecule. In the latter case, only virtual photons are produced by
the supertip; this gives an excitation transfer tip (`exciton tip') and only sample luminescence is detected. Supertip development will be the key to scanning probe microscopy which relies on quantum optics mechanisms, such as Foërster energy transfer [ 48 ] or Kasha effect [ 49 ]. These interactions occur at the interface of the tip ( its active center ) and the sample ( which are quantum mechanically coupled ). For the highest resolution, this active center would consist of a single molecule, or molecular cluster, that does the imaging. This molecule is then the energy donor site for the Foërster energy transfer, or the spin-orbit interaction site for the Kasha effect. 10.1. Single dendrimer photon antenna
Kopelman et al. designed a supertip made of a single symmetric macromolecule by using newly developed dendrimer supermolecules [ 46 ]. The so-called starburst phenylacetylene dendrimers include the largest so far synthesized structurally ordered molecule ( D127, see Fig. 8 ). These fractal, tree-like supermolecules have spatially localized eigenfunctions, and, in particular, localized electronic states. Furthermore, in the so-called SYNDROME family of dendrimers [ 50 ], simple theory leads to selectively lower excitation energies at the central locus of the ordered macromolecule, with energies increasing towards the rim. This is borne out experimentally by the vibronic spectra of the entire family of molecules ( D-2 to D-127 ), with full internal consistency in the observed electronic energies ( red shifts ), vibrational quanta, Franck^Condon factors, overall transition moments and picosecond spectral diffusion. The architecture of this series of dendrimers is controlled by organic synthetic methods. For example, the overall shape of a `D-127' molecule is bowl-like with a molecular size î . It can thus act as both optical and force around 125 A active center. The large `rim' is bound to the tip by cumulative van der Waals bonding or covalent bonds. D-127 may be used in supertip preparation in two ways. The ¢rst is to make supertips in the range of î . In this case, D-127 is an energy transfer 100 A acceptor. It traps most of the energy quanta from the bulk of the optical probe. D-127 is the light emitting active center; The second way D-127 can be used for î resolusupertip preparation is to achieve about 10 A tion. To further demonstrate the `energy funnel' model a partial dendrimeric wedges with ( and without ) an excitation acceptor, a perylene derivative pendant, at the locus has been synthesized, shown in Fig. 8. As expected, the energy transfer from the large antenna
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Fig. 8. Dendrimer molecule ( top ) and Nano-star molecule ( bottom ).
(`tree canopy') to the small acceptor center is indeed dramatic. The presence of the antenna ( 39 phenyl groups ) increases the yield of the yellow perylenic emission by three orders of magnitude for a given excitation wavelength. This molecule is an ordered
supermolecule transducer of absorbed radiation (STAR ) [ 46 ] in analogy to the primary excitation energy collecting antenna of some natural photosynthetic systems. Overall, such a photonic subwavelength nano-lens may play an important role in devel-
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oping molecular excitonics, including luminescent optical nanoprobes, scanning exciton tunneling microscopy [ 2 ], and nanometer-scale ¢ber optic chemical and biochemical sensors. 10.2. Single dye molecule probe
Another effort in making smaller optical probes is the direct immobilization of one molecule onto a NFO probe. Advances in sensitive optical measurement schemes have led to the detection and the physical and chemical characterization of individual molecules. To exploit the advances in single molecule detection, it is thus feasible to prepare single molecule probes based on near-¢eld optical nanoprobes. Very recently, a single carbocyanine dye C18 ( DiI ) molecule was immobilized on a near-¢eld optical ¢ber probe, as shown in Fig. 9 [ 51 ]. The single DiI molecule probe's optical properties have been characterized. Photobleaching of the single DiI molecule on the probe occurs as a discrete and total extinction of its £uorescence. A simple optical method is used to differentiate single molecule probes from multiple molecule probes. Using the optical intensities of a probe before and after molecular immobilization, an optical intensity ratio can be calculated and used to identify single molecule probes with 99% accuracy. The single molecule light sources are just the ¢rst step in the efforts to developing a variety of single biomolecule probes ( such as single antigen probe, single DNA probe and single ligand-receptor probe ) for single molecule optical microscopy and single molecule interaction studies with extremely high spatial resolution and sensitivity.
Fig. 9. A schematic drawing of a single molecule probe. The single DiI molecule is immobilized onto a PMMA thin ¢lm on a NFO ¢ber probe. The DiI molecule emits a different wavelength of light when excited by a laser beam.
11. Summary and outlook NFO has generated considerable interest and has been successfully applied in microscopy, spectroscopy, single molecule localization, photo-nanofabrication and subwavelength optical biochemical probes and sensors. NSOM has led to a revolution in physical science by providing optical resolution at the level of tens of nanometers. Single molecules have been localized and individual molecules' photochemical and photophysical properties have been studied. Laser desorption and Raman studies have been carried out using NFO probes. Biological applications have also been attempted. In addition, a novel nanotechnology, photo-nanofabrication, has been developed for the preparation of nanometer biochemical probes. Despite NFO's success in the physical sciences, the constraints of imaging biological samples prevent NSOM from being applied to living cells ef¢ciently. The complexity and fragility of biological systems have hampered the application of NFO in biology. There are a few dif¢culties, such as scanning feedback, signal collection, biological sample thickness and cell fragility. So far NFO technology has been mainly developed for rigid solid surfaces. Most of the feedback mechanisms cannot be applied to soft biological membranes and cannot function well inside the aqueous milieu of cells. The dif¢culty associated with smaller biological sample has increased `exponentially' with the decrease in sample size. There are some recent efforts towards NFO's application in biology. It is expected that NFO's potential will be fully explored for biological applications. Many theoretical and technical problems have still to be solved ^ from understanding the contrast mechanism to the nanofabrication of molecular sized probes to the control of photo-bleaching ( a standard problem in £uorescence microscopy ). The mechanism of light^matter interaction may be different in the farand near-¢eld regimes, leading to different spectral selection rules and in particular to an enhanced cross-section of light absorption ( and thus £uorescence ) [ 2,52 ]. However, the future looks bright. Growth in NFO research in its early days has not been as spectacular as that of its SPM predecessors, STM and AFM. On the other hand, NFO is increasingly making its appearance in new areas of research as well as in established areas as its capabilities expand: the number of NFO microscopes, worldwide, has risen in ¢ve years from about 5 to about 300. The ability to image in-solution environments has been a major research focus in the last few years
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and is being demonstrated, and the combination of better resolution and biochemical identi¢cation capabilities seems ideally suited to a number of biomedical investigations. Biosamples, including living cells and even ion channels, could be imaged down to the molecular level and analyzed spectroscopically by either NFO probes or biochemical sensors. The intracellular molecular dynamics of organogenesis, metabolism, splitting and biochemical damage could be followed in vivo and in real time. At the same time DNA could be sequenced in situ or even repaired by the right probe at the right location. The key to all these will be the further development of NFO probes which will have high light throughput, excellent reproducibility and are easy to prepare. In addition, functionalized NFO probes will play a more and more important role in NFO application.
Acknowledgements I thank my group members for their important contribution. This work is supported by NSF Grant CHE9733650 and by a Beckman Young Investigator Award from the Arnold Beckman Foundation.
References [ 1 ] M. Paesler and P. Moyer, Near-¢eld Optics: Theory, Instrumentation and Applications, John Wiley and Sons, New York, 1996. [ 2 ] W. Tan and R. Kopelman, Nanoscopic imaging and sensing by near ¢eld optics, in X. Wang and B. Herman ( Editors ), Fluorescence Imaging Spectroscopy and Microscopy, John Wiley and Sons, New York, 1996, p. 407.. [ 3 ] E. Abbeé, Arch. Mikrosk. Anat. 9 ( 1873 ) 413. [ 4 ] A. Lewis, M. Isaacson, A. Muray, A. Harootunian, Ultramicroscopy 13 ( 1984 ) 227. [ 5 ] D.W. Pohl, W. Denk, M. Lanz, Appl. Phys. Lett. 44 ( 1984 ) 651. [ 6 ] E.H. Synge, Phil. Mag. 6 ( 1928 ) 356. [ 7 ] J.A. O'Keefe, J. Opt. Soc. Am. 46 ( 1956 ) 359. [ 8 ] E.A. Ash, N. Nichols, Nature 237 ( 1972 ) 510. [ 9 ] G. Binnig, H. Rohrer, Ch. Gerber, E. Weibel, Phys. Rev. Lett. 49 ( 1982 ) 57. [ 10 ] G. Binnig, C.F. Quate, Ch. Gerber, Phys. Rev. Lett. 56 ( 1986 ) 930. [ 11 ] R. Brunner, A. Bietsch, O. Hollricher, O. Marti, Rev. Sci. Instrum. 68 ( 1997 ) 1769. [ 12 ] K. Lieberman, S. Harush, A. Lewis, R. Kopelman, Science 247 ( 1990 ) 59. [ 13 ] E. Betzig, J.K. Trautman, T.D. Harris, J.S. Weiner, R.L. Kostelak, Science 251 ( 1991 ) 1468. [ 14 ] W. Tan, Z.-Y. Shi, S. Smith, D. Birnbaum, R. Kopelman, Science 258 ( 1992 ) 778.
[ 15 ] D. Zeisel, B. Dutoit, V. Deckert, T. Roth, R. Zenobi, Anal. Chem. 69 ( 1997 ) 749. [ 16 ] T. Matsumoto, M. Ohtsu, J. Lightwave Technol. 14 ( 1996 ) 2224. [ 17 ] E. Betzig, J.K. Trautman, Science 257 ( 1992 ) 189. [ 18 ] X. Xie and R.C. Dunn, Brookhaven Natl. Lab., BNL, BNL 61733, Proceedings of the Nineteenth DOE Solar Photochemistry Research Conference, 1995, p. 60. [ 19 ] E. Betzig, R.J. Chichester, F. Lanni, D.L. Taylor, Bioimaging 1 ( 1993 ) 129. [ 20 ] E. Monson, G. Merritt, S. Smith, J. Langmore, R. Kopelman, Ultramicroscopy 57 ( 1995 ) 257^262. [ 21 ] Th. Enderle, T. Ha, D. Ogletree, D. Chemla, C. Magowan, S. Weiss, Proc. Natl. Acad. Sci. USA 94 ( 1997 ) 520. [ 22 ] S. Iwabuchi, H. Muramatsu, N. Chiba, Y. Kinjo, Y. Murakami, T. Sakaguchi, K. Yokoyama, E. Tamiya, Nucleic Acids Res. 25 ( 1997 ) 1662. [ 23 ] S.K. Kook, R. Kopelman, J. Phys. Chem. 96 ( 1992 ) 10672. [ 24 ] D. Birnbaum, S.K. Kook, R. Kopelman, J. Phys. Chem. 97 ( 1993 ) 3091. [ 25 ] R.D. Grober, T.D. Harris, J.K. Trautman, E. Betzig, W. Wegscheider, L. Pfeiffer, K. West, Appl. Phys. Lett. 64 ( 1994 ) 1421. [ 26 ] J.K. Trautman, J.J. Macklin, L.E. Brus, E. Betzig, Nature 369 ( 1994 ) 40. [ 27 ] T.D. Harris, D. Gershoni, L. Pfeiffer, M. Nirmal, J.K. Trautman, J.J. Macklin, Semiconductor Sci. Technol. 11 ( 1996 ) 1569. [ 28 ] E. Betzig, R.J. Chichester, Science 262 ( 1993 ) 1422. [ 29 ] W.P. Ambrose, P.M. Goodwin, J.C. Martin, R.A. Keller, Science 265 ( 1994 ) 364. [ 30 ] X. Xie, R.C. Dunn, Science 265 ( 1994 ) 361. [ 31 ] P.G. Haydon, S.P. Marchese-Ragona, T.A. Basarsky, M. Szulczewski, M. McCloskey, J. Microsc. 182 ( 1996 ) 208. [ 32 ] S. Nie, S.R. Emory, Science 275 ( 1997 ) 1102. [ 33 ] S. Emory, S. Nie, Anal. Chem. 69 ( 1997 ) 2631. [ 34 ] W. Tan, L. Bang, J. Cordek, X. Liu, SPIE-Int. Soc. Opt. Eng. 3273 ( 1998 ) 45. [ 35 ] W. Tan, Z.-Y. Shi, R. Kopelman, Anal. Chem. 64 ( 1992 ) 2985. [ 36 ] W. Tan, Z.-Y. Shi, R. Kopelman, Sensors Actuators B Chem. 28 ( 1995 ) 157. [ 37 ] M. Shortreed, R. Kopelman, M. Kuhn, B. Hoyland, Anal. Chem. 68 ( 1996 ) 1414. [ 38 ] M.R. Shortreed, E. Bakker, R. Kopelman, Anal. Chem. 68 ( 1996 ) 2656. [ 39 ] M. Shortreed, E. Monson, R. Kopelman, Anal. Chem. 68 ( 1996 ) 4015. [ 40 ] Z. Rosenzweig, R. Kopelman, Anal. Chem. 67 ( 1995 ) 2650. [ 41 ] Z. Rosenzweig, R. Kopelman, Anal. Chem. 68 ( 1996 ) 1408. [ 42 ] D. Uttamchandani, S. McCulloch, Adv. Drug Deliv. Rev. 21 ( 1996 ) 239. [ 43 ] S. McCulloch, D. Uttamchandani, IEE Proc. Optoelectron. 144 ( 1997 ) 162. [ 44 ] A. Song, S. Parus, and R. Kopelman ( 1997 ) Anal, Chem. 69 ( 1997 ) ( 1997 ) 863. [ 45 ] J. Cordek, X. Wang and W. Tan ( 1998 ) Anal. Chem. submitted.
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[ 46 ] R. Kopelman, M. Shortreed, Z. Shi, W. Tan, Z. Xu, J. Moore, A. Bar-Haim, J. Klafter, Phys. Rev. Lett. 78 ( 1997 ) 1239. [ 47 ] E.B. Fauman, R. Kopelman, Mol. Cell. Biophysics 6 ( 1989 ) 47. [ 48 ] M. Pope and E. Swenberg, Electronic Processes in Organic Crystals, Oxford University Press, New York, 1982.
[ 49 ] M. Kasha, J. Chem. Phys. 20 ( 1952 ) 71. [ 50 ] Z. Xu, Z. Shi, W. Tan, R. Kopelman, J. Moore, Polymer Preprints 33 ( 1993 ) 130. [ 51 ] W. Tan and X. Wang, Development of a single molecule optical probe, Thin Solid Film ( 1998 ) in press. [ 52 ] R. Kopelman, W. Tan, Science 262 ( 1993 ) 1382.
Elemental analysis by diode laser spectroscopy A. Zybin
Institute of Spectrochemistry and Applied Spectroscopy ( ISAS ), Bunsen-Kirchhoff-Strasse 11, D-44139 Dortmund, Germany
C. Schnuë rer-Patschan
LaserSpec Analytik GmbH, Frankfurter Ring 193a, D-80807 Muë nchen, Germany
M.A. Bolshov, K. Niemax*
Institute of Spectrochemistry and Applied Spectroscopy ( ISAS ), Bunsen-Kirchhoff-Strasse 11, D-44139 Dortmund, Germany After about 25 years of development of laser analytical spectroscopy, laser spectrochemical instruments at present work ef¢ciently only in research laboratories. There are practically no commercial laser spectroscopic instruments for routine elemental analysis. The only laser-based instruments are Raman spectrometers with stable cw laser sources for molecular analysis and pulsed lasers with ¢xed wavelengths which are used for laser ablation ( LA ) ^ laser sampling of solid materials. The main dif¢culties of laser spectroscopic techniques are: relatively high cost, insuf¢cient reliability, and the necessity of quali¢ed personal. Semiconductor laser diodes are mass produced for compact disc players, laser printers, optical data storage systems and telecommunication equipment. A number of these laser diodes have excellent spectroscopic properties, which make them attractive sources for spectrochemical analysis. Presently, laser analytical instruments *Corresponding author.
based on laser diodes have the highest potential for transfer from research laboratories to routine practice. Recently, the excellent analytical capabilities of diode laser spectrometry in the detection of low concentrations of analytes have been well documented. Recent advances in diode lasers and analytical trends are discussed below. z1998 Elsevier Science B.V. All rights reserved. Keywords: Diode laser spectroscopy; Laser ablation; Semiconductor laser diodes in spectroscopy; Micormachining
1. Atomic absorption spectroscopy with diode lasers For analytical applications, diode lasers ( DLs ) are most widely used as resonance light sources for atomic absorption spectrometry ( AAS ) ^ ( DLAAS ). Generally, tunable lasers like dye lasers or optical parametric oscillators (OPO ) have a reputation as powerful, but unstable light sources, and are therefore not well suited for atomic absorption measurements where the stabil-
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Optical measurements on the nanometer scale Weihong Tan 1
Department of Chemistry and The UF Brain Institute, University of Florida, P.O. Box 117200, Gainesville, FL 32611-7200, USA Near-¢eld optics has seen a variety of novel applications, including ultramicroscopy, microspectroscopy, photofabrication, single molecule studies and biochemical sensing. Intracellular calcium responses upon drug stimulation have been monitored in single living cells. Single molecule optical probes have been nanofabricated, and these probe's optical and spectroscopic properties have been characterized. Ultrasensitive detection schemes, using near-¢eld optical probes, have been developed for ultratrace level analysis. Biomolecule immobilization has been carried out on optical ¢ber probes for the preparation of sensitive biochemical sensors. These research activities will further enrich the ¢eld of near-¢eld optics, and it is expected that near ¢eld optics' potential will be fully explored for a variety of applications. z1998 Elsevier Science B.V. All rights reserved. Keywords: Near-¢eld optics; Single molecule detection; Biochemical sensors; Single molecule probes; Biomolecule immobilization; Imaging; Microspectroscopy; Calcium measurement; In-vivo monitoring; Glutamate; Lactate; Optrode
1. Introduction Laser technology has been the driving force for the development of many analytical and biophysical techniques. These techniques can carry out precise and accurate determinations with excellent detection limits and reliability for a variety of samples. One of the most important recent advances, scanning probe microscopy (SPM ), has also bene¢ted from laser technology. There are many different types of SPMs, among which an optical microscope, near-¢eld scanning optical microscopy (NSOM ), has been depend1
Tel.: +1 (352) 846-2410.
ent on lasers. NSOM, based on near-¢eld optics (NFO ), has begun to gain recognition in several ¢elds, including microscopy, spectroscopy, photofabrication, single molecule studies and biochemical sensing [ 1,2 ].
2. Near-¢eld optics (NFO ) The wealth of optical phenomena offers many opportunities for the investigation of individual nanometer-sized structures if the relevant radiation can be con¢ned to a suf¢ciently small extension. However, in most conventional optical techniques, the standard rules of interference and diffraction lead to the Abbeé diffraction limit [ 3 ] on the resolution of optical microscopes. This limit is approximately V / 2, where V is the wavelength of light. No classical optical microscope can overcome this diffraction barrier. Electron and Xray microscopes do not overcome it either, except that their V is signi¢cantly shorter. Their better absolute resolution comes, however, at the price of their use of highly ionizing radiation, which may cause severe damage to some samples. Through the years, as technological and scienti¢c studies required ¢ner and ¢ner resolution, the bounds imposed by far-¢eld diffraction pushed the optical microscope to its fundamental limits. The search for better resolution has led to the concept of near-¢eld optics [ 4,5 ]. NFO has enabled researchers to examine optically a variety of specimens without being limited in resolution to one half the wavelength of light. NFO is generating considerable interest and has been applied in microscopy, spectroscopy, single molecule studies, biochemical analysis and photonanofabrication. NFO enables us to bypass the optical diffraction limit by utilizing a small light source which effectively focuses photons through a tiny aperture that may be as small as V / 50. The principle underlying this concept is schematically shown in Fig. 1. The near-¢eld apparatus consists of a near-¢eld light source, with the sample in the near-¢eld and a far-¢eld detector. To form a
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only in the 1980s was the principle followed by optical experiments. Major advances in SPM that provide sample and probe manipulation with subnanometer precision and for three-dimensional computer imaging from a series of line scans have facilitated the development of visible NSOM. Today's NSOM has indeed borrowed some of the electromechanical and computer control scanning techniques from STM and AFM.
3. Near-¢eld optical probes
Fig. 1. Schematic drawing of near-¢eld optics.
subwavelength optical probe, light is directed to an opaque screen containing a small aperture. The radiation emanating through the aperture and into the region beyond the screen is ¢rst highly collimated, with dimension equal to the aperture size, which is independent of the V of the light employed. The region of collimated light is known as the `near-¢eld' region. The highly collimated emissive photons only occur in the near-¢eld regime. To generate a high-resolution image, a sample has to be placed within the near¢eld region of the illuminated aperture. The aperture then acts as a subwavelength-sized light probe which can be used as a scanning tip to generate an image. That is why this optical microscopy is called near-¢eld scanning optical microscopy. As with many scienti¢c developments, the expression of the fundamental concepts behind NFO considerably predated their successful implementation. The NFO principle has been discovered and rediscovered several times [ 4^8 ]. In the 1920s, Edward Hutchinson Synge discussed an instrument very similar to today's NSOM with Albert Einstein, and published his ideas in The Philosophical Magazine. His basic vision included the design of a subwavelength aperture scanned in the near-¢eld of a sample. Synge's ideas were independently rediscovered several times in the 1950s but it was not until 1972 that we saw the ¢rst experimental demonstration of near-¢eld scanning microscopy by Ash and Nichols. They used microwaves ( wavelength 3 cm ) to obtain a subwavelength line scan. The photon `scanning probe technique' preceded all other scanning probe microscopies, such as scanning tunneling microscopy (STM ) [ 9 ] and atomic force microscopy ( AFM ) [ 10 ]. However,
The major technical challenge in NFO is that we need a small subwavelength light source with enough intensity. Another is that the sample has to be scanned closely and quickly. The latter requirement is not too dif¢cult nowadays ( magnetic memories are scanned extremely quickly at even closer distances ). There are other problems, such as a `feed-back' mechanism to avoid physical contact and damage to the source. There are also wonderful recent solutions [ 11 ], like combined NFO and force, or combined NFO and AFM operation. These further enrich the contrast mechanisms of near-¢eld optics: refraction, re£ection, polarization, luminescence, lateral force interactions, and so on. However, the ¢rst requirement of a tiny but intense and scannable light source has been much more challenging. NFO is realized by subwavelength optical probes. Light can be apertured down to sizes
Fig. 2. Scanning electron microscopy micrograph of a subwavelength optical ¢ber probe.
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much smaller than its wavelength, with no obvious theoretical limit until one reaches the size of an atom. Subwavelength probe development has been the key to the advances in NFO instrumentation and application. Originally, tiny nanofabricated ori¢ces in a thin metal sheet or ¢lm were used as light sources. However, the photon throughput was very limited. This led, among others, to the idea of a glass micropipette which has been used for intracellular measurements. Further development has led to the advent of active subwavelength light sources ( ¢ber optic tips and luminescent probes ) [ 12^14 ]. Now glass micropipette and optical ¢ber tips are both used for NFO applications, with the most frequently used being the ¢ber optic tip, as shown in Fig. 2. There are two techniques in NFO ¢ber probe fabrication: optical ¢ber micro-pulling and etching. In NFO probe nanofabrication process, the ¢rst step is the preparation of ¢ber optic tips of appropriate size and shape. For example, a ¢ber tip is produced by drawing an optical ¢ber in the puller with appropriate heating by a CO2 laser and pulling force. The second step is the metal coating of such tips. The optical ¢ber tip is coated with aluminum, by vapor deposition, to form a small aperture. Pulling such ¢bers is mostly done today with a commercial puller ( such as Sutter Instrument Corp., Novato, CA, P-97 or P-2000 ). One can now reproducibly pull robust and ef¢cient ¢ber optic tips with ori¢ces as small as 20 nm, optically streamlined and clean enough on the outside to facilitate metal coating. Fiber optic tips can also be prepared by etching with chemical solutions [ 15 ]. There
is a large difference in light throughput between the pulled and the etched optical ¢ber probes. Etched tips may provide two to four orders of magnitude higher light throughput. There are also other recent efforts in preparing better NFO probes. For example, a new technique based on photolithography has been developed to fabricate a ¢ber probe with a nanometric protruding tip. An optical ¢ber is ¢rst sharpened by chemical etching and coated with metallic ¢lm. Then, it is coated with photoresist and its apex region is selectively exposed to an evanescent wave. Finally, the metallic ¢lm at the apex region is etched away. The tip diameter of the NFO probe fabricated by this method is about 30 nm [ 16 ]. One of the major concerns troubling today's NFO technology is the quality of NFO tips. The major problems in this area are: reproducibility, limited light throughput for probes with sizes smaller than 50 nm, and leakage of light through pinholes.
4. Near-¢eld scanning optical microscopy and spectroscopy NSOM belongs to the SPM family, sometimes referred to as the `children of the STM'. However, the rich contrast mechanisms in optics and the ability to image a broad range of samples in a variety of environments make NSOM unique. The very same tip can be scanned over the same sample with an alternation of the contrast mechanism ( for example, £uorescence and shear force ), yielding images with a high degree
Fig. 3. NSOM and shear force images of a latex sphere sample. The sample was obtained from TopoMetrix, and prepared by coating latex spheres on a glass slide. The glass slide was then coated with a thin layer of aluminum. The latex spheres were washed away, and holes were left on the glass surface. Left: topographic image obtained through shear force. Right: optical image by NFO probe. All features ( such as A ) correspond well to each other in these two images. A is the metal coating site: it is higher in the topographic image ( bright ), and does not transmit light ( dark ).
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of ¢delity, as well as additional information content ( this is like adding the natural color to a three-dimensional topographic map ). One of the major advantages of NSOM is its ability to obtain simultaneous optical and topographic images. The optical image is obtained through the optical signal, while the topographic image is obtained through shear force, shown in Fig. 3. This is potentially very useful for a variety of studies where the functionality is closely linked to the sample's morphological conditions. NSOM has led to a revolution in physical science by providing optical î with rigid samples resolution at the level of 120 A [ 17 ]. Very impressive biological images with tens of nanometer resolution have been obtained for cytoskeletal actin, blood cells, DNA molecules and photosynthetic units [ 18^21 ]. Simultaneous topographic and optical images of human chromosomes have been obtained using a sharp and bent optical ¢ber probe. These images have provided useful information on native chromosome structure [ 22 ]. Near-¢eld scanning optical spectroscopy (NSOS ) is based on near-¢eld optical microscopy. It basically adds one more dimension, spectroscopy, to NSOM and can be used to obtain the spectrum of various nanostructures, such as subcellular organelles and quantum wells. NSOS inherits all the advantages of NSOM: its non-invasive nature, its ability to look at non-conducting and soft surfaces, and the addition of the spectral dimension. The ability to obtain spectroscopic information with a nanometer-sized resolution makes NSOS very promising for a wide variety of biomedical and chemical research. Examples include the detection of £uorescent labels on biological samples and isolating local nanometer-sized heterogeneity in microscopic samples. Using NSOS, tetracene and perylene doped in polymethylmethacrylate as well as microscopic crystals have been studied, which demonstrates that nanoscopic inhomogeneity can be detected in what might at ¢rst appear to be homogeneous [ 23,24 ]. Near-¢eld optical spectra have been obtained for single molecules and quantum wells [ 25,26 ]. Low-temperature near-¢eld scanning optical microscopy was used for spectroscopic studies of single, nanometer-dimension, cleaved edge overgrown quantum wires [ 27 ]. A direct experimental comparison between a two-dimensional system and a single genuinely one-dimensional quantum wire system, inaccessible to conventional far-¢eld optical spectroscopy, is enabled by the enhanced spatial resolution. The photoluminescence of a single quantum wire is easily distinguished from that of the surrounding quantum well. Near-¢eld microscopy / spectroscopy
provides a means to obtain optical information from a variety of samples on a nanometer scale.
5. Single molecule detection and localization Molecular structure and dynamics have traditionally been inferred through averaging techniques, such as X-ray crystallography, electron diffraction, and various spectroscopies. Near-¢eld optical microscopy and spectroscopy are new tools providing highly improved imaging and localization techniques for single molecule studies. Among the most recent exciting advances in NFO research are studies of single molecules [ 28^30 ]. Using NFO to localize and detect single molecules has its unique advantages. Actually, there is no need for a molecular size NFO probe for single molecule localization and detection. The size of the probe can be quite large compared to that of one molecule ( 100 nm vs. 1 nm ). Single molecule localization is not single molecule imaging. The sample is prepared with a very low surface concentration of the molecule of interest. The reason a 100 nm probe can localize one molecule ( 1 nm ) [ 28 ] is that there are no other molecules within this 100 nm area. Therefore, one molecule can be studied. Very elegant observations of single molecules together with their optical spectroscopy have been performed by a variety of techniques. Individual carbocyanine dye molecules are localized with NSOM [ 28 ]. The imaging resolution is about 50 nm, and the molecular location is resolved within about 25 nm in the horizontal plane and 5 nm in the vertical direction. About two dozen isolated dye molecules are imaged within minutes. Information has also been obtained on the orientation of these individual molecules. Along with single molecule localization, studies of single molecule spectroscopy, dynamics, photochemistry and photophysics have also been performed by NFO. The ability to observe the optical spectrum of a single molecule or, alternatively, of molecular aggregates can afford insights into the interactions that distinguish one molecular environment from another [ 26 ]. Near-¢eld spectroscopy of single molecules has been performed in air at room temperature. In this experiment, time-dependent emission spectra of a single molecule have been obtained. The spectra of individual molecules exhibit wavelength shifts from, and have typically smaller line widths than, those from the bulk material, as is expected since the spectral lines from the bulk material will be broadened by the range
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Fig. 4. A NFO probe inserted into a vascular smooth muscle cell. Measurements are done by monitoring £uorescence intensity of a calcium ion dye excited by light exiting from the NFO probe. Very localized quantitative measurements are due to the near¢eld optical excitation of the probe.
of local environments of the individual molecules. NFO has also been used to study the molecular dynamics of individual molecules dispersed on a glass surface. The £uorescence lifetimes of single molecules are measured on a nanosecond time scale, and their excited-state energy transfer to the aluminum coating of the near-¢eld probe is characterized [ 30 ]. The feasibility of £uorescence lifetime imaging with single molecule sensitivity, picosecond temporal resolution and a spatial resolving power beyond the diffraction limit has been demonstrated. NFO has opened a new avenue in single molecule studies. This may facilitate the manipulation of single molecules in the basic physical sciences as well as in medical diagnostics and in biotechnology.
6. Subcellular monitoring with NFO probes for single point monitoring NFO has been applied to study biological samples by monitoring intracellular analyte changes. There are two approaches for cellular monitoring: park the probe outside on the cell membrane and insert the probe into a cell. In the ¢rst approach, a new feedback method, photon density feedback, has been developed to mon-
itor the registration of a near-¢eld illumination probe with living cell membranes [ 31 ]. In this method, the £uorescence intensity of a uniformly distributed £uorochrome is monitored while the sample is moved in the z-axis towards the probe. Upon contact between the cell membrane and the near-¢eld probe a maximum intensity is detected. Using this method, physiological properties of neurons, astrocytes or mast cells have been monitored, indicating that this high-resolution optical detection method will permit a new approach to the study of molecular distribution and action within living specimens. Another method of intracellular monitoring is to insert the probe inside a living cell. For example, NFO probes are used to penetrate vascular smooth muscle cells (VSMC ) to monitor Ca2 £uctuations during cell stimulation ( J. Bui, T. Zelles, H. Lou, V. Gallion, I. Phillips and W., Tan, submitted ). As shown in Fig. 4, a NFO probe has entered a single VSMC, without any visible damage or leakage. Fluorescence images of these cells have con¢rmed that the cells are still alive after the penetration of the probe. The penetration of the probe into a cell is very similar to cell microinjection. The NFO ¢ber probe is controlled by a micromanipulator. The tip of the NFO probe de¢nes the spatial resolution of the localized subcellular measurements. Single cell
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responses to neurohormone stimulation which contracts VSMC by increasing intracellular Ca2 have been observed. The results indicate that this technique is useful for detecting resting Ca2 and Ca2 increases after drug administration. Different response pro¢les of intracellular Ca2 in£ux have been observed after ionomycin and bradykinin administration. Further, responsive heterogeneities due to ionomycin among different cells of the same type have been detected. NFO probes make possible real-time visualization of intracellular events during drug administration by probing nanoscopic cellular regions.
7. Laser desorption, Raman studies and ultrasensitive detection Laser desorption and Raman studies have also been carried out using NFO probes [ 15,32,33 ]. Near-¢eld Raman spectra have been obtained from single Ag colloidal nanoparticles with excitation intensities as low as 10 nW. Far-¢eld confocal studies have been carried out on spatially isolated, single Ag nanoparticles. A direct comparison of the near-¢eld and confocal results shows that near-¢eld optical excitation does not lead to signi¢cantly different selection rules in Raman spectroscopy. This work further demonstrates that single metallic nanoparticles can be attached to the tip of a near-¢eld probe for potential application in surface-enhanced optical microscopy and nanofabrication [ 33 ]. As mentioned in Section 3, etched NFO probes have much higher light throughput. Thus the rapid heating of these probes with pulsed laser radiation can be used for thermal desorption of organic crystals and for modi¢cation of polymer surfaces. A lateral resolution of 75 nm is achieved in these studies [ 15 ]. In addition, NFO has also been used for ultrasensitive detection of dye molecules in extremely small samples [ 34 ]. A NFO probe is inserted into an aqueous sample contained in a 5 Wm diameter pore for a speci¢c duration. The optical signal from the £uorophores, physically immobilized onto the probe, is monitored by an avalanche photon diode, and is related to the concentration of the £uorophores. Rhodamine 6G (R6G ) molecules and R6G-labeled DNA molecules have been tested. Excellent linearity has been observed for a wide range of concentrations. NFO probes are able to detect R6G concentrations down to 10315 M. It is expected that this technique will be extremely useful for measurements of intracellular biomolecules.
Fig. 5. Schematic drawing of near-¢eld nanofabrication: photo-nanofabrication of a nanometer biochemical sensor probe. The probe is prepared with a near-¢eld optics controlled biochemical immobilization process. Depending on the molecules or biomolecules in the solution, different types of probes can be prepared with a variety of biochemical sensitivities.
8. Photo-nanofabrication and ultrasmall biochemical sensors NFO enables a major advancement in nanofabrication techniques for nanometer devices. Microfabrication based on conventional optics has been widely used in photolithography. However, it has not been able to produce nanometer structures and devices due to the diffraction limit. Using near-¢eld optics, a novel nanofabrication technology for the preparation of nanometer structures has been developed. The nanostructures or probes can be converted to sensors for highly localized biochemical measurements by attaching the appropriate chemical indicators to the probe tips. 8.1. Photo-nanofabrication
Photo-nanofabrication is a new and controllable nanofabrication technology [ 14,35 ]. Using the near¢eld optics principle, photo-nanofabrication controls the size of the luminescent material grown at the end of an optical ¢ber probe, by photochemical reactions. These reactions are initiated and driven by an appropriate wavelength of light. The luminescent material is formed ( synthesized ) only in the presence of light and is `bonded' only to the area where light is emitted. The
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Fig. 6. Microphotograph of a NFO biochemical sensor. It is prepared by photo-nanofabrication. Bright tip area shows that sensing molecules are attached to the tip end surface.
key to photo-nanofabrication is a near-¢eld photochemical reaction, in which the electromagnetic waves of the light source are mapped by the photochemical process. Thus the size of the luminescent probe is de¢ned by the light emitting aperture and is independent of the wavelength of the light used to promote the chemical reaction. The photochemical reaction only occurs in the near-¢eld region, where the photon £ux and the absorption cross-section are the highest [ 2 ]. NFO has thus revolutionized nanofabrication techniques for ultrasmall nanometer devices and patterns. The NFO based nanofabrication process is shown in Fig. 5. To illustrate the principle of photo-nanofabrication, here we describe the near-¢eld photopolymerization process, by which submicrometer optical ¢ber pH sensors have been prepared. The metal-coated ¢ber tip is ¢rst silanized, then is used for photopolymerization which is controlled by the light emanating from the near-¢eld light source. The size of the light source and the near-¢eld evanescent photon pro¢le control the size and shape of the immobilized photoactive polymer. The pH sensors are prepared by incorporating £uoresceinamine derivative, acryloyl£uorescein, into an acrylamide-methylenebis( acrylamide ) copolymer that is attached covalently to a silanized ¢ber tip surface by photopolymerization. The rate and size of polymer formation is controlled by visually monitoring the distal tip through a microscope. This process enables the incorporation of pH sensitive dye molecules covalently bonded to the optical ¢ber sur-
face and the production of nanometer biochemical sensors [ 35 ]. The size of the polymer grown on the aperture of the optical ¢ber tip is equal to or smaller than that of the aperture. By photo-nanofabrication, one is able to produce submicrometer structures at the end of NFO probe, as shown in Fig. 6. 8.2. Miniaturized biochemical sensors
Photonanofabrication produces nanometer sized optical probes with or without a speci¢c biochemical sensitivity. Probes with a speci¢c biochemical sensitivity are nanometer biochemical sensors. The reduced size of the light sources, together with the near-¢eld enhanced molecular excitation cross-section and the good spectral and time resolution have enabled the development of rugged, ultra-small, ultra-sensitive and ultra-fast ¢ber-optic biochemical sensors. The size of the sensors ranges from 50 nm to a few micrometers, and no mechanical con¢nement is used. Thus the analytes have immediate access to the dye on the sensor tip. This gives the NFO sensors the shortest response times among any reported optical ¢ber sensors. These nanometer intracellular sensors require only attoliters ( 10318 liter ) of sample, zeptomoles ( 10321 mole ) of unknown and milliseconds or microseconds of response times. The sensing occurs in the near-¢eld regime of the optical excitation, thus highly increasing the sensitivity per photon and per sensor molecule. This has decreased the volume needed for ( non-destructive ) analysis. Since the initial pH sensor
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research in 1992, many different optical sensors have been successfully prepared for direct, real-time chemical analysis of intracellular processes and other studies. These sensors monitor pH, calcium, sodium, potassium, chloride, oxygen and glucose [ 14,35^45 ]. Even smaller probes, so called PEBBLE ( probes encapsulated by bio-listic embedding ) optochemical sensors, have been recently developed ( Clark and Kopelman, submitted ). These ¢berless probes have all their dimensions in the nanoscale range and typically are spheres with a radius of 100 nm. These probes have been applied to monitor biological samples, such as single mammalian cells. PEBBLE probes are introduced into a cell with a `gene gun', i.e., a particle delivery system usually used for the `dry' insertion into viable cells of DNA molecules.
9. Biomolecule probes for biochemical sensing In addition to the above photonanofabrication method for nanometer sensors, there have also been development in direct covalent immobilization techniques for biomolecule sensors. A variety of biomolecules ( enzymes, antibody, antigen, proteins and
DNA ) can be immobilized onto solid supports for the study of biomolecule interactions. These solid supported biomolecules have proved to be useful for many biological studies. However, these solid supports cannot be used to study biological processes at the subcellular level. The NFO probes should easily allow preparation techniques for biomolecule probes suf¢ciently small for studies in nanoscopic regions. Photo-nanofabrication is one such preparation technique for ultrasmall biochemical sensors. However, to have biomolecules covalently bonded to a NFO tip, special functional groups, such as vinyl monomer, are required. A generalized immobilization technique for most biomolecules has been developed recently by creating a procedure applicable to the synthesis of various nanometer biomolecule probes ([ 45 ]; Liu and Tan, submitted ). New immobilization procedures, including tip silanization, conjugation, and chemical binding of these compounds, are being created to coordinate the NFO tip's characteristics with the speci¢c properties of biological compounds. As shown in Fig. 7, biomolecules with free amine functional groups can be immobilized to NFO probes, thus preparing NFO biosensors. These sensors operate by detecting distinct changes in optical properties of the speci¢c biomolecules adhered to the tips.
Fig. 7. GDH immobilization on NFO ¢ber probes.
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Using this method, a glutamate dehydrogenase ( GDH ) probe has been prepared and used to detect glutamate ( Glu ). By covalent bonding, GDH enzymes can be immobilized on a ¢ber tip surface. The ¢ber is ¢rst silanized with trimethylacylpropyldiethylenetriamine to cover the tip with amine groups. As amines are found in most enzymes as well as in other biomolecules, it is then necessary to incorporate a homobifunctional crosslinker as a conjugate reagent between the enzyme molecules and the amine groups on the ¢ber tip surface. Therefore, the carboxyl group will conjugate to the amino acid side chains of the enzyme. This approach has been successful for the immobilization of GDH on optical ¢ber probes. Further miniaturization of GDH probes to approximately 100 nm will increase the probe's sensitivity and the ability to work with minute biological samples. The GDH probe has been used to detect Glu ef¢ciently, down to 0.2 WM. The GDH is highly selective in detecting Glu due to the speci¢city of the GDH enzymatic reaction. The miniaturization of Glu sensor has also resulted in fast response times.
10. Single molecular probes For better spatial resolution and higher sensitivity in biochemical analysis, smaller probes are required and are in the making. There are recent exciting new developments in optical supertips [ 2,46 ]. These probes can be developed as a single molecule sensor. Potential applications, which are unique to single molecule probes, include sequencing DNA, probing nanometer scale environments, monitoring individual molecule reactivities, studying the variability of molecular conformations, detecting disease infection at an early stage, and devising nanostructures and molecularscale imaging probes for single molecule microscopy. Existing designs for optical supertips are based on the same principle as the green plant photosynthetic system [ 47 ]. A submicrometer antenna collects the photons by absorption and transfers the excitation energy to a single active center [ 2 ]. From there the energy is either ( i ) radiated as a photon or ( ii ) transferred to the sample in an energy transfer process ( Foërster^Dexter ) [ 48 ]. In either case the result is generally affected by the nearby sample molecule. ( i ) The radiated excitation may be affected, e.g., by intermolecular spin-orbit coupling ( Kasha effect, external heavy atom effect ) [ 49 ]. ( ii ) The energy transfer results in a £uorescence or phosphorescence typical of the sample molecule. In the latter case, only virtual photons are produced by
the supertip; this gives an excitation transfer tip (`exciton tip') and only sample luminescence is detected. Supertip development will be the key to scanning probe microscopy which relies on quantum optics mechanisms, such as Foërster energy transfer [ 48 ] or Kasha effect [ 49 ]. These interactions occur at the interface of the tip ( its active center ) and the sample ( which are quantum mechanically coupled ). For the highest resolution, this active center would consist of a single molecule, or molecular cluster, that does the imaging. This molecule is then the energy donor site for the Foërster energy transfer, or the spin-orbit interaction site for the Kasha effect. 10.1. Single dendrimer photon antenna
Kopelman et al. designed a supertip made of a single symmetric macromolecule by using newly developed dendrimer supermolecules [ 46 ]. The so-called starburst phenylacetylene dendrimers include the largest so far synthesized structurally ordered molecule ( D127, see Fig. 8 ). These fractal, tree-like supermolecules have spatially localized eigenfunctions, and, in particular, localized electronic states. Furthermore, in the so-called SYNDROME family of dendrimers [ 50 ], simple theory leads to selectively lower excitation energies at the central locus of the ordered macromolecule, with energies increasing towards the rim. This is borne out experimentally by the vibronic spectra of the entire family of molecules ( D-2 to D-127 ), with full internal consistency in the observed electronic energies ( red shifts ), vibrational quanta, Franck^Condon factors, overall transition moments and picosecond spectral diffusion. The architecture of this series of dendrimers is controlled by organic synthetic methods. For example, the overall shape of a `D-127' molecule is bowl-like with a molecular size î . It can thus act as both optical and force around 125 A active center. The large `rim' is bound to the tip by cumulative van der Waals bonding or covalent bonds. D-127 may be used in supertip preparation in two ways. The ¢rst is to make supertips in the range of î . In this case, D-127 is an energy transfer 100 A acceptor. It traps most of the energy quanta from the bulk of the optical probe. D-127 is the light emitting active center; The second way D-127 can be used for î resolusupertip preparation is to achieve about 10 A tion. To further demonstrate the `energy funnel' model a partial dendrimeric wedges with ( and without ) an excitation acceptor, a perylene derivative pendant, at the locus has been synthesized, shown in Fig. 8. As expected, the energy transfer from the large antenna
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Fig. 8. Dendrimer molecule ( top ) and Nano-star molecule ( bottom ).
(`tree canopy') to the small acceptor center is indeed dramatic. The presence of the antenna ( 39 phenyl groups ) increases the yield of the yellow perylenic emission by three orders of magnitude for a given excitation wavelength. This molecule is an ordered
supermolecule transducer of absorbed radiation (STAR ) [ 46 ] in analogy to the primary excitation energy collecting antenna of some natural photosynthetic systems. Overall, such a photonic subwavelength nano-lens may play an important role in devel-
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oping molecular excitonics, including luminescent optical nanoprobes, scanning exciton tunneling microscopy [ 2 ], and nanometer-scale ¢ber optic chemical and biochemical sensors. 10.2. Single dye molecule probe
Another effort in making smaller optical probes is the direct immobilization of one molecule onto a NFO probe. Advances in sensitive optical measurement schemes have led to the detection and the physical and chemical characterization of individual molecules. To exploit the advances in single molecule detection, it is thus feasible to prepare single molecule probes based on near-¢eld optical nanoprobes. Very recently, a single carbocyanine dye C18 ( DiI ) molecule was immobilized on a near-¢eld optical ¢ber probe, as shown in Fig. 9 [ 51 ]. The single DiI molecule probe's optical properties have been characterized. Photobleaching of the single DiI molecule on the probe occurs as a discrete and total extinction of its £uorescence. A simple optical method is used to differentiate single molecule probes from multiple molecule probes. Using the optical intensities of a probe before and after molecular immobilization, an optical intensity ratio can be calculated and used to identify single molecule probes with 99% accuracy. The single molecule light sources are just the ¢rst step in the efforts to developing a variety of single biomolecule probes ( such as single antigen probe, single DNA probe and single ligand-receptor probe ) for single molecule optical microscopy and single molecule interaction studies with extremely high spatial resolution and sensitivity.
Fig. 9. A schematic drawing of a single molecule probe. The single DiI molecule is immobilized onto a PMMA thin ¢lm on a NFO ¢ber probe. The DiI molecule emits a different wavelength of light when excited by a laser beam.
11. Summary and outlook NFO has generated considerable interest and has been successfully applied in microscopy, spectroscopy, single molecule localization, photo-nanofabrication and subwavelength optical biochemical probes and sensors. NSOM has led to a revolution in physical science by providing optical resolution at the level of tens of nanometers. Single molecules have been localized and individual molecules' photochemical and photophysical properties have been studied. Laser desorption and Raman studies have been carried out using NFO probes. Biological applications have also been attempted. In addition, a novel nanotechnology, photo-nanofabrication, has been developed for the preparation of nanometer biochemical probes. Despite NFO's success in the physical sciences, the constraints of imaging biological samples prevent NSOM from being applied to living cells ef¢ciently. The complexity and fragility of biological systems have hampered the application of NFO in biology. There are a few dif¢culties, such as scanning feedback, signal collection, biological sample thickness and cell fragility. So far NFO technology has been mainly developed for rigid solid surfaces. Most of the feedback mechanisms cannot be applied to soft biological membranes and cannot function well inside the aqueous milieu of cells. The dif¢culty associated with smaller biological sample has increased `exponentially' with the decrease in sample size. There are some recent efforts towards NFO's application in biology. It is expected that NFO's potential will be fully explored for biological applications. Many theoretical and technical problems have still to be solved ^ from understanding the contrast mechanism to the nanofabrication of molecular sized probes to the control of photo-bleaching ( a standard problem in £uorescence microscopy ). The mechanism of light^matter interaction may be different in the farand near-¢eld regimes, leading to different spectral selection rules and in particular to an enhanced cross-section of light absorption ( and thus £uorescence ) [ 2,52 ]. However, the future looks bright. Growth in NFO research in its early days has not been as spectacular as that of its SPM predecessors, STM and AFM. On the other hand, NFO is increasingly making its appearance in new areas of research as well as in established areas as its capabilities expand: the number of NFO microscopes, worldwide, has risen in ¢ve years from about 5 to about 300. The ability to image in-solution environments has been a major research focus in the last few years
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and is being demonstrated, and the combination of better resolution and biochemical identi¢cation capabilities seems ideally suited to a number of biomedical investigations. Biosamples, including living cells and even ion channels, could be imaged down to the molecular level and analyzed spectroscopically by either NFO probes or biochemical sensors. The intracellular molecular dynamics of organogenesis, metabolism, splitting and biochemical damage could be followed in vivo and in real time. At the same time DNA could be sequenced in situ or even repaired by the right probe at the right location. The key to all these will be the further development of NFO probes which will have high light throughput, excellent reproducibility and are easy to prepare. In addition, functionalized NFO probes will play a more and more important role in NFO application.
Acknowledgements I thank my group members for their important contribution. This work is supported by NSF Grant CHE9733650 and by a Beckman Young Investigator Award from the Arnold Beckman Foundation.
References [ 1 ] M. Paesler and P. Moyer, Near-¢eld Optics: Theory, Instrumentation and Applications, John Wiley and Sons, New York, 1996. [ 2 ] W. Tan and R. Kopelman, Nanoscopic imaging and sensing by near ¢eld optics, in X. Wang and B. Herman ( Editors ), Fluorescence Imaging Spectroscopy and Microscopy, John Wiley and Sons, New York, 1996, p. 407.. [ 3 ] E. Abbeé, Arch. Mikrosk. Anat. 9 ( 1873 ) 413. [ 4 ] A. Lewis, M. Isaacson, A. Muray, A. Harootunian, Ultramicroscopy 13 ( 1984 ) 227. [ 5 ] D.W. Pohl, W. Denk, M. Lanz, Appl. Phys. Lett. 44 ( 1984 ) 651. [ 6 ] E.H. Synge, Phil. Mag. 6 ( 1928 ) 356. [ 7 ] J.A. O'Keefe, J. Opt. Soc. Am. 46 ( 1956 ) 359. [ 8 ] E.A. Ash, N. Nichols, Nature 237 ( 1972 ) 510. [ 9 ] G. Binnig, H. Rohrer, Ch. Gerber, E. Weibel, Phys. Rev. Lett. 49 ( 1982 ) 57. [ 10 ] G. Binnig, C.F. Quate, Ch. Gerber, Phys. Rev. Lett. 56 ( 1986 ) 930. [ 11 ] R. Brunner, A. Bietsch, O. Hollricher, O. Marti, Rev. Sci. Instrum. 68 ( 1997 ) 1769. [ 12 ] K. Lieberman, S. Harush, A. Lewis, R. Kopelman, Science 247 ( 1990 ) 59. [ 13 ] E. Betzig, J.K. Trautman, T.D. Harris, J.S. Weiner, R.L. Kostelak, Science 251 ( 1991 ) 1468. [ 14 ] W. Tan, Z.-Y. Shi, S. Smith, D. Birnbaum, R. Kopelman, Science 258 ( 1992 ) 778.
[ 15 ] D. Zeisel, B. Dutoit, V. Deckert, T. Roth, R. Zenobi, Anal. Chem. 69 ( 1997 ) 749. [ 16 ] T. Matsumoto, M. Ohtsu, J. Lightwave Technol. 14 ( 1996 ) 2224. [ 17 ] E. Betzig, J.K. Trautman, Science 257 ( 1992 ) 189. [ 18 ] X. Xie and R.C. Dunn, Brookhaven Natl. Lab., BNL, BNL 61733, Proceedings of the Nineteenth DOE Solar Photochemistry Research Conference, 1995, p. 60. [ 19 ] E. Betzig, R.J. Chichester, F. Lanni, D.L. Taylor, Bioimaging 1 ( 1993 ) 129. [ 20 ] E. Monson, G. Merritt, S. Smith, J. Langmore, R. Kopelman, Ultramicroscopy 57 ( 1995 ) 257^262. [ 21 ] Th. Enderle, T. Ha, D. Ogletree, D. Chemla, C. Magowan, S. Weiss, Proc. Natl. Acad. Sci. USA 94 ( 1997 ) 520. [ 22 ] S. Iwabuchi, H. Muramatsu, N. Chiba, Y. Kinjo, Y. Murakami, T. Sakaguchi, K. Yokoyama, E. Tamiya, Nucleic Acids Res. 25 ( 1997 ) 1662. [ 23 ] S.K. Kook, R. Kopelman, J. Phys. Chem. 96 ( 1992 ) 10672. [ 24 ] D. Birnbaum, S.K. Kook, R. Kopelman, J. Phys. Chem. 97 ( 1993 ) 3091. [ 25 ] R.D. Grober, T.D. Harris, J.K. Trautman, E. Betzig, W. Wegscheider, L. Pfeiffer, K. West, Appl. Phys. Lett. 64 ( 1994 ) 1421. [ 26 ] J.K. Trautman, J.J. Macklin, L.E. Brus, E. Betzig, Nature 369 ( 1994 ) 40. [ 27 ] T.D. Harris, D. Gershoni, L. Pfeiffer, M. Nirmal, J.K. Trautman, J.J. Macklin, Semiconductor Sci. Technol. 11 ( 1996 ) 1569. [ 28 ] E. Betzig, R.J. Chichester, Science 262 ( 1993 ) 1422. [ 29 ] W.P. Ambrose, P.M. Goodwin, J.C. Martin, R.A. Keller, Science 265 ( 1994 ) 364. [ 30 ] X. Xie, R.C. Dunn, Science 265 ( 1994 ) 361. [ 31 ] P.G. Haydon, S.P. Marchese-Ragona, T.A. Basarsky, M. Szulczewski, M. McCloskey, J. Microsc. 182 ( 1996 ) 208. [ 32 ] S. Nie, S.R. Emory, Science 275 ( 1997 ) 1102. [ 33 ] S. Emory, S. Nie, Anal. Chem. 69 ( 1997 ) 2631. [ 34 ] W. Tan, L. Bang, J. Cordek, X. Liu, SPIE-Int. Soc. Opt. Eng. 3273 ( 1998 ) 45. [ 35 ] W. Tan, Z.-Y. Shi, R. Kopelman, Anal. Chem. 64 ( 1992 ) 2985. [ 36 ] W. Tan, Z.-Y. Shi, R. Kopelman, Sensors Actuators B Chem. 28 ( 1995 ) 157. [ 37 ] M. Shortreed, R. Kopelman, M. Kuhn, B. Hoyland, Anal. Chem. 68 ( 1996 ) 1414. [ 38 ] M.R. Shortreed, E. Bakker, R. Kopelman, Anal. Chem. 68 ( 1996 ) 2656. [ 39 ] M. Shortreed, E. Monson, R. Kopelman, Anal. Chem. 68 ( 1996 ) 4015. [ 40 ] Z. Rosenzweig, R. Kopelman, Anal. Chem. 67 ( 1995 ) 2650. [ 41 ] Z. Rosenzweig, R. Kopelman, Anal. Chem. 68 ( 1996 ) 1408. [ 42 ] D. Uttamchandani, S. McCulloch, Adv. Drug Deliv. Rev. 21 ( 1996 ) 239. [ 43 ] S. McCulloch, D. Uttamchandani, IEE Proc. Optoelectron. 144 ( 1997 ) 162. [ 44 ] A. Song, S. Parus, and R. Kopelman ( 1997 ) Anal, Chem. 69 ( 1997 ) ( 1997 ) 863. [ 45 ] J. Cordek, X. Wang and W. Tan ( 1998 ) Anal. Chem. submitted.
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[ 46 ] R. Kopelman, M. Shortreed, Z. Shi, W. Tan, Z. Xu, J. Moore, A. Bar-Haim, J. Klafter, Phys. Rev. Lett. 78 ( 1997 ) 1239. [ 47 ] E.B. Fauman, R. Kopelman, Mol. Cell. Biophysics 6 ( 1989 ) 47. [ 48 ] M. Pope and E. Swenberg, Electronic Processes in Organic Crystals, Oxford University Press, New York, 1982.
[ 49 ] M. Kasha, J. Chem. Phys. 20 ( 1952 ) 71. [ 50 ] Z. Xu, Z. Shi, W. Tan, R. Kopelman, J. Moore, Polymer Preprints 33 ( 1993 ) 130. [ 51 ] W. Tan and X. Wang, Development of a single molecule optical probe, Thin Solid Film ( 1998 ) in press. [ 52 ] R. Kopelman, W. Tan, Science 262 ( 1993 ) 1382.
Elemental analysis by diode laser spectroscopy A. Zybin
Institute of Spectrochemistry and Applied Spectroscopy ( ISAS ), Bunsen-Kirchhoff-Strasse 11, D-44139 Dortmund, Germany
C. Schnuë rer-Patschan
LaserSpec Analytik GmbH, Frankfurter Ring 193a, D-80807 Muë nchen, Germany
M.A. Bolshov, K. Niemax*
Institute of Spectrochemistry and Applied Spectroscopy ( ISAS ), Bunsen-Kirchhoff-Strasse 11, D-44139 Dortmund, Germany After about 25 years of development of laser analytical spectroscopy, laser spectrochemical instruments at present work ef¢ciently only in research laboratories. There are practically no commercial laser spectroscopic instruments for routine elemental analysis. The only laser-based instruments are Raman spectrometers with stable cw laser sources for molecular analysis and pulsed lasers with ¢xed wavelengths which are used for laser ablation ( LA ) ^ laser sampling of solid materials. The main dif¢culties of laser spectroscopic techniques are: relatively high cost, insuf¢cient reliability, and the necessity of quali¢ed personal. Semiconductor laser diodes are mass produced for compact disc players, laser printers, optical data storage systems and telecommunication equipment. A number of these laser diodes have excellent spectroscopic properties, which make them attractive sources for spectrochemical analysis. Presently, laser analytical instruments *Corresponding author.
based on laser diodes have the highest potential for transfer from research laboratories to routine practice. Recently, the excellent analytical capabilities of diode laser spectrometry in the detection of low concentrations of analytes have been well documented. Recent advances in diode lasers and analytical trends are discussed below. z1998 Elsevier Science B.V. All rights reserved. Keywords: Diode laser spectroscopy; Laser ablation; Semiconductor laser diodes in spectroscopy; Micormachining
1. Atomic absorption spectroscopy with diode lasers For analytical applications, diode lasers ( DLs ) are most widely used as resonance light sources for atomic absorption spectrometry ( AAS ) ^ ( DLAAS ). Generally, tunable lasers like dye lasers or optical parametric oscillators (OPO ) have a reputation as powerful, but unstable light sources, and are therefore not well suited for atomic absorption measurements where the stabil-
0165-9936/98/$ ^ see front matter PII: S 0 1 6 5 - 9 9 3 6 ( 9 8 ) 0 0 0 6 3 - 6
ß 1998 Elsevier Science B.V. All rights reserved.
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