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tructures within aircraft jet tur bines, nuclear reactors, and the human body are difficult to ob serve and analyze in situ. These hard-toreach locations and hostile environments have motivated the development of a wide variety of industrial and medical en doscopes, optical aids for examining inter nal cavities that usually take the form of flexible or rigid tubes. In most cases, the heart of an endoscope is an imaging fiber made up of thousands of individual optical fibers melted and drawn together in a co herent manner to carry and maintain an image from one end to the other. Although one picture can convey a thousand words, the visual information ac quired from these endoscopes is only one part of a total analysis—chemical and physical characterizations are needed as well. Fiber-optic sensors can be used to monitor a wide range of physical and
Paul Pantano David R. Walt Tuffs University 0003-2700/95/0367-481 A/$09.00/0 © 1995 American Chemical Society
we will look at remote viewing in Optical imaging Report, struments, review progress in the develop of imaging-fiber chemical sensors, sensors can providement and examine possible analytical uses in the ability to combine visual and images and chemicalwhich chemical information can be advanta information from geous. Optical fiber configurations hard-to-reach An opticalfiberis a filament made of lightguiding dielectric material that works by locations and hostiletotal internal reflection. It consists of an in core of high refractive-index material environments ner and an outer region of low refractive-index
chemical parameters, such as tempera ture, pressure, viscosity, humidity, and chemical concentrations. Remarkably, very few endoscopes can provide visual as well as chemical or physical data. Recently, coherent imagingfibershave been used to constructfiber-opticchemi cal sensors that can acquire both visual and chemical information and transmit it through the same imagingfiber.In this
material called the clad. This configura tion enables light rays entering the core within a certain critical angle to be con fined by total internal reflection at the core-clad interface. An optical fiber's in put and output behaviors are character ized by its numerical aperture, which de pends on the difference in the refractive in dices of the core and clad. The aperture defines the angle of the acceptance cone for light propagation along the fiber. Opticalfiberscan be divided into vari ous categories depending on the refracAnalytical Chemistry, August 1, 1995 481 A
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tive index profile of the core and the num ber of modes the fiber can support. Sin gle strands of transparent materials such as glass, plastic, or fused silica can propa gate light over long distances with mini mal attenuation. Because light undergoes thousands of reflections per meter as it proceeds down the length of the fiber, the position at which an individual ray exits the fiber will not correspond to the posi tion at which it entered the fiber. As a re sult, a single optical fiber cannot be used to transmit an image (1-4). Individual optical fibers can be fused into a bundle. Both rigid and flexible fiber optic bundles are available in many lengths, diameters, geometries, and multifurcated combinations. Bundles can be housed in special jackets for use in unusual environ ments, and their relatively low cost makes them disposable. Fiber- optic bundles are commonly used to provide high-intensity, uniform illumination of UV, visible, or IR radiation for equipment used in diagnos tic and surgical procedures such as head light systems for surgeons and illuminators for remote viewing instruments. In addi tion, laser delivery systems increasingly use fiber-optic bundles to carry high-output light energy to remote targets, for example to cure dental polymers and fixatives and for medical operative procedures (5). Most light-delivery bundles contain un aligned optical fibers and are used only to transmit energy. In contrast, imaging fi bers are bundles in which the position of each optical fiber is the same at the input and output ends. In these bundles, each optical fiber acts like a pixel, and the size of the pixel determines the resolution of the transmitted image. A modern highresolution imaging fiber consists of 3000100,000 individually clad optical fibers, each 3-10 pm in diameter (Figure 1). (For information on fabricating optical fibers, see References 6-8.) One of the first dem onstrations using a bundle of unclad fi bers to transmit an image took place ~ 40 years ago at Imperial College in London (9), and much of the pioneering work with clad imaging fibers is attributed to Will Hicks at American Micro-Optical. Remote viewing instruments What makes fiber optics ideal for endos copy are the high numerical apertures that allow elevated levels of illumination from 482 A
Individually clad optical fibers
Silica jacket
Imaging fiber
Figure 1 . A coherent fiber-optic bundle. (Left) An imaging fiber is composed of thousands of micrometer-sized optical fibers fused together in a single flexible-fiber format. (Right) Image of a 350-pm-diameter imaging fiber with ~ 6000 optical fibers, each with a diameter of 3-4 pm, taken by a CCD camera. Far-field viewing of 6-point text ("TUFTS") is made possible by a distal GRIN lens.
an external light source as well as the flex GRIN lenses have different fractional pitches so that the distance for far-field ibility of the bundles. The basic compo viewing can be preselected (e.g., 5-100 nents of afiber-opticendoscope are a co herent fiber for image transmission, a non mm from the face of the lens). In addition coherentfiber-opticbundle for illumination, to forward viewing, miniaturized mir rors and prisms can be coupled to the a proximal objective, a proximal eyepiece GRIN lens to allow observation from other and/or video camera, and a distal gradient perspectives. index (GRIN) lens (2). Endoscopes are classified by the opti GRIN lenses are short, small-diameter cal apparatus used to provide the image glass rods that can be mounted onto and and by the degree of flexibility. The most optically aligned with the imaging fiber. common rigid endoscopes are based on The index of refraction within a GRIN the Hopkins rod-lens system (10). These lens is parabolic in that the light follows a sinusoidal path. The pitch (i.e., the • telescopic endoscopes do not use an imag ing fiber; instead, light is transmitted by length of rod required for one sinusoidal cycle) determines the imaging characteris glass columns and refracted through inter vening air lenses. This system transmits tics of the lens so that a real image can be images with the utmost resolution, clarity, formed at the lens surface. Different
Analytical Chemistry, August 1, 1995
and brightness required for the most tax ing of inspection needs. Although such en doscopes can be used to view regions through cavities as small as 1 mm in diam eter, their lack of flexibility limits their usefulness. Nonetheless, there are hundreds of ap plications in which rigid endoscopes are used, such as fuel lines, air ducts, sewer pipes, oil and gas lines, nuclear reactors, gun barrels, power station rotors, and jet and piston engines. As evidenced by this list, one of the major advantages of endo scopes is that they permit remote viewing of hostile environments up to 100 m from the observer. They can be used under high and low temperatures and pres sures, intense electric and magnetic fields, and in the presence of hazardous chemi cals and ionizing radiation. Flexible endoscopes use the same dis tal lens and fiber-optic bundle illumination systems asrigidtelescopic endoscopes. However, in flexible endoscopes image transmission is accomplished by a coher ent imaging fiber or a distal charge-cou pled device (CCD) (10). The resolution of flexiblefiber-opticand electronic endo scopes depends on the size of the indi vidual optical fibers or semiconductor pix els, respectively. In a modern flexiblefiber-opticendo scope, up to 40,000 bundled fibers convey the image. As long as the fibers in the bundle are collimated, an image can be transmitted along the bundle regardless of twists and turns. Until recently, all flexi ble endoscopes usedfiber-opticbundles for both illumination and image transmis sion. However, flexible endoscopes that use a CCD for image transmission have now been introduced. These electronic en doscopes have a miniature, high-resolu tion, color video camera and lens located at their distal tips. The choice between using a flexible fi ber-optic orflexibleelectronic endoscope is based on its outer diameter, typically 0.7-8 mm for afiber-opticendoscope and 6-16 mm for an electronic endoscope. In addition, the temperature of the sample's environment is a factor when using an electronic endoscope, because it affects the dark current of the distally sited CCD. In both cases, the ability to support a wide variety of surgical tools and manipu lative instruments has enabled both
rigid and flexible endoscopes to be used in hundreds of different medical proce dures. Imaging-fiber chemical sensors
Most fluorescence-based fiber-optic chemical sensors use an optical fiber as a light conduit to detect an analyte through a change in optical properties (e.g., inten sity, wavelength, or lifetime). In one con figuration, a light source is coupled to the proximal end of afiberand excitation en ergy is propagated to the fiber's distal tip, where a fluorescent indicator is immobi lized. The indicator-analyte interaction modulates the fluorescence intensity, and a portion of the isotropically emitted light returning through the samefiberis directed to a suitable detector after filter ing out any residual excitation light (11).
Λ modified fiber can detect and spatially resolve localized changes in the concentration of an analyte. Traditionally,fiber-opticchemical sen sors have been fabricated using either a single fiber or a noncoherentfiberbundle. Bundles have the advantage of high lightgathering power and, as a result, the thick ness of the sensing layer can be mini mized to improve the temporal response of the sensor (3). Recently, we have used coherent imaging fibers to make fiber optic chemical sensors. In one approach, imagingfibersare used to fabricate array sensors that can concurrently view a sam ple and detect a single analyte. In a sec ond approach, sensors are made that have spatially discrete sensing sites for multianalyte determinations. Combined imaging and chemi cal sensing. Despite many innovations and developments infiber-opticchemical sensors,fibershave not been used to view a sample and concurrently detect an ana
lyte. Although chemical sensors using a single opticalfiberor a non-coherent fiber optic bundle have been applied to a wide variety of analytical determinations (12), they cannot be used for imaging. Simi larly, imaging fibers and endoscopes in general are used only for their originally intended purpose, image transmission. Combined imaging and chemical sensing requires that an analyte-sensitive indicator be immobilized in a thin layer on the distal surface of the fiber in such a way that it does not compromise the fi ber's imaging capabilities. Polymeric methods of immobilization are especially suitable because a sufficiently thin (i.e., < 5 pm), uniform, planar optically trans parent sensing layer can be spin coated di rectly onto the distal surface of an imag ingfiber,creating thousands of microsensors capable of simultaneously measuring chemical concentrations over tens of thousands of square micrometers. Be cause the image-carrying capabilities of the fiber are preserved, the operator can position the sensor and correlate chemical measurements with visual information. A modified epifluorescence micro scope has been used for both fluores cence measurements and imaging. The microscope is capable of making continu ous ratiometric measurements using a CCD camera and computer-controlled fil ter wheels and shutters. By combining the distinct optical pathways of the imaging fiber with the spatial discrimination of a CCD, both visual and fluorescence mea surements can be obtained with 4-pm spatial resolution (13) limited only by the size of the individualfiberscomprising the bundle. Whenfluorescencemeasurements are taken, images are captured by the CCD camera with the filter wheels positioned at the dye's excitation and emission max ima. During viewing, the sample is illumi nated by an external source, and the im ages are captured by the same CCD cam era with the excitation shutter closed and the emission filter wheel moved to a neutral densityfilter.Alternatively, a beam splitter can be used with a second CCD camera so that simultaneous viewing and chemical sensing are possible. Thefirstdemonstration of this tech nique used a 350-pm-diameter imaging fi ber coated with a pH-sensitive or an acetyl-
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choline (ACh)-sensitive polymer layer. The pH-sensitive polymer layer was immo bilized by spin coating an N-fluoresceinylacrylamide/hydroxyethylmethacrylate polymer onto the distal face of an imaging fiber. The ACh-sensitive layer was cre ated by co-immobilizing acetylcholinester ase (AChE) in a water-soluble functionalized prepolymer known as poly(acrylamide-co-yV-acryloxysuccinimide) (PAN). The AChE-derivatized PAN was spin coated onto an imaging fiber and allowed to react with fluorescein isothiocyanate (FITC) to incorporate the pH-sensitive dye into the polymer layer. AChE cata lyzes the hydrolysis of ACh to choline and acetic acid; the dissociated protons from the enzyme-generated acetic acid react with the immobilized FITC to provide a pH-sensitive fluorescent signal propor tional to the ACh concentration. These acetylcholine biosensors have a detection limit on the order of 35 μΜ and a re sponse time < 1 s (13). The ability of a modified imaging fiber to detect and spatially resolve localized changes in the concentration of an analyte
is shown in the images in Figure 2. The ex perimental setup shown in Figure 2a con sists of an AChE-modified imaging fiber, a picospritzer, and a pulled-glass capillary tube filled with 1.0 mM ACh. The tip of the tube was submerged in buffer and posi tioned normal to the distal surface of a ver tically held AChE-modified imaging fi ber. Figure 2b is an image of a glass pi pette illuminated with white light, viewed through the AChE-modified imaging fiber and positioned within the imaging fiber's field of view. Figures 2c-g show a sequence of fluo rescence images from the AChE-modified imaging fiber before and after a 200-ms pulse of ACh was delivered to the fiber. The ACh sensitivity of the AChE/FITC/ PAN layer is evidenced by the dark circu lar area that forms (as the FITC fluores cence is quenched by the enzyme-gener ated acid) only in the region of the fiber where ACh was locally injected. This demonstrates that successive two-dimen sional profiles of a heterogeneous sample, such as a single cell, can be acquired by a single modified-imaging fiber sensor.
Picospritzer (a)
Τ
ACh solution x-y-z micropositioner
.AChE/FITC/PAN imaging fiber
Figure 2. Detecting and resolving localized changes in analyte concentration. (a) Pulled-glass capillary tube containing ACh, positioned normal to the distal surface of an AChE-modified imaging fiber submerged in phosphate buffer at pH 7.0. (b) Pulled-glass capillary tube image acquired with a CCD camera viewed through a 350-pm-diameter AChE-modified imaging fiber, (c) Fluorescence image at 530 nm taken with 490-nm excitation from an AChE-modified imaging fiber before and (d-g) after a 200-ms pulse of 1.0 mM ACh was delivered onto the distal tip of the modified imaging fiber. All measurements were acquired with a 50-ms CCD acquisition time at 200-ms intervals. Red indicates high fluorescence intensities. 484 A
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In this configuration, the thin analytesensitive polymer is coated directly on the distal face of the imaging fiber and ob jects are in focus only when they contact the face of the fiber. As described above, imaging fibers can also be fitted with a distally sited GRIN lens. In this configura tion, the thin polymeric sensing layer can be coated directly onto the GRIN lens sur face to permit far-field viewing of the sam ple and its environment, with the out-offocus fluorescence signal collected and correlated to analyte concentration (14). With this simple polymer-dye-enzyme chemistry, any commercially available en doscope can be modified to have both chemical sensing and imaging capabilities over small distances (without a GRIN lens) or over longer distances (with a dis tal GRIN lens).
Multianalyte optical
sensors.
The ideal chemical sensor measures the in situ concentration of analyte continu ously, reversibly, inexpensively, and rap idly. In most situations, it is important to measure several parameters simulta neously. One of the benefits of using an imaging fiber to make a multianalyte fiber optic chemical sensor is that the sepa rate optical pathways of the imaging fiber can be used to carry signals simulta neously from multiple indicators immobi lized at different locations on the fiber tip. We have developed a quick photoinitiated polymerization reaction whereby dis crete sensing regions can be immobi lized in precise locations on the distal face of a single optical imaging fiber (15,16). Photodeposition of an indicator-polymer matrix is performed using a UV source (e.g., a mercury-xenon lamp). Light with the appropriate wavelength is directed through a pinhole and focused onto the proximal face of an imaging fiber using a microscope objective. The light travels through the imaging fiber to the distal face, which is submerged in a polymeriza tion solution (i.e., monomer, initiator, and indicator). A small area (radius of 6-25 pm) is illuminated for a predetermined time during which polymer growth occurs only at the illuminated region on the dis tal face (Figure 3, top). After illumination, the residual monomer is removed by rinsing and the initiation light is focused onto a different region of the imaging fi ber. The process may be repeated, or a
different dye-polymer combination can be used. Many distinct sensing regions can be photodeposited onto a single imaging fi ber, with the size of the pinhole and the ob jective's magnification controlling the size of each sensing region. Using a CCD, the fluorescence from each of the differ ent sensing regions immobilized on the imaging fiber can be spatially resolved (Figure 3, bottom). The creation of spa tially discrete sensing sites on a single optical sensor solves many of the prob lems associated with designing multianalyte optical sensors, such as spectral over lap of multiple indicators and the need to use individual optical fibers for each analyte. In addition, when these discrete sensing regions are immobilized around the periphery of the imaging fiber's distal face, the bare central region can be used to view the sample and its environment as described above. Simultaneous measurements are espe cially important in a number of clinical, bi ological, and environmental applications, especially when the dynamics of different analytes are closely interrelated (e.g., pH, pC0 2 , and p0 2 ). A multianalyte imaging fiber sensor that allows simultaneous pH, C0 2 , and 0 2 sensing has recently been fabricated in our laboratory (17). The pHsensitive polymer matrix is created by covalentiy immobilizing a pH indicator (acryloyl fluorescein) in a polymer hydrogel. The fluorescence intensity of the pH indicator is enhanced by deprotonation. The C0 2 measurement is made ac cording to the principles of a Severinghaus electrode, in which a pH sensor (also acryloyl fluorescein in a polymer hydrogel) is used to detect H 2 C0 3 production from the reaction of C0 2 and H 2 0 after the C0 2 traverses a gas-permeable mem brane (18). A pH-sensitive polymer ma trix is overcoated with a gas-permeable photopolymerizable siloxane membrane that blocks the transport of hydrogen ions across the membrane but allows C0 2 to cross and react with water to form H 2 C0 3 . This reaction increases the acidity in the microenvironment of the polymer and thus decreases the fluorescence inten sity of the pH indicator proportional to the C0 2 concentration. Finally, the 02-sensitive polymer ma trix is deposited by entrapping a transition
>4|() Focused initiation light
Imaging fiber
pH 8.0
Activated area
Capillary tube
Functionalized distal face of fiber
Monomer solution
pH 7.0
Figure 3. Immobilizing sensing regions on an optical fiber. (Top) Setup of the photopolymerization procedure used to fabricate multicomponent fiber-optic chemical sensors. (Bottom) Fluorescence images of 13 pH-sensitive matrices immobilized on the distal face of a 500^m-diameter imaging fiber in pH 8.0 (left) and pH 7.0 phosphate buffer (right). All polymer-dye matrices appear as distinct fluorescent regions and respond to pH changes in a similar fashion.
metal complex, such as Ru(Ph2phen)|+, in a photopolymerizable siloxane mem brane (19). This transduction mechanism is based upon 0 2 collisional quenching of the ruthenium complex fluorescence. When the distinct optical pathways of the modified imaging fiber are combined with the spatial discrimination of a CCD, the fluorescence intensity of each region can be monitored simultaneously, as shown in Figure 4. C0 2 , 0 2 , and pH sensors can be con verted into biosensors by coupling indica tor dyes with enzyme reactions in which transducing species such as protons, C0 2 , or oxygen are liberated or consumed (20). However, the transducing species must be measured independently or kept constant. To account for nonenzymatic fluctuations of pH, C0 2 , and 0 2 in the sample matrix, these biosensors typically use a second sensor as a reference. The site-selective photopolymerization method allows separate enzyme-containing matri ces and pH, C0 2 , and 0 2 sensitive matri ces to be immobilized in proximity to each other on the distal face of a single imag ing fiber for simultaneous and indepen dent measurements of each entity.
We recently described the fabrication and performance of a biosensor that per mits the simultaneous measurement of penicillin and pH (21). This biosensor is based on the enzyme penicillinase and was fabricated by site-selective photodeposition of penicillin- and pH-sensitive poly mer matrices on a 350-pm-diameter imag ing fiber. The penicillin-sensitive matri ces were fabricated by entrapping the lyophilized enzyme as micrometer-sized particles in a polymer hydrogel contain ing the covalentiy bound pH indicator ac ryloyl fluorescein. Dissociated protons from enzyme-generated penicilloic acid decrease the fluorescence intensity of the pH indicator in proportion to the penicil lin concentration. This imaging fiber sensor detects peni cillin in the range 0.25-10.0 mM (in the pH range 6.2-7.5) with a penicillin detection limit of 100 μΜ. The ability to monitor the pH of the medium in conjunction with the penicillin concentration should allow this sensor to monitor penicillin produc tion in complex sample matrices such as fermentation broths, where dramatic pH changes can occur during the fermenta tion process (22). Analytical Chemistry, August 1, 1995 485 A
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Figure 4. Simultaneous fluorescence measurements of 0 2 , CO a , and pH using a 350-Mm-diameter multianalyte imaging fiber sensor. All measurements were made in 0.1 M phosphate buffers (pH 5.0 or 7.2) that were air saturated (0.03% C02/21% 0 2 ), oxygen saturated (100% 0 2 ), or bubbled with a 15% CCy85% N2 gas mixture. The sensor was placed in the specified buffer for 60 s and fluorescence images were taken every 10 s.
The same differential measurement strategy has been applied to oxygendependent biosensors. Members of our group have fabricated a fiber-optic sensor for the continuous and simultaneous deter mination of both glucose and oxygen with separate glucose and oxygen sensing sites photoimmobilized in distinct positions on the distal face of a 35(Him-diameter imag ing fiber. The oxygen-sensing site is com posed of an oxygen-sensitive ruthenium complex entrapped in a hydrophobic gaspermeable copolymer. The glucose-sensing site is made by coating an oxygen-sensing site with a second polymeric layer contain ing glucose oxidase. Glucose is quantitated via the enzymatic consumption of oxygen with a response time of 30 s. This fiber optic biosensor should be ideal for in situ glucose measurements where oxygen ten sions fluctuate.
entifically important experiments and clini cal measurements. Preliminary work in our laboratory to image biological sam ples with planar, polymer-modified imag ing fibers has included sea urchin eggs (50-100 μπι diameter) (14) and mouse fi broblast cells (10-20 pm diameter) (13). Potential applications include measuring cell surface chemistries to correlate posi tional and morphological information with chemical distributions (e.g., monitoring in situ neurochemical dynamics). Modified imaging fibers may allow chemical concentrations at surfaces to be measured on scales smaller than a single cell and ultimately may provide informa tion such as cellular surface concentration gradients. In some cases, extracellular measurements may provide an alternative to intracellular measurements when changes in intracellular chemical concen trations appear as complementary extra cellular changes via membrane transport Applications Combining viewing and chemical sensing pumps. The physical flexibility of imaging fibers may also allow them to be used for with imaging fiber chemical sensors of diagnosis in which cell-by-cell histological fers a significant advance in studying fun damental properties on a cellular level and examination can be combined with sur face chemical determinations. should open the door for a variety of sci 486 A
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Other combinations of imaging and spectroscopic determinations could be used for diagnosis. Feld showed that near-IR Raman spectra can be used to dif ferentiate diseased and normal regions in a human aorta (23) and McCreery used a fiber-optic probe to obtain near-IR Raman spectra from breast tissue in a clinical set ting (24). The spectral or chemical sens ing and morphological examination of cel lular-scale features could be acquired concurrently via optical imaging fibers with the ultimate goal of creating a system for rapid, minimally invasive diagnostic techniques, eliminating the need to take a specimen from the patient by biopsy. The union of fluorescence imaging and endoscopy also shows great promise for the diagnosis of medical conditions. The localization and early identification of dis eased tissue can be accomplished either by tissue autofluorescence or by the characteristic fluorescence signature ob tained when an administered porphyrin derivative is selectively retained by ma lignant tissue. Such information can be used to direct laser irradiation or surgical instruments to only those tissue regions requiring treatment. Lam has exploited the difference in autofluorescence between diseased and normal tissue to develop an imaging sys tem based on a CCD camera attached to the eyepiece of a conventional flexible fi ber-optic endoscope (25). The endo scope's noncoherent fiber bundle deliv ers 442 nm excitation, and the resulting tis sue autofluorescence is collected through the coherent imaging fiber. A computercontrolled filter wheel permits the sequen tial acquisition of green (480-520 nm) and red (> 630 nm) fluorescence images at 115-ms intervals. Preliminary results have shown significant improvement over conventional white-light endoscopy for detecting dysplasia and carcinoma in lung lesions. These conditions are difficult to detect in situ because the lesions are only a few layers thick and a few millimeters wide. Recently, a multispectral fluorescence imaging system was reported by Andersson-Engels in which fluorescence images are acquired simultaneously from three different spectral regions and displayed on a CCD camera (26). Fluorescence im ages from tumors on the hind legs and in
the brains of rats injected with a porphyrin derivative were clearly improved by multicolor processing. Work is in progress to adapt the system for transmitting fluorescence images through an imaging fiber endoscope. Improvements in the specificity of autofluorescence measurements have been made by Feld, who developed a diagnostic algorithm based on excitationemission matrices (27). Using an in vitro model, fluorescence emission spectra from 370 nm excitation differentiates between normal and diseased colon tissues in 96% of 26 cases tested. Similar results were also obtained when the emission spectra were collected in real time from patients by using an optical fiber introduced through an endoscopic channel (28).
Combining viewing and chemical sensing offers a significant advance. In all of these applications, the advantages of delivering and collecting light from the endoscope's coherent imaging fiber are numerous and significant. Reducing the number of operative channels means that endoscopes will have a smaller diameter, thereby decreasing the size of the incision or puncture needed and/or increasing the number of the internal regions that can be accessed by endoscopic examination. We gratefully acknowledge the National Institutes of Health forfinancialsupport, and our coworkers, especially Brian G. Healey, Lin Li, and Suneet Chadha, for their scientific contributions.
I.; Wilson, G. S., Eds.; Oxford University Press: Oxford, 1987; pp. 638-54. (4) Seitz, W. R. CRC Crit. Rev. Anal. Chem. 1988,19,135-73. (5) Andersson-Engels, S.; Johansson, J.; Svanberg, S.; Svanberg, K. Anal. Chem. 1989, 61, 1367 A-1373 A and 1990, 62,19 A27 A. (6) Tsumanuma,T., etal. SPIEProc. 1988, 906, 92-96. (7) Mogi, M.; Yoshimura, K. SPIE Proc. 1989,1067,172-81. (8) Chigusa, Y; Fujiwara, K.; Hattori, Y; Matsuda, Y Optoelectronics 1986,1,203-16. (9) Hopkins, H. H.; Kapany, N. S. Nature 1954,173,39-41. (10) Melzer, A; Buess, G.; Cuschieri, A In Operative Manual of Endoscopic Surgery; Cuschieri, A; Buess, G.; Perissat, J., Eds.; Springer-Verlag: Berlin, 1992; pp. 14-36. (11) Arnold, M. A. Anal Chem. 1992, 64, 1015A-25A (12) Biosensors with Fiberoptics; Wise, D. L.; Wingard, L. B., Jr., Ed.; Humana: Clifton, NJ, 1991. (13) Walt, D. R.; Bronk, K. S.; Pantano, P.; Michael, K. L. SPIE Proc. 1995,2388, 554-57. (14) Walt, D. R.; Bronk, K. S. U.S. Patent 5 298 741,1994. (15) Barnard, S. M.; Walt, D. R Nature 1991, 353,338-40. (16) Bronk, K. S.; Walt, D. R. Anal. Chem. 1994, 66, 3519-20. (17) Healey, B. G.; Walt, D. R. SPIE Proc. 1995,2388, 519-23. (18) Severinghaus, J. W.; Bradley, A F.J. Appl. Physiol. 1958,13,515-20. (19) Bacon, J. R.; Demas, J. N. Anal. Chem. 1987,59,2780-85. (20) Arnold, M. A; Meyerhoff, M. E. CRC Crit. Rev. Anal. Chem. 1988,20,149-96. (21) Healey, B. G.; Walt, D. R. SPIE Proc. 1995,2388, 568-73. (22) Agayn, V. I.; Walt, D. R. Bio/Technology 1993,11, 726-29. (23) Rava, R P.; Baraga, J. J.; Feld, M. S. Spectrochim. Acta 1991, 47A, 509-12. (24) Frank, C. J.; Redd, D.C.B.; Gansler,T. S.; McCreery, R. L.Anal. Chem. 1994, 66, 319-26. (25) Lam, S., etal./. Thorac. Cardiovasc. Surg. 1993,105,1035-40. (26) Andersson-Engels, S.; Johansson, J.; Svanberg, S.Appl. Opt. 1994,33,8022-29. (27) Feld, M. S., et al. Photochem. Photobiol. 1991,53,777-86. (28) Feld, M. S., et al. Gastrointest. Endosc. 1990,36,105-11.
Paul Pantano is a postdoctoral scientist whose research interests include development of enzyme-modified microelectrodes andfiber-opticchemical sensors for biochemical analyses. David R. Walt is profesReferences sor and chair of the Department of Chem(1) Wolfbeis, 0. S. In Molecular Luminescence Spectroscopy: Methods and Applications;istry. His research interests include optical sensors, biomaterials, neurochemistry, and Schulman, S. G., Ed.; John Wiley and Sons: New York, 1988; pp. 129-281. microfabrication techniques. Address corre(2) Hopkins, H. H. In Endoscopy; Berci, G., spondence to Walt at Tufts University, Max Ed.; Appleton-Century-Crofts: New York, Tishler Laboratory for Organic Chemistry, 1976; pp. 27-63. Department of Chemistry, 62 Talbot Ave(3) Schultz, J. S. In Biosensors: Fundamentals and Applications; Turner, A.P.F.; Karube, nue, Medford, MA 02155.
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