AU3524900A

AU3524900A - An image guide and method for sub-micron imaging and picosecond timing

Info

Publication number: AU3524900A
Application number: AU35249/00A
Authority: AU
Inventors: Jacob R. Tymianski; James K. Walker
Original assignee: University of Florida
Current assignee: University of Florida
Priority date: 1999-03-09
Filing date: 2000-03-09
Publication date: 2000-09-28
Also published as: CA2358558A1; EP1159602A1; WO2000054033A1

Description

WO 00/54033 PCT/US00/06344 DESCRIPTION AN IMAGE GUIDE AND METHOD FOR SUB-MICRON IMAGING AND PICOSECOND TIMING 5 Background of the Invention Image guides are bundles of optical fibers which convey optical images. Because each optical fiber of an image guide transmits only a minute discrete portion of the image, it is of course necessary for each end of the image guide to be coherently related to the other end such 10 that the image exiting the image guide is identical to that which enters the multiplicity of fibers. Image guides are used in a variety of industrial and medical imaging scopes. For example, endoscopes utilize image guides to convey images of human and/or animal vessels and internal cavities. Additionally, image guides are also used in industrial borescopes used for many types of industrial imaging. 15 Image quality is critical to the performance of image guides. Specifically, resolution, brightness, and contrast sensitivity are a few important performance characteristics which affect image quality. Resolution can be expressed as the measure of the image guide's ability to separate images of two neighboring object points. Improved image resolution can be obtained by having a larger number of optical fibers, in the bundle, per unit area. The brightness of an 20 image guide is a measure of the ratio of the amount of light exiting the output end of the image guide to the amount of light incident to the input end of the image guide. The brightness of an image guide can be improved by, for example, increasing the portion of the image guide end available for light transmission, increasing the numerical aperture (NA), and/or decreasing the transmission loss of the image guide. The contrast sensitivity is a measure of the ratio of the 25 amount of light, comprising the image, exiting the output end of the image guide to the total amount light exiting the output end of the image guide. The light exiting the output end of the image guide, and not contributing to the image, reduces the contrast sensitivity. Depending upon the intended use of the image guide, other characteristics such as flexibility may also be important. For example, it is often advantageous for image guides to 30 have great flexibility to reach otherwise inaccessible locations such as coronary vessels. In other applications, such as laparoscopy, a more rigid image guide is preferred. The subject invention concerns, in one aspect, improved image guides which result in endoscopes and borescopes with highly advantageous characteristics. One specific embodiment of the subject invention is the use of improved image guides 35 in angioscopes. Angioscopy is a specific type of endoscopy which uses a flexible angioscope WO 00/54033 PCT/US00/06344 2 to transmit images from the heart and the coronary tree. Angioscopes are valuable tools for use in the investigation and treatment of heart and vascular disease. In various studies, atheromatous plaque rupture and splitting, endothelial exfoliation, and thin mural thrombi that could not be detected by angiography were able to be detected by angioscopy (Ushida, Y. et al. [1989] Am. 5 Heart Journal 117(4):769-776). Unfortunately, angioscopes, which are typically between 1.0 and 1.5 mm in diameter, are not small enough to access the entire coronary tree. The image guide of existing angioscopes typically has a diameter of about 0.27 mm and is surrounded by fibers arranged circumferentially to provide uniform illumination of the inner lumen. Figure 1 is a schematic structure of an angiofiberscope image guide. An angioscope 10 image guide is typically a hexagonal array of about 2000 fibers of the step index type. A step index (SI) optical fiber is one in which a fiber is composed of a core surrounded by a cladding where the refractive indices of the core and cladding are n, and n, respectively, where n , > n,. Typically, this SI optical fiber is glass but, as discussed below, SI polymer optical fiber is also known. Light at less than the critical angle, which is transmitted down the core experiences 15 internal reflection with very high efficiency at the core/cladding interface. Although the light reflects efficiently at the boundary, a small fraction of the light temporarily penetrates the cladding in the form of evanescent waves before returning to the core. If the cladding is not thick enough, these evanescent waves can pass through the cladding causing some of this light to leak out, or tunnel, through the cladding into the adjacent fiber. This causes a reduction in 20 resolution and a reduction in contrast sensitivity. If the core diameter is reduced, at fixed cladding thickness, less light is transmitted and the image loses brightness. On the other hand, if the cladding thickness is reduced, for a fixed core diameter, more leakage, or tunneling, occurs. Hence, there is an optimum fiber core diameter and cladding thickness. This optimization process has been studied experimentally (Tsumanuma, T. et al. [1988] Proc. SPIE 25 906:92-96). Tsumanuma et al. determined that a core diameter of 3 ptm and cladding thickness of 1 pm was optimal. With a core diameter of 3 .im and a cladding thickness of 1 rtm, only 36% of the light which hits the end of the step-index glass fiber image guide actually strikes the area defined by the cores of the microfibers. Most of the available light is lost on the cladding area. Since it is 30 only light striking the core area which can contribute to image brightness, only a marginal reduction in microfiber diameter can be made without significant brightness reduction. The resolution of an image guide is dependent on the number of microfibers per unit cross-sectional area. For example, existing angioscope image guides cannot be increased significantly in diameter to incorporate more microfibers, due to the dimensions of the vascular 35 system, and the diameter of the presently employed microfibers cannot be reduced in size WO 00/54033 PCT/US00/06344 3 without significant brightness reduction. Therefore, it is difficult to improve the resolution of existing angioscopes. Another important characteristic of flexible image guides is flexibility as measured by the minimum bend radius of the image guide. The flexibility of existing angioscopes is typically 5 limited by the stiffness of the image guide. For example, the typical minimum bend radius is about 8 mm, which makes procedures difficult in some regions of the coronary tree. This degree of flexibility has been achieved by acid leaching of the image guide to divide it into several separate units, except for its ends where the microfiber spatial coherence is mandatory. Further subdivision of the glass image guide would increase flexibility, but at the expense of rapidly 10 increasing the fragility of the microfibers. There is already a fairly rapid deterioration of image quality due to microfiber breakage which shows up as black spots on the image. In addition, coloration of the transmitted image of glass endoscopes has been observed (Tsumanuma et al., supra) when the endoscope is subjected to severe bending as occurs in angioscopy. This can cause loss of spectroscopic information in angioscopic clinical diagnosis due to wavelength 15 dependent light leakage from the fiber cores. The subject invention can also be utilized for submicron imaging, such as morphological submicron imaging, functional submicron imaging, and tissue type (optical biopsy) submicron imaging through fluorescence lifetime measurement. In a specific application, instrumentation in accordance with the subject invention can be utilized for in vivo micro-imaging of brain 20 structure and function at the subcellular level. Instrumentation capable of assisting researchers seeking a better understanding of the structure, function and tissue type of in vivo neuronal processes can be of great value from both a fundamental, clinical, and commercial viewpoint. Currently, the most widely used in vitro techniques in biomedicine are optical microscopy and spectroscopy. Both conventional far-field 25 and near-field optical microscopy have been used with resolutions of approximately ?J2 and of the order of )J50, respectively, where , is the wavelength of light used. However, these techniques cannot be used with respect to submillimeter probes for deep insertion into the brain. In order to probe deep into the brain, fiber optics can be used to eliminate the requirement that the sample be placed on the stage of a microscope. 30 Over the course of the last ten years, there have been many efforts to develop advanced bioanalytical techniques for the monitoring of a variety of biomolecules. For example, single or incoherent fibers have been used for a variety of chemical sensors. Likewise, coherent imaging fibers have been employed for image transmission with respect to, for example, endoscopy. Recently, the concept of using a coherent imaging fiber for investigating remote 35 samples and measuring surface chemical concentrations has been introduced. This method is WO 00/54033 PCT/US00/06344 4 based on the placement of a uniform layer of biosensor on the distal surface of an optical fiber. In this way, morphological visual information from a remote sample with 4 p/im spatial resolution was obtained. In addition, the device can be used to acquire chemical information from across the sample with the same spatial resolution. This technique also allowed for the 5 temporal response of the biochemical reactions to be recorded at 30 frames per second. Furthermore, the sensing layer can be changed to facilitate measurement of various analytes of interest providing a powerful and general method. It has recently been shown that tissue fluorescence lifetime differences of about 10 picoseconds can distinguish between collagen, elastin, keratin and artery. The fluorescence 10 lifetimes of these tissues appear to have at least two exponential components with lifetimes of a few hundred picoseconds and a few nanoseconds, respectively. Accordingly, these different types of tissue can, in theory, be distinguished based on measurement of different fluorescence lifetime. Fluorescence lifetime probes exist for various types of measurements, having timing ability and imaging capability. However, existing fiberoptic imaging fibers have an image time 15 dispersion of about 100 times too great to permit their applicability for tissue identification. Accordingly, an imaging optical fiber with low time dispersion would be very helpful in improving this technique. Brief Summary of the Invention 20 The subject invention pertains to image guides having highly advantageous optical and physical characteristics. The excellent characteristics of the image guides of the subject invention result, at least in part, from the use of gradient-index (GRIN) optical fiber. A further aspect of the subject invention concerns novel manufacturing processes used to produce image guides. 25 The image guides of the subject invention are highly advantageous because of their small diameter, greater flexibility, and excellent image quality. These image guides are useful in a wide variety of industrial and medical applications. Specifically exemplified herein are endoscopes for use in medical diagnostic procedures such as angioscopy and neurological imaging. Also exemplified are borescopes for use in industrial imaging. In general, the image 30 guides of the subject invention can be used in virtually any imaging scope used to examine locations which are inaccessible to the human eye. Such scopes may be used to visualize locations ranging from blood vessels to jet engine blades or high pressure pipes. Such scopes are also used in non-destructive testing procedures. The image guides of the subject invention achieve substantial improvements in 35 performance compared to existing image guides. Specific advantages that can be achieved WO 00/54033 PCT/US00/06344 5 utilizing the subject invention include: (1) a brighter image; (2) improved resolution; (3) a smaller diameter so as to be able to pass through narrower openings; (4) greater flexibility; and (5) less expense. By adjusting the dimensions and materials of the image guides of the subject invention, these performance characteristics can be optimized for a particular application. The 5 image guides of the subject invention are particularly advantageous for applications which necessitate the use of a guide having a very small diameter or an image guide which requires a very high resolution for a fixed diameter. In a specific embodiment, the subject invention pertains to an approximately 0.5 mm diameter angiofiberscope with enhanced flexibility and improved image quality. In the specific 10 application of angioscopy, the subject invention provides access to essentially 100% of the vascular tree with unprecedented image quality. Scopes of a variety of sizes can be manufactured with this new technology. In one embodiment, these scopes can be plug-in compatible with the already-installed base of electronic cameras, illumination systems, and monitors. In addition to the small diameter endoscope for 15 angioscopy, the subject invention can also be utilized for other applications in medical endoscopy and industrial imaging. In addition, the subject invention relates to an imaging optical fiber having a low image transmission time dispersion. In a specific embodiment, an image time dispersion of approximately 5 picoseconds can be obtained. Accordingly, the fiberoptic probe of the subject 20 invention can yield morphology, functional analysis, and tissue type information, simultaneously. This instrumentation can assist researchers to understand the molecular physiology of nervous system cells, their tissue environment, as well as the manner in which this physiology is affected by, for example, disease, pharmacologic agents, and/or development. The subject instrument can permit studies to be performed with respect to the biology of the brain 25 and other organs. The subject invention also pertains to a method and instrumentation for in vivo micro imaging of structure and function at the subcellular level. The subject method can, for example, be utilized for in vivo sub-micron imaging of structure, function, and tissue type deep within the brain. The subject instrumentation can utilize a sub-millimeter diameter fiber optic probe having 30 a spatial resolution on the order of 2 pm and time dispersion for image transmission on the order of 5 picoseconds. The distal tip of the probe can be designed to provide magnification. In a specific embodiment, a factor of ten magnification can be used. A variety of disposable probes can be utilized, each one suitable for investigating a specific neural process. Preferably, the probes can be compatible in size with existing sterotactic biopsy needles. Different 35 immobilization methods for the preparation of a variety of biosensor probes can be utilized for WO 00/54033 PCT/US00/06344 6 attachment to the subject probes. For example, enzyme, ligand and DNA molecules can be immobilized on probes and provide highly sensitive measurements for a variety of extracellular in vitro studies. Advantageously, the subject instrument can provide morphological sub-micron imaging, functional sub-micro imaging, and/or tissue type sub-micron imaging through 5 fluorescence lifetime measurement. In addition, the subject device can enable histological sampling to be correlated with in vivo biochemistry. Brief Description of the Drawings Figure 1 is a schematic structure of an angiofiberscope image guide. 10 Figure 2 shows the structures of the two basic types of optical fibers. Figure 3 shows the trajectories of typical light rays in SI and GRIN fibers. Figure 4 shows brightness of state of the art glass image guide versus a GRIN guide with the same size microfibers (5.0 im). Figure 5 illustrates, schematically, a specific embodiment of the subject method for 15 continuous production of GRIN fibers made by using two miscible optical polymers with different refractive indices whose relative concentrations vary radially to produce the required refractive index profile. Figure 6 is a schematic of the GRIN die block (GDB). Figure 7 is a schematic of the flow pattern in the feed chamber. 20 Figures 8A and 8D illustrate a longitudinal cross section and a transverse cross section, respectively, of the distal end of a sheath designed to fit over a plastic optical fiber image guide. Figures 8B and 8E illustrate a longitudinal cross section and a transverse cross section, respectively, of the distal end of a sheath designed to fit over a plastic optical fiber image guide, wherein the sheath comprises an illumination fiber. 25 Figures 8C and 8F illustrate a longitudinal cross section and a transverse cross section, respectively, of the distal end of a sheath designed to fit over a guiding means incorporated with a plastic optical fiber image guide, wherein the sheath comprises an illumination fiber. Figure 9 shows a general schematic of a bioprobe system in accordance with the subject invention. 30 Figure 10 shows a schematic of a specific probe in accordance with the subject invention, illustrating extrema light rays which show the object and image sizes. Figure 11 shows measured refractive index data points with respect to a GRIN plastic optical fiber, with an approximate refractive index profile. Figure 12 shows measured spatial resolution data for a plastic GRIN image guide and 35 a SI image guide.

WO 00/54033 PCT/US00/06344 7 Figures 13A and 13B illustrate measurements of the time dispersion of a pulse traveling through 100 m of SI fiber and 68 m of GRIN fiber, respectively. Figure 14 illustrates a specific set-up for adjusting the GRIN lens location during the manufacturing process in accordance with the subject invention. 5 Figure 15 illustrates schematically a set-up for measuring the MTF of a subject probe. Detailed Disclosure of the Invention A general layout of a specific embodiment of a bioprobe system 1 in accordance with the subject invention is shown in Figure 9. The subject bioprobe system 1 can utilize a probe 10 tip 4 held in a fixed position to an image guide 5 by a structural means which can allow access to objects under study. In a preferred embodiment, probe tip 4 and image guide 5 can be housed inside a standard sized stereotactic biopsy needle. The combination of probe tip 4 and image guide 5 can be referred to as a probe 2. Preferably, probe 2 can be disposable to reduce or eliminate the costs of sterilization. In the example shown in Figure 9, the probe 2 is 15 approximately 15 cm long. The sensor surface 3 can be located on the surface of probe tip 4, where probe tip 4 is designed to provide magnification. In the embodiment shown in Figure 9, probe tip 4 provides a factor of ten magnification. Preferably, spherical, chromatic, and other aberrations are controlled such as to achieve diffraction limited resolution at the sensor surface 3. The image plane produced by probe tip 4 can be incident on, and transmitted by, a graded 20 index (GRIN) optical image guide 5. In a preferred embodiment, a plastic graded-index optical image guide is used. More preferably, image guide 5 can comprise GRIN microfibers having, for example, 2 to 3 ,m diameters. A second, preferably flexible, GRIN image guide 6 can be used to transmit the image from the probe 2 to instrumentation 7 for viewing the image, permitting location of the 25 instrumentation out of the sterile field. Flexible GRIN image guide 6 can be interfaced to the probe 2 via an optical magnification section 8, for example a x10 magnification section. The design and structure of magnification section 8 can be similar to the design and structure of the magnification section of probe tip 4. In a specific embodiment, magnification section 8 comprises a lens which is in contact with image guide 5 and spaced from the distal end of image 30 guide 6 such that the image of the object is incident on the distal end of image guide 6. Other lens assemblies can also be utilized. The use of 5 -m diameter GRIN microfibers in the GRIN image guide 6 contributes minimally to degradation of the image resolution. Preferably, a plastic GRIN image guide 6 is utilized to allow greater flexibility and lower production costs. The instrumentation system utilized can perform at least three functions. A first 35 function is subcellular imaging, which can utilize a white light source 9. Traditionally, this WO 00/54033 PCT/US00/06344 8 white light was provided by a xenon arc lamp. However, new solid state arrays of different wavelength laser diodes can also be used to provide an alternative white light source. The white light can reflect off a dichroic mirror 11 onto the proximal end 12 of GRIN image guide 6. This light can then be transmitted to the distal tip 4 of the probe where it can illuminate the sensing 5 area and the contiguous tissue. The reflected light from the tissue can then return through the probe, and be transmitted through the dichroic mirror and detected by, for example, an intensified, back illuminated charge coupled device (CCD) camera 14. Functional imaging can also be accomplished with the subject instrumentation, using light whose wavelength is at the absorption maximum of a fluorophore incorporated in the 10 sensor 3 of the probe. Such light can be provided, for example, by using filters with a xenon light source, or by using an appropriate laser diode. The fluorescent light can be transmitted through the probe and, after filtering at the fluorophore emission wavelength, detected by CCD camera 14. In addition, tissue type imaging can be performed with the subject instrumentation 15 system by, for example, utilizing fluorescence imaging. Fluorescence imaging can yield information on the local chemical and/or structural environment, via the impact of the chemical and/or structure on the non-radiative decay rate of an excited fluorophore. Since the non radiative decay rate affects the characteristic fluorescence decay time, measurement of the latter quantity with the subject instrumentation can yield information about the tissue type. Imaging 20 of fluorescence lifetime can provide a reliable means of acquiring spatially resolved information on the local environment of distributed fluorophore in tissue. This procedure has been referred to as fluorescence lifetime imaging (FLIM). Fluorescence lifetime imaging has been used to measure [Ca 2 ], [02], and pH. However, until recently, most FLIM systems were limited to the nanosecond scale. New results have been reported with a system having a temporal system 25 response of less than 100 ps and a capacity to image differences in fluorescence lifetime of less than 10 ps. These new results were obtained in vitro using a microscope and suggest it is possible to distinguish between, for example, collagen, elastin, aorta wall, and plaque. Advantageously, the subject invention can allow the use of FLIM with respect to tissue in vivo. The wavelength of the excitation light may be adjusted to control its penetration depth into the 30 tissue thereby controlling the required depth of field of the instrumentation. A schematic of a specific embodiment of a probe in accordance with the subject invention is shown in Figure 10. The tissue being investigated can be placed in contact with the sensor surface which is located on the distal surface of a gradient index (GRIN) lens 4. In the embodiment shown in Figure 10, the GRIN lens is 1.39 mm long. An inverted, magnified image 35 can then be formed at a distance of 5.0 mm from the lens 4, on the distal surface of a GRIN WO 00/54033 PCT/US00/06344 9 zimage guide. In this embodiment, a 500 4m diameter plastic GRIN image guide 5 composed of 2.0 km microfibers is used. Accordingly, for a magnification of ten, the region of sensor surface which can be viewed is 50 4m diameter. Figure 10 shows typical extrema light rays for the system, illustrating the object and image sizes as well as the small radial extent, 0.25 mm for 5 a specific embodiment. of the GRIN lens which is used. Preferably, the subject system is designed to maintain all aberrations down to the level where the resolution is diffraction limited. Existing endoscopes do not have diffraction limited resolutions because the GRIN lens is typically connected directly to an image guide, the normal endoscope acceptance angle, 26, is typically about 70', and the resolution is normally determined by the diameter of the microfibers 10 in the endoscope. Furthermore, while the full radial extent of the GRIN lens is used in existing endoscopes, the subject scope can be designed to use a much lower percent of radial extent of the lens. For example, only about 10% of the full radial extent is used in the embodiment of Figure 10. Accordingly, the effect of some lens aberrations is much less with respect to the subject invention as compared with typical endoscopes. A standard endoscope GRIN lens may 15 be adequate for some applications of the subject invention. However, more complex lens systems employing one or more lenses can also be utilized. Given below, is a first order calculation of the preferred properties of a lens for use with the subject invention. The following equation describes the refractive index distribution of a GRIN lens. 20 N,(r) = No,k + N,,,r 2 + N,,xr 4 + where No, is the refractive index at wavelength k on the axis, r is the radial distance from the axis and Ni,x are the constants which describe the index gradient at a particular wavelength k. To a first approximation, for small r, all terms higher than r 2 may be neglected. It is worth noting that an axially symmetric GRIN lens manufacturing process can only have Nx(r) 25 represented by a constant plus a term proportional to r 2 . A linear term in r would give rise to a discontinuity at r = 0 which is not physically possible given the manufacturing process. Hence, this first approximation calculation is well justified for small values of r. Table 1 shows parameters of a specific embodiment of the subject system. 30 Table 1. Some Parameters of the GRIN Lens xlO Magnifier. Field of View GRIN Lens Gradient Magnification Image Distance on Surface of Diam. Length Constant From Lens Lens N, (mm 2 ) 50 um diameter 500 4m 1.30 mm 1.18 10.0 5.0 mm WO 00/54033 PCT/US00/06344 10 The optical design of the GRIN lens system can be thoroughly modeled by any one of several commercial software packages available from various companies such as Stellar Software, Berkeley, CA; Sinclair Optics, Fairport, NY; and Optikos Co., Cambridge, MA. There 5 are at least two companies, NSG America Inc. and Gradient Lens Corp., New York, which make glass based GRIN lenses. There is at least one company, Nanoptics, Inc., Florida, which makes plastic GRIN lenses. All three of these companies have the capacity to fabricate a lens with appropriate specifications for use with the subject invention. In addition, to minimize aberrations, the end surfaces of the GRIN lens can be fabricated to be non planar. In the above 10 discussion, a probe having magnification of 10, field of view of 50 /m in diameter and 0.5 ~m resolution was described. However, other combinations can also be utilized. These can include, for example, a magnification of 5, field of view of 100 /m diameter and 0.8 pm resolution. The image from the GRIN lens system can then be formed on the distal surface of the plastic GRIN image guide. In an alternative embodiment, the GRIN lens magnification system can be 15 removed, such that the sensor can be utilized directly on the distal surface of the plastic GRIN image guide, providing a 500 /m field of view and 2 4m resolution. Plastic optical fiber has been made for about 40 years. Until recently, it has been fabricated with a step index (SI) of refraction. A cross section of such a fiber is shown in Figure 2. Both polymer and glass SI fibers are constructed with a core and cladding with refractive 20 indices n, and n, respectively, where n, > n,. A fundamental requirement for SI fiber is that the thickness of the cladding must be X to minimize light leakage and maintain contrast. The area presented by the cladding does not contribute to image brightness, and also limits the number of fibers which can be used in a given diameter image guide and hence, limits the resolution. A second type of fiber is known as gradient-index or graded-index (GRIN) fiber can also be 25 made with polymer or glass. The GRIN structure is also shown in Figure 2. In comparing the SI structure with the GRIN structure, it is noted that there are different trajectories of light rays in these two fiber structures. This is shown schematically in Figure 3. Within SI fiber, the light travels in straight lines. At angles less than the critical angle of internal reflection, the light is reflected at the core cladding interface. At angles greater than the critical 30 angle, the light is refracted into the cladding from which it travels into the adjacent fiber in the SI image guide. This large angle light traverses the various fibers in the image guide until it reaches the side of the image guide and is absorbed. In contrast, within GRIN fiber, the light travels in a curved trajectory, always being refracted back towards the axis of the fiber. At angles less than the critical angle, light never reaches the outer edge of the fiber. At angles 35 greater than the critical angle, the light exits the fiber similar to the case of the SI image guide.

WO 00/54033 PCT/US00/06344 11 Figure 11 shows measured refractive index data points and an approximate refractive index profile for a communication type GRIN plastic optical fiber produced by Nanoptics, Inc., Florida, wherein the data points were measured by Lucent Technologies. The subject invention utilizes gradient-index (GRIN) optical fiber to produce image 5 guides for use in angioscopes, endoscopes, borescopes, other imaging scopes and devices. The subject invention achieves substantial improvements in performance compared to existing image guides, including: (1) a brighter image; (2) improved resolution; (3) a smaller diameter to pass through narrower openings; (4) greater flexibility; and (5) less expense. By adjusting the dimensions and materials of the image guides utilizing the teachings of the subject invention, 10 these characteristics can be optimized, to facilitate the use of the image guides for a wide variety of applications. Thus, the image guides of the subject invention can be used in medical endoscopy as well as in industrial imaging. In a specific embodiment, the subject invention utilizes GRIN plastic optical fiber (POF), to make a major advance in angioscopy. The subject invention further pertains to new manufacturing processes useful in the production of the 15 improved image guides described herein. The refractive index of a GRIN optical fiber can be generally represented by the axi symmetric index profile n(r) = n, [1 - 2Af(r)] ' for r a where f(0) = 0, f(a) = 1, a is the radius of the core, and 2 2 A -1 2 1 2 2n 2 nl 2 2n 1 20 wherein n, and n, are the values of refractive index at r-0 and r=a, respectively. In a specific embodiment, r g fir) = (-) a where g is the profile parameter which, for g=2, yields a parabolic profile. In a preferred embodiment, in order to minimize time dispersion, g preferably lies between 1.5 and 2.5 and, 25 more preferably, between 1.75 and 2.25. The most preferred value of g depends on the choice of material composing the GRIN optical fiber. Although maintenance of the GRIN structure cross the entire diameter of the fiber can be preferred for brightness and time dispersion purposes, a thin step-index section can be WO 00/54033 PCT/US00/06344 12 incorporated at the outer most portion of the fiber. This step-index outer section can enhance image contrast. In a specific embodiment, a thin extra murial absorber can be incorporated at the outer edge of the fiber to absorb cross-talk, thus enhancing contrast. Preferably, the outer step-index portion should be less than about 0.5 p/m, and more preferably less than about 0.25 5 p/m. In this way, a large portion of the fiber is still available for receiving and carrying light. Accordingly, it is preferred to design the width of this outer step-index section such as to optimize the combination of time dispersion, contrast, and brightness. Suitable polymers containing light absorbing compounds and having good adherence to the GRIN fiber can be used for this outer layer of fiber. 10 There are substantial advantages of the GRIN imaging guide of the subject invention compared to the SI image guide. One of the advantages pertains to the brightness of the images which can be obtained. The brightness, B. of the image transmitted by a guide is defined by the equation: a-L B = S*NA 210 10 where S is the ratio of the total cross-sectional area of the cores of SI fibers (or the total cross 15 sectional area of GRIN fibers) to the total cross-sectional area of the image guide. NA is the numerical aperture of the fibers, defined NA s F 2 NASI V1 -n2 NAGRIN /2 2 where NAs, and NAGE are the numerical apertures for SI and GRIN optical fiber, respectively. The light attenuation, a, is given in units of dB/meter, and L is the length of the image guide in units of meters. The approximate values of these parameters for 5 pm outer diameter 20 fibers are given in Table 2.

WO 00/54033 PCT/US00/06344 13 Table 2. Values of parameters describing optimized SI angioscope image guide, and the GRIN guide Image guide S NA t(dB/m) SI (glass) 0.36 0.43 1.0 5 GRIN (plastic) 1.0 0.46 0.2 Because the GRIN fiber has no cladding, 100% of the GRIN image guide cross-sectioned surface area is available to transmit light compared to 36% for the optimized SI glass image guide. The numerical aperture of the two types of optical fiber are comparable. In the case of 10 glass (Tsumanuma et al., supra), the high value of NA = 0.43 is achieved by appropriate ionic doping at the expense of deteriorating the transmission to a value of a = 1.0 dB/m. A 5.0 ptm diameter GRIN-POF has been measured (Koike, Y. et al. [1993] In Design Manual and Handbook and Buyers Guide, Information Gatekeepers, Inc., Boston, p. 19) to have an attenuation of 0.2 dB/m. There are at least three ways to produce GRIN-POF, namely, using two 15 or more miscible polymers, using a copolymer with monomer subunits, or by doping a polymer with a low molecular weight additive. For GRIN-POF, a value of NA = 0.46 can be achieved with available polymers by making the GRIN-POF using two or more miscible polymers with different refractive indices. The relative concentrations of the two or more miscible polymers vary radially to produce the required GRIN profile. Alternatively, a copolymer can be used in 20 which the ratio of monomer subunits change as a function of radius in a manner such as to produce the required GRIN profile. Most available GRIN-POF, which is produced by radial dependently doping a given polymer with a low molecular weight additive, has NA in the range 0.1 to 0.22. This NA is adequate for the 100 m lengths used in digital transmission for local area networks. By changing the ratio of the polymer components in the fiber, instead of doping with 25 an additive, the NA can be increased to 0.46 or more. In fact, certain copolymer blends can achieve an NA of at least 0.67. The brightness, B, of the transmitted image has been evaluated using the parameters given in Table 2, and is plotted in Figure 4. It can be seen that the GRIN-POF image guide is about twice as bright as the existing state of the art SI glass optical fiber (GOF) image guides. 30 We shall assume that the working length of the angioscope is about 1.3 m, and the total length is 3.3 m. A second major advantage of GRIN image guides is the potential for improving the spatial resolution. The resolution of an image guide can be improved if a larger number of microfibers is used per unit area. An image guide made of GRIN-POF has 100% of the cross 35 sectional area of each fiber end available to transmit light. For fundamental physical reasons, WO 00/54033 PCT/US00/06344 14 the diameter of an optical fiber can only be reduced to about 1.0 gm without losing the ability to transmit light. Therefore, there is much potential improvement in resolution from the existing state of the art of a 5 pm microfiber diameter down to the fundamental limit of 1.0 Im. Thus, while the brightness of the GRIN-POF guide is about double that of the SI-GOF 5 guide, the improvement in resolution using the GRIN-POF guide is up to a factor of five. These improvements in image quality are substantial and demonstrate the advantages of the GRIN-POF technology of the subject invention. The flexibility of glass guides is limited to about 8 mm minimum bending radius. This is not adequate for some branches of the coronary tree. In addition, coloration of the transmitted 10 image of glass endoscopes has been observed (Tsumanuma et al., supra) when an endoscope is subjected to severe bending as occurs in angioscopy. The endoscopes of the subject invention can have a factor of about five or more greater flexibility, which can provide important advantages particularly in angioscopic applications. If flexibility is not an important criterion in a specific application, then a glass image guide made of GRIN fiber can be used and would 15 offer higher resolution than the existing glass image guides made of SI fibers. The enhanced flexibility of the subject plastic GRIN fibers is due to the mechanical properties of polymers, which depend upon their processing history. Molecular orientation, such that the polymer chains are aligned along the axial direction of the image guide, produces macroscopic anisotropy. An excellent modem review of this subject is provided by Struik, 20 L.C.E. [1990] Internal Stresses, Dimnensional Instabilities, and Molecular Orientations in Plastics, John Wiley & Sons Ltd., Chichester, England. The properties of the chain segments measured in the direction of the polymer chains (and image guide) are determined by strong covalent chemical bonds, whereas weak Van der Waals forces are operating in the transverse direction of the image guide. As a direct result of molecular orientation, there is: (a) increased 25 axial strength of the guide and (b) increased axial strain to break of the guide; and, therefore, enhanced flexibility. In a preferred method for inducing molecular orientation in a polymeric GRIN image guide, the image guide fiber is stretched, at an appropriate temperature, at low strain rate. This gives the required enhancement in mechanical properties without reduction in optical 30 transmission. In the subject invention, by using the technique of molecular alignment, a 270 pm diameter polymethylmethacrylate based POF image guide has exhibited unlimited 180' flexing cycles with a bending radius of 1.5 mm. This is to be compared with an 8 mm bending radius limit for the SI glass guide.

WO 00/54033 PCT/US00/06344 15 The enhanced image quality and flexibility of the angioscopic image guide of the subject invention represents an additional major advance in this type of instrumentation. The subject GRIN-POF image guide is brighter, higher resolution, more flexible, and lower cost than the existing image guides. The GRIN-POF scopes of the subject invention are improved over 5 existing SI-GOF scopes by, for example: 1. For fixed microfiber diameter of about 5 4m and an image guide width of about 270 jim, the GRIN-POF scopes are at least about 50% brighter than SI-GOF scopes. 2. The resolution of the subject GRIN-POF scopes for a fixed guide width of 270 10 pm are at least 50% higher than SI-GOF scopes. 3. The flexibility of the subject GRIN-POF scopes are at least about three times higher than the existing SI-GOF guide. In a specific embodiment, the imaging scopes of the subject invention can be inserted into a plastic tube (sheath), which can have a transparent end plate. This combination can then 15 be used for imaging internal body structures. The image viewed through the end plate is unimpaired by the sheath or end plate. The advantage of this sheath is that it is disposable and allows the imaging scopes to be reused with minimal sterilization. In a preferred embodiment, the sheath can have at least one internal, or external, illuminating optical fiber(s) which transmits light to illuminate the internal body structure to be 20 imaged. Additionally, it is preferred but not essential, that there be no transparent end plate at the distal end of the illuminating optical fiber(s) to avoid the illuminating light reflecting at such a plate and impairing the quality of the image. A longitudinal cross section and a transverse cross section of a sheath comprising an external illumination fiber are shown in Figures 8B and 8E, respectively. 25 When performing, for example, endoscopy or angioscopy, a flexible guiding means, typically made of metal, is often incorporated to facilitate guiding the endoscope or angioscope within the body. A longitudinal cross section and a transverse cross section of a sheath comprising an external illumination fiber, where the sheath is designed to fit over a guiding means incorporated with a plastic optical fiber image guide, are shown in Figures 8C and 8E, 30 respectively. In this case, the sheath and illuminating fiber could be regarded as disposable after a single use. Accordingly, the scope and/or sheath of the subject invention can comprise such a flexible guiding means, such that many combinations of imaging scope, guiding means, illuminating fiber(s), and sheath are possible.

WO 00/54033 PCT/US00/06344 16 Spatial resolution is usually represented by the modulation transfer function (MTF) of the image guide. Figure 12 shows MTF data for a 10 cm long plastic GRIN image guide with 5 ptm microfibers and a 10 cm long plastic GRIN image guide with 2.8 im microfibers. The 5.0 /m GRIN guide has an MTF very similar to that of the Japanese researchers (Tsumanuma et al., 5 supra) using optimized 5.0 im step-index glass fiber. The similarity in resolution is to be expected because the same fiber diameter is used. Having verified the present measurement technique, the 5.0 gm MTF data was compared with that from the 2.8 p/m GRIN image guide. A major improvement can be seen in resolution for the 2.8 ptm diameter fiber compared to the 5.0 4m diameter fiber. The shapes of the two (5.0 and 2.8 gm) MTFs are somewhat different, 10 making it difficult to give a quantitative measure of the difference. However, the difference is large, and appears related to the difference in fiber diameters. Accordingly, a plastic GRIN image guide offers advantages compared to a step-index glass or plastic image guide. Even though it is possible to make a glass GRIN image guide, the cost is much higher than for plastic GRIN image guides because the production of the necessary large numerical aperture glass 15 GRIN material is by ionic diffusion in salt solution at high temperature. Since this process works only for small (<4 mm) diameter glass rods, it is an inherently costly, low volume process. Another advantage of GRIN image guides relates to the extremely small time dispersion associated with GRIN fibers. The development of GRIN optical fiber (glass and polymeric) was, 20 in part, motivated by a desire to produce a very high bandwidth communication medium. With SI fiber, the distance traveled by the light depends on the angle of its trajectory along the fiber. Since the speed of light is a constant in the SI fiber, there is a variation, or dispersion, of the arrival times of the light at the end of the fiber due to the different angles of the light trajectory. In the case of GRIN fiber, the path length of the light also increases with increasing angle of its 25 trajectory. However, with GRIN fiber the speed of the light increases at larger radius due to the decreasing refractive index. The GRIN profile of the fiber can be arranged to minimize the time dispersion to be, for example, about 200 times less than that produced by SI fiber. This effect is independent of the fiber diameter. Measurements of the time dispersion of pulses traveling down SI and GRIN plastic 30 optical fiber (POF) are shown in Figures 13A and 13B. For a 1 meter fiber length, the SI and GRIN fibers give time dispersions of about 1 nanosecond, and 5 picoseconds, respectively. Referring to Figure 9, time dispersion can also exist in parts of the subject invention other than the GRIN image guide. For example, time dispersion can be generated in the initial xlO magnification tip 4, the xl0 magnification interface 8 between the disposable probe and the 35 flexible image guide 6, and the interface between the proximal end 12 of image guide 6 and the WO 00/54033 PCT/US00/06344 17 camera 14. An estimate of the time dispersion contributions of these three are < 1 ps, <4 ps, and < 5 ps, respectively. The time dispersion from all sources in the embodiment shown in Figure 9 is preferably less than 10 ps. Accordingly, the GRIN image guide can enable the subject invention to provide 2 4m resolution imaging and allow for distinguishing between different 5 types of tissue from measurement of fluorescence lifetime in the range of a few picoseconds to a few nanoseconds. In a preferred embodiment of the subject invention, a sub-millimeter diameter probe can be designed, utilizing GRIN optical fiber, to provide sub-micron morphological, functional and tissue-type imaging. For many high resolution optical systems, structural integrity and tolerances of 10 manufacture are important to the performance of the system. This is true of the subject probe as well. In addition, the subject probe can be extremely small. Also, it is preferred to manufacture the subject probe as inexpensively as possible, while still maintaining sufficient structural integrity and proper tolerances, in order to make the subject probe disposable. The glass GRIN lenses currently available, for example those produced by NSG (SELFOC lenses), 15 can have gradient index constants, N,, which vary by as much as ± 10%. Although this is of little consequence with respect to their use in endoscopes, their use with the subject probes can necessitate the individual adjustment of the precise position of the lens in each probe during the fabrication process. The subject invention also relates to a method and means for accurately, rapidly, and 20 inexpensively locating the GRIN lens 4 relative to the distal surface of the GRIN image guide. This method and means can allow the achievement of, for example, optimal resolution. Figure 14 illustrates a schematic of a specific set-up for positioning the GRIN lens, relative to the GRIN image guide. A piezo-driven scanning stage 15 (Polytec P.I.) can be connected to the GRIN lens such as to control the position of the GRIN lens over a range of 200 im to an accuracy of one 25 hundredth of a micron. In the embodiment shown in Figure 14, the piezo-driven scanning stage 15 is connected to the GRIN lens 4 via an acrylic rod 16. A Ronchi resolution pattern can be attached on the face of the acrylic connecting rod such as to be in direct contact with the GRIN lens 4. The CCD camera can then record the image of the resolution pattern and quantify the measured resolution as a function of the position of the GRIN lens. Once optimum position of 30 the GRIN lens is determined, an ultra-violet light 17 can be used to illuminate u.v. curable epoxy 18 to fix the GRIN lens 4 at the desired position, for example within a tube 19. In a preferred embodiment, tube 19 can be made of stainless steel. This procedure, including u.v. curing epoxy surrounding a GRIN lens inside a stainless steel tube, can provide an accurate positioning of the GRIN lens in a rapid and inexpensive way. In a preferred embodiment, the stainless steel tube 35 19 can be, for example, a sterotactic biopsy needle. It is also envisioned that, a person skilled WO 00/54033 PCT/US00/06344 18 in the art having the benefit of the subject disclosure can accomplish the positioning of the GRIN lens in a variety of ways using a variety of housing means. The overall image quality of the subject probe can depend on several factors such as error in the position of the GRIN lens relative to the GRIN image guide, unevenly polished 5 surfaces of the image guide, misalignment of the optic axes, and aberrations. There are also limiting factors to the image quality such as inter-fiber distance in the image guide and cross-talk between fibers in the image guide. Cross-talk between fibers in the image guide can reduce the contrast of the image. In addition, for image guides made of individual fibers having diameters less than about 1.5 p/m, the limited number of electromagnetic modes of transmission may 10 induce some artificial coloration of the image. Accordingly, the image guide can preferably be made of individual fibers having diameters of at least about 2 pm. Animportantquantifier of image quality is a measurement of the modulation transfer function (MTF). A set-up for measuring the MTF of the sub-micron probe is shown schematically in Figure 15. Referring to Figure 15, a linear attenuator 30 can be used to control the intensity of a 15 laser beam 31 from a He:Ne laser 32 (He:Ne at 632.8 nm, 3 mW) whose output can be measured by a photodiode 33. The beam can be enlarged by a spatially filtered beam expander 34 which reduces the beam angular divergence, for example, to 10' rad. The expanded beam 35 is allowed to strike a mask 36 (sharp edge) which is fixed in space. The distal tip 37 of the subject probe can be held by a manually controlled xyz translation stage 38. In order to measure the 20 MTF of the probe, the translation stage 38 can be adjusted until the distal surface of GRIN lens 4 is within 1000 p/m from the mask edge 36. The diffraction limited image of the edge can then be recorded by the camera 39. A precise calibration of the linearity of the entire system can be performed by varying the beam attenuator 30. A measurement of the MTF of the system can be obtained from the edge response function by taking a one dimensional Fourer transform, as 25 known in the art. The measurement can be performed at increments of 0.5 4m by moving the translation stage in one dimension in 0.5 kzm increments over a total distance (about 2.0 4m), corresponding to the repetitive structure of the GRIN image guide (2.0 4m diameter microfibers). These MTF measurements can then be averaged to obtain the system MTF in one dimension. The system MTF can then be measured in the same way in the orthogonal direction. 30 Preferably, the GRIN microfibers of the subject probe can be arranged in rows during the manufacturing process. In this case, the system MTF can therefore be affected by the orientation of the probe relative to the mask edge. To account for this orientation dependence, the system MTF can be measured for various angular orientations of the probe to mask edge. For example, the system MTF can be measured at 300 angular steps. After averaging the 35 measurements from the various angular orientations, the final overall system MTF can be WO 00/54033 PCT/US00/06344 19 obtained. Performing these MTF measurements with low beam divergence minimizes fiber cross talk. Preferably, the components of the subject probe are designed such that the results are invariant with respect to the bending of the probe. Other measurements, such as using a diffraction limited focused beam incident on a I 1m diameter pinhole can also be performed. 5 In particular, such measurements with different focusing lens apertures can explore a range of modal excitation in the GRIN microfibers which can affect the MTF. In addition, these measurements can explore the phenomena of radiative and leaky modes which tend to deteriorate the image quality by lowering the contrast. Accordingly, the results of these measurements can be used as feed back to optimize the design of the subject probe. 10 Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. Example 1 - Production of GRIN Fiber 15 General processes for fabricating a plastic GRIN optical fiber are known to those skilled in this art. These processes can produce plastic GRIN fiber wherein the refractive index varies in a controlled way as a function of radius. Typically, the refractive index varies parabolically as a function of the radius. The varying refractive index can be achieved by, for example, radial dependently doping a given polymer with a low molecular weight additive. Alternatively, in a 20 preferred embodiment of the present invention, plastic GRIN fiber is made by using two miscible polymers with different refractive indices whose relative concentrations vary radially to produce the required refractive index profile. Figure 5 illustrates, schematically, a specific embodiment of the subject method for continuous production of GRIN fibers made by using two miscible optical polymers with 25 different refractive indices whose relative concentrations vary radially to produce the required refractive index profile. Two optical polymers (materials Ma and Mb) with different refractive indices are introduced to the GRIN die block (GDB) through separate feed channels, A and B, by two extruders, Xl and X2. The GDB is shown, schematically, in more detail in Figure 6. Material Ma, which is fed to the channel A, flows into the mixing chamber D through the 30 channel Cl 1, whereas the material Mb flows from channel B to a mixing chamber D through the channel C2. By varying the gap, G, or length, L, of the channels C1 and C2, the flow rate of each material can be varied in the axial, or z-direction (see Figure 7). Consequently, a blend with a gradually varying composition in the z-direction can be prepared in the mixing chamber

D.

WO 00/54033 PCT/US00/06344 20 Since the refractive index of the polymer blend depends on the ratio of component polymers in the blend composition, the blended material in the mixing chamber D can have a gradually varying refractive index along the z-direction. While the rotating mixer blade Dl located in the middle of the mixing chamber D provides uniform mixing of the two materials 5 Ma and Mb at each location of z, axial mixing in the z-direction does not occur since there is essentially no pressure gradient in the z-direction. The axially varying blend prepared in the mixing chamber D is then fed through the channel E to the feed chamber F which houses a rotating cone Fl 1. As used herein, reference to a cone refers to any tapering cylindrical form. The taper can be, but does not have to be, at a 10 constant angle. While the material is flowing from D toward the die exit H through E and F, the axial variation of the blend composition in the mixing chamber D is converted to a radial variation, thus creating the gradient-index fiber. In Figure 7, the flow pattern of the polymer blend is shown schematically. Since the material fed to the feed chamber F at a downstream location near the die exit H is swept by the 15 upstream material, it is positioned away from the rotating cone F 1. The flow patterns 1, 2, and 3 of Figure 7 show such positioning of materials schematically. Due to the rotating cone Fl, the materials in the feed chamber F follow a helical stream line pattern. For simplicity, however, only the axial and radial components of the flow pattern are depicted in Figure 7. The rotating cone is for the uniform positioning of the material in the circumferential direction so that the 20 axisymmetry of refractive index can be ensured while creating radially varying refractive index. The rotation speed of FI should be sufficiently high to ensure the axisymmetry of refractive index, preferably taking into account the residence time of the material in the feed chamber F. When the material leaves the die exit H, the circular strand has a refractive index 25 decreasing with the radial position and a gradient-index optical fiber is formed when the strand is pulled off. Example 2 - Production of Small Diameter GRIN Fiber Image Guide The subject plastic GRIN image guide can be produced by thermal processing of the 30 original GRIN fiber. Examples of thermal processing include fusing, drawing, and stretching. Many original GRIN fiber sections can be fused together to produce a multifiber image guide. This multifiber image guide can be drawn to reduce the diameter of the image guide and each individual fiber contained therein. Finally, the image guide can be stretched to increase the flexibility.

WO 00/54033 PCT/US00/06344 21 At elevated temperature, low molecular weight additives have enhanced diffusivity. As a result, plastic GRIN fiber made with additives may end up with a degraded refractive index profile. Therefore, in a preferred embodiment, the plastic GRIN image guide should be made with fiber composed entirely of at least two miscible polymers or layers of polymers of different 5 refractive index where there will be no degradation of the profile. In one embodiment, approximately 0.5 mm diameter GRIN fiber is cut into 1.0 m length sections, and approximately 10,000 fiber sections are bundled together in a 100 x 100 square. This bundle is set within a 50 mm x 50 mm cross-section stainless steel square tube. The tube is placed in a heated oven and the fibers are subjected to pressure at an appropriate temperature 10 to make a fused boule of solid polymeric fibers. A square tube is preferred because it is easier to apply pressure to the bundle of fibers, although a round tube can also be used. The solid square boule may be machined into a round boule if a round image guide, rather than a square image guide, is desired. The solid boule is then placed in the heating chamber of a drawing tower, in which the lower part of the boule is continuously heated and drawn down to a uniform 15 diameter multi-microfiber image guide. As those skilled in the art would readily appreciate, it is important when manufacturing image guides that the ends of the image guide be coherently related to each other. Image guides can be produced with outer diameters as small as approximately 0.5 mm down to approximately 0.1 mm containing microfibers of approximately 5 gm down to 20 approximately 1 gm, respectively. This range of microfiber diameters extends from the existing glass microfiber diameter (5 pm) of small diameter fiberscopes down to the fundamental limit (approx. 1.0 pm). The image guide may have an outer protective cladding of polymer extruded upon it in the conventional cross-head die method, or by solution cladding if desired. The image guide is 25 cut to length, the ends are polished and, if desired, a gradient-index rod lens is attached, preferably glued, onto one end. The resulting fiberscope can be sterilized with solution, or ETO prior to use and thereafter, to facilitate reuse, if desired. Example 3 - Production of Large Diameter GRIN Fiber Image Guide 30 A GRIN fiber image guide can be made with either GRIN-GOF or GRIN-POF. If made with glass, the image guide will be more rigid and can have application where a more rigid image guide is preferred. If made with polymer, the image guide will be flexible and can be used in applications where flexibility is desired. A 3 mm diameter GRIN POF image guide can be made with the high resolution of 1000 x 1000 corresponding to the high definition television 35 (HDTV) standard. Image guide with a 100 x 100 array of 3 pm diameter microfibers is WO 00/54033 PCT/US00/06344 22 produced as described in Example 2. This square image guide, 0.3 mm on a side, are arranged in a 10 x 10 array with dimensions of 3 mm x 3 mm of the desired length of say 1 m. Low viscosity epoxy is used at each end (within 1 cm of the end) of the array to secure the positions of the fibers. When the epoxy is cured the ends are cut and polished. The guide may be given 5 a GRIN lens at one end, and protective cladding as described in Example 2. The resulting fiberscope guide has a higher resolution than heretofore achieved. Example 4 - Production of a 3 mm Diameter Fiber Optic Taper When an image is presented at one end of existing fiber optic tapers, the output face of 10 the taper displays an image which is larger or smaller than the input image. The ratio of image sizes is equal to the ratio of the dimensions of the input and output faces of the taper (called the taper ratio). Existing tapers are based on glass SI fiber and have typical taper ratios of 2:1 or 3:1. At the small end of the taper, the microfiber is usually no less than 5 pm, for the important reasons discussed earlier. Therefore, the microfiber dimension at the large end of the taper will 15 be 15 pm, for a taper ratio of 3:1. The spatial resolution of existing tapers is, thus, severely limited. This example describes a fiber optic taper which has about twice the resolution of the existing tapers. An image guide is made in the manner described in Example 2 with dimensions 0.75 mm x 0.75 mm, and containing 7.5 pm microfiber. A 20 x 20 array of sections of this 20 image guide, 20 cm long, is packed into a square cross section stainless steel tube. The assembly is placed into a vacuum oven. After a vacuum is established, the temperature is increased. At a temperature, about 200 - 600C above the polymer glass transition temperature, the fibers fuse into a solid mass. The oven is allowed to cool to room temperature, and the fused boule is removed from the fixture. The boule is placed in a stretching machine, whose design is identical 25 to the machines used for producing glass fiber optic tapers. Each end of the boule is held by a rotating chuck. The center of the boule is heated by a cylindrical heater. When the boule has reached the appropriate temperature, the chucks are both retracted in unison. The boule is stretched and forms an hour glass shape. Heat is turned off and the boule is permitted to cool while still rotating. The boule is removed from the fixture, and cut at the locations required for 30 the desired taper ratio of 3:1. Two symmetric tapers are produced from each stretched boule. The microfiber dimensions at the large and small ends are 7.5 Pm and 2.5 jIm respectively. This GRIN based fiber optic taper has twice the resolution of existing glass tapers.

WO 00/54033 PCT/US00/06344 23 Example 5 - Production of a Fiber Optic Plate Fiber optic plates are frequently used to attach to the surface of an opto-electronic device such as a charge coupled device (CCD). As dimensions of the structures of these semiconducting devices continue to decrease, it is increasingly important to use a fiber optic 5 plate with microfibers less than 5 pm in size. In this way, the resolution of the system will be improved. This example concerns the production of a fiber optic plate containing 2.5 gm microfibers. Image guide fiber was produced, as in Example 2, with dimensions 250 Pm x 250 jim. One meter lengths of this fiber are packed into a 25 mm x 25 mm fixture to form an array 10 of 1000 x 1000 fibers. The fibers are fused as before to form a solid boule. Next, this solid boule is cut into sections and a 25 by 25 bundle of these solid boule sections is placed within a 1 inch x 1 inch stainless steel square tube. The tube is placed in a heated oven and the solid boule sections are subjected to pressure at an appropriate temperature to make a second fused boule of solid polymeric fibers, said second boule being approximately 15 1 inch by 1 inch. Finally, a section is cut off the end of the boule and the ends of the boule section are polished to make a fiber optic plate. Example 6 Referring to Figures 8A-8F, this example provides three illustrative combinations of 20 image guide, illuminating fiber, guiding means, and/or sheath. Figures 8A and 8D illustrate a longitudinal cross section and a transverse cross section, respectively, of the distal end of a sheath designed to fit over a plastic optical fiber image guide. The sheath covers the distal tip of the image guide with a transparent end plate. In this case, any illuminating fibers and/or guiding means would not be enclosed within this sheath, although they could have their own 25 sheaths. Figures 8B and 8E illustrate a longitudinal cross section and a transverse cross section, respectively, of the distal end of a sheath designed to fit over a plastic optical fiber image guide, wherein the sheath comprises an illumination fiber. The distal end of the illumination fiber is not covered by the sheath, in this example, so as to not impair the image. Accordingly, the 30 illuminating fiber can be disposed of with the sheath. In this embodiment, the sheath acts to attach and position the illumination fiber with respect to the image guide. Figures 8C and 8F illustrate a longitudinal cross section and a transverse cross section, respectively, of the distal end of a sheath designed to fit over a guiding means incorporated with a plastic optical fiber image guide, wherein the sheath comprises an illumination fiber. The 35 distal end of the illumination fiber is not covered by the sheath, but the distal end of the image WO 00/54033 PCT/US00/06344 24 guide plus guiding means is covered. In this embodiment, the guiding means can be reused along with the image guide. Other geometrical arrangements of the guiding means, image guide, and illumination fiber are obviously possible. 5 Example 7 - Morphological Imaging Fine detail, for example sub-cellular, endoscopic morphological imaging can be accomplished with the subject scope. An example is the imaging of the peripheral nerve. Realignment of the fine nerve fascicles after nerve transection remains a major difficulty for ligation procedures and successful function recovery. Alignment of fascicles during ligation 10 procedures can be aided by insertion of the subject scope within the distal nerve stump to view microstructure (nerve sheaths) at the surgical interface. Also, excision of tumors growing in association with peripheral nerves often leads to major functional deficits and even mortality. Insertion of the subject scope can assist in distinguishing the tumor-nerve interface (aided by fluorescent cell labeling) to precisely guide excision and avoid incidental nerve damage. 15 The subject probe can be utilized to permit both remote visualization of cells and image chemical concentrations using an analyte-sensitive material on the distal tip. By this means, the user is able to both position the distal tip at the desired location and, most importantly, correlate the chemical measurements with the visual information. Photographs of in vivo tissue samples can also be recorded. Accordingly, the sub-micron resolution of the subject probe can reveal 20 the morphology and chemical correlations with excellent detail. Example 8 - Functional Imaging Sub-cellular functional imaging can be accomplished with the subject scope using, for example, substrates of a family of extracellular matrix-degrading enzymes, the matrix 25 metalloproteinases (MMPs). These enzymes are involved in the ability of cells to infiltrate tissues (invade or metastasize). Secreted as inactive proenzymes and regulated by endogenous inhibitors, a major concern is to localize the active enzymes. Peptide substrates (which fluoresce when degraded) for MMPs can be immobilized onto a GRIN lens incorporated with the subject scope. Localization of enzymatic activity can be examined in relation to cell surfaces and 30 extracellular matrix structures (including vasculature). The sub-cellular functional imaging with the subject probe can also be used to study glutamate, a major excitatory neurotransmitter. Important for this functional imaging is the ability to immobilize the appropriate enzyme, ligand, etc. (in this case it is glutamate dehydrogenase) onto the distal surface of the GRIN lens used for magnification. The GRIN lens 35 material may be glass or acrylic type polymer.

WO 00/54033 PCT/US00/06344 25 With respect to immobilization on a glass lens, the technique for bridging the inorganic to-organic interface requires several steps. A specific technique for covalently bonding glutamate dehydrogenase onto a glass surface has been known for at least twenty years. However, the process involves baking the glass at 120'C for at least 4 hours. Accordingly, there 5 is some concern that there may be some diffusion of the metallic ion concentration which makes the GRIN profile in the glass lens. Also, it is noted that 120'C is above the glass transition temperature of the nearby polymeric GRIN image guide. Accordingly, careful heat sinking is required. The immobilization of bio-molecules onto polymeric surfaces in accordance with the 10 subject invention can involve the technique of covalently bonding heparin onto the surface of intra-ocular acrylic lenses. In this method, a polymeric amine compound, polyethyleneimine is irreversibly absorbed onto the surface of the acrylic. The surface amine groups are made to react with carboxyl groups of bi-functional linker, gluteraldehyde, followed by a final attachment of the glutamate dehydrogenase to the gluteraldehyde. In the subcellular imaging 15 of glutamate, measurements of enzymatic activity, response time, stability, selectivity, and sensitivity can be accomplished. Example 9 - Method and Device for Utilizing Fluorescence to Study Tissue This example describes a method and device which take advantage of the properties of 20 GRIN optical fiber. At least one GRIN fiber can be used to study the fluorescence characteristics of an object under study. The GRIN fiber utilized in this example can comprise, for example, glass or plastic. A first GRIN fiber can be used to excite tissue with short pulses of light, for example in the picosecond range. A second GRIN fiber can then detect fluorescence from the excited tissue, in the picosecond to several nanosecond range. In an alternative 25 embodiment, a single GRIN fiber can be used to carry the light pulses to an area under study and detect the fluorescence from the excited tissue. An endoscope can be used in conjunction with the GRIN fibers, for example to locate organs or tissue for study. The endoscope can incorporate a SI or GRIN image guide coupled to a GRIN lens in the standard endoscope design. The GRIN fiber or fibers may be inserted into 30 the working channel of the endoscope such that a user may utilize the endoscope for correct placement of the tips of the GRIN fibers with respect to the tissue or object of study. If desired, multiple GRIN fibers can be used for either carrying the light pulses to the area under study and/or for detecting the fluorescence from the excited tissue. In addition, a sub-cellular imaging scope (daughter scope) in accordance with the subject 35 invention can be inserted into the working channel of an endoscope (parent scope) such that the WO 00/54033 PCT/US00/06344 26 user may utilize the parent scope to locate tissue of interest and the daughter scope to investigate the tissue. In a specific embodiment, the daughter scope can then be inserted further until it is in contact with the subject tissue. Contacting the tissue of interest can be done utilizing the view from the parent scope to assist the user. The daughter scope can then be used to conduct ultra 5 fast (picosecond to nanoseconds) fluorescence lifetime imaging (FLIM). In this way the type of tissue may be identified from measurement of the fluorescence lifetime. Example 10 - Production of a Fiber Image Guide Which Provides Stereo Vision In many applications, an image guide's diameter is limited by the constraints of the 10 application, for example the internal diameter of the lumen to be imaged or other apertures through which the image guide must pass. Accordingly, there can be a limit to the number of microfibers across each transverse dimension of an image guide for a fixed minimum diameter of step index microfiber, for example microfibers with diameters of about 5.0 microns. An imaging scope utilizing two GRIN image guides, each with microfibers having half the diameter 15 of the original step index microfibers, 2.5 microns in this example, permits the use of a side-by side pair of image guides. Each image guide of the pair can have the same resolution as the original step index guide. In this example, two transmitted images of an object may be utilized from two different locations corresponding to the separate axes of the two image guides. These two images can be used to provide a stereoscopic vision of the subject being viewed. 20 Advantageously, the spatial resolution of the stereo image can be at least as good as the original step index image guide and the transverse dimensions of the stereo image guide can be the same as the original image guide. In certain applications, it may be preferable to utilize three, four, or even more GRIN image guides in a single imaging scope. 25 It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

Claims

Claims 1. A method of studying the fluorescence characteristics of an object, comprising the following steps: transmitting a pulse of light via at least one excitation gradient-index optical fiber to an object; and detecting fluorescence from the object, after excitation of the object by said pulse of hght, via at least one detection gradient-index optical fiber.
2. The method according to claim 1 , wherein said at least one excitation gradient- index optical fiber and said at least one detection gradient-index optical fiber are the same single gradient-index optical fiber
3. The method according to claim 1 , wherein said at least one excitation gradient-index optical fiber and said at least one detection gradient-index optical fiber are the same plurality of gradient-index optical fibers.
4. The method according to claim 1, further compnsing the step of positioning said excitation fiber and said detection fiber with respect to the object while viewing the vicinity of the object via an imaging scope.
5. The method according to claim 1, wherein the fluorescence lifetime is less than or equal to 5 nanoseconds m duration.
6. The method according to claim 5, wherein the fluorescence lifetime is less than 500 picoseconds in duration.
7. The method according to claim 6, wherein the fluorescence lifetime is less than 50 picoseconds in duration.
8. A method of studying an object, compnsing the steps of: receiving an image of an object onto a distal end of an image guide compnsing gradient index optical fibers; transmitting the image of the object from the distal end to a proximal end of the image guide; and detecting the image of the object exiting the proximal end of the image guide.
9. The method according to claim 8, wherein the distal end of said image guide is in contact with said object.
10. The method according to claim 8, wherein the time dispersion introduced to the image from reception to detection is less than 500 picoseconds.
11. The method according to claim 10, wherein the time dispersion introduced to the image from reception to detection is less than 50 picoseconds.
12. The method according to claim 8, wherein before the step of transmitting the image of the object via said image guide, further compnsing the step of: magnifying the received image via a magnifying means located at an appropnate distance from the distal end of the image guide such that a magnified image of the object is incident of the distal end of the image guide.
13. A method of studying an object, compnsing the steps of: receiving an image of an object onto a distal end of an image guide comprising a plurality of step index optical fibers, wherein said distal end of said image guide is m contact with the object; transmitting the image of the object from the distal end to a proximal end of the image guide; and detecting the image of the object exiting the proximal end of the image guide.
14. The method according to claim 13, wherein before the step of receiving an image of an object onto a distal end of an image guide, comprising the step of: magnifying the image of the object with a lens, where the lens is attached to the distal end of the image guide and the distal end of the lens is in contact with the object.
15. The method according to claim 12, wherein the step of magnifying the received image is accomplished by a gradient-index lens.
1 16. The method according to claim 12, wherein the step of magnifying the received

? image is accomplished by a lens system
1 17. The method according to claim 9, wherein before the step of detecting the image

2 of the object exiting the gradient-index image guide, further comprising the steps of

3 magnifying the image exiting the gradient-index image guide; and

4 transmitting the magnified image of the object via an additional image guide compnsing

5 at least one gradient-index optical fiber.
1 18. The method according to claim 17, wherein the step of magnifying the image

2 exiting the gradient-index image guide is accomplished by a gradient index lens
1 19. The method according to claim 17, wherein said additional gradient-index image

2 guide comprises a plurality of plastic optical fibers.
1 20. The method according to claim 8, wherein before the step of receiving an image of

2 an object, further comprising the step of:

3 irradiating the object with light.
1 21. The method according to claim 20, wherein the step of irradiating the object is

? accomplished by transmitting white light to the object via said gradient-index image guide.
1 22. The method according to claim 20, wherein the step of irradiating the object is

2 accomplished by transmitting light, having a wavelength near the absorption maximum of a

3 fluorophore in a sensor attached to a distal end of the image guide, to the object.
1 23. The method according to claim 8, wherein the image guide compnses gradient-

2 index optical fibers having a diameter of between 1.5 μm and 15 μm.
1 24. The method according to claim 23, wherein the image guide compnses gradient-

2 index optical fibers having a diameter of between 1.5 and 5 μm.
1 25. The method according to claim 16, wherein the additional gradient-index image

2 guide is compnses gradient-index optical fibers having a diameter of between 5 and 30 μm.
26. The method according to claim 15, wherein said gradient-mdex lens is approximately 1.39 mm long and positioned approximately 5 mm from a distal end of said image guide.
27. The method according to claim 15. wherein said gradient-index lens has a magnification of between 3 and 30.
28. The method according to claim 17, wherein the method is used to perform subcellular morphological imaging of tissue in contact with the distal end of said image guide.
29. The method according to claim 17, wherein the method is used to perform subcellular functional imaging of tissue, wherein a layer of biosensor is located on the distal end of the image guide and the tissue is in contact with the layer of biosensor.
30. The method according to claim 17, wherein the method is used for typing of tissue, wherein the tissue is m contact with the distal end of the image guide and the tissue is illuminated with a pulse of light such that an image of fluorescence from the tissue is measured, wherein a fluorescence lifetime of the tissue is used to type the tissue.
31. A device for studying the fluorescence characteπstics of an object compnsing: at least one excitation gradient-index optical fiber. a means for launching a pulse of light into said at least one excitation gradient-mdex optical fiber; at least one detection gradient-index optical fiber; and a means for detecting light.
32. The device according to claim 31, wherein the fluorescence of an object can be studied by exciting said object with a pulse of light launched into, and transmitted to the object by, said at least one excitation gradient-mdex optical fiber such that fluorescence from the excited object is received, and transmitted to the means for detecting light, by said at least one detection gradient-index optical fiber, wherein companson of the launched pulse of light and the detected fluorescence from the object can provide information about the object.
33 The device according to claim 31, wherein said at least one excitation gradient- index optical fiber and said at least one detection gradient-mdex optical fiber are a single gradient- dex optical fiber.
34 The device according to claim 31, further comprising an imaging scope, wherein the imaging scope is connected to the excitation fiber and detection fiber such that a view of the vicinity of an object is provided by the imaging scope can be used to position the excitation fiber and detection fiber relative to the object.
35. The device according to claim 34, wherein said imaging scope compnses step-index fibers
36 The device according to claim 34, wherein said imaging scope compnes GRIN fibers.
37. The device according to claim 34 wherein the detection can be moved relative to the positioning scope to contact the object while holding the scope in position.
38. The device according to claim 31 , wherein said means for launching a pulse of light launches pulses of light less than or equal to 1 nanosecond in duration.
39. The device according to claim 38, wherein said means for launching a pulse of light launches pulses of light less than or equal to 100 picoseconds in duration.
40. The device according to claim 39, wherein said means for launching a pulse of light launches pulses of light less than or equal to 10 picoseconds m duration.
41. The device according to claim 32, wherein said means for detecting light can detect the time lag of the fluorescence with respect to the excitation of the object to within 1 nanosecond
42. The device according to claim 32, wherein said means for detecting light can detect the time lag of the fluorescence with respect to the excitation of the object to within 100 picoseconds 43 The device according to claim 32. wherein said means for detecting light can detect the time lag of the fluorescence with respect to the excitation of the object to within 10 picoseconds

44 An image guide, comprising a plurality of gradient-mdex optical fibers, wherein when an input image is incident on a first end of said image guide, each of said plurality of gradient-index optical fibers transmits a pixel of the input image from the first end of the image guide to the second end of the image guide, wherein an output image is formed at the second end of the image guide and wherein said gradient index profile of said optical fibers extends for at least 90% of the cross-sectional area of the fiber

45 The image guide according to claim 44, wherein said gradient-mdex profile of said optical fibers extends for at least 95% of the cross-sectional area of the fiber.

46 An imaging scope compnsing an image guide, wherein said image guide compnses a plurality of gradient-index optical fibers, wherein when an input image is incident on a first end of said image guide, each of said plurality of gradient-index optical fibers transmits a pixel of the input image from the first end of the image guide to the second end of the image guide, wherein an output image is formed at the second end of the image guide, and wherein said gradient-mdex profile of said optical fibers extends for at least 90% of the cross-sectional area of the fiber

47 The image scope according to claim 46, wherein said gradient-mdex profile of said optical fibers extends for at least 95% of the cross-sectional area of the fiber