WO2003010561A2

WO2003010561A2 - Optical coupler for measuring tissue fluorescence

Info

Publication number: WO2003010561A2
Application number: PCT/US2002/023412
Authority: WO
Inventors: Robert G. Messerschmidt; Jim Childs; Pierre Trepagnier; James Mansfield; Jenny E. Freeman; Sean Toy; Britton Chance
Original assignee: Argose, Inc.
Priority date: 2001-07-25
Filing date: 2002-07-24
Publication date: 2003-02-06
Also published as: AU2002322606A1; WO2003010561A3

Abstract

The present invention is directed to a device for efficiently optically coupling an area of tissue to a spectroscope. The optical coupler preferably comprises a rigid partial enclosure having an inside wall that reflects electromagnetic radiation, an opening on the enclosure for the passage of light and one or more mirrors and light detector pairs. The coupler may be a component of a tissue spectroscopic system comprising one or more of the coupler, a spectroscope, and interface medium, a detector and a light source. The method involves detecting optical properties of a tissue and preferably the skin.

Description

OPTICAL COUPLER FOR MEASURING TISSUE FLUORESCENCE

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a device, system, and method for collecting spectral data from tissue. Particularly, the invention relates to optically coupling an area of tissue with a spectroscope.

2. Description of Background

Many attempts have been made to develop a painless, non-invasive external device to monitor physiological parameters, e.g., glucose levels, of tissue. Various approaches have included spectroscopic technologies, such as near-infrared spectroscopy and Raman spectroscopy.

Commercial spectrofluorometer products are available for taking skin fluorescence spectra. These devices often comprise an excitation light source and a detector for acquiring spectra of light emitted, e.g., fluorescence light, from skin upon excitation. A fiber optic probe, i.e., contact probe, is used to couple the skin with the excitation and emission optics by simply pressing the probe against the skin. However, bringing the probe into direct contact with the skin has various disadvantages. For example, the surface of the skin is not perfectly smooth, but contains small hills and valleys, due to pores, wrinkles, hair follicles, and other surface irregularities. These irregularities can lead to small air pockets between the probe and the skin surface. The presence of air pockets may lead to reflection losses and additional scattering due to index mismatch at the probe/air and air/skin interfaces. These losses can induce higher variation in the spectra than would otherwise be the case, thereby generating unnecessary noise.

Another disadvantage is leakage of a portion of the excitation light directly to the detector. Particularly, a portion of the detected light is excitation light that did not interact with the target, e.g., skin, or interacted in an undesirable way such as, e.g., specular reflection off the skin surface. This "cross-talk" is primarily caused by the proximity of the excitation and detection fiber optics.

Another disadvantage with conventional fiber-based devices is the detection of extraneous light from outside light sources. Fluorescence and/or diffusely reflected emission of light from skin can be of very low intensity. Therefore outside light sources can significantly interfere with the weak signals of interest.

Another disadvantage with fiber probes arises from skin heterogeneity. Sub-surface heterogeneity can give rise to variance in emission that is dependent on fiber-probe geometry and probe location. In particular, the average size and number of the fibers will affect measurements taken from skin that is generally heterogeneous due to variations in pigment, wrinkles, or hair density, or anomalies such as scarring. These inhomogeneities can be a problem in vivo fluorescence, for example, since the spectroscopic details of the various anomalies are not of particular interest and, in fact, may obscure the desired information contained in the spectra, hi this case, many spectra at different sites must be obtained and averaged in order to lessen the dependencies on the skin.

The foregoing disadvantages complicate the interpretation of spectral data and can lead to inaccurate measurements of physiological parameters. Furthermore, careful and complicated feedback and control of the fiber probe contact is required to avoid non-repeatable pressure, mechanical shear, and torque forces on skin that are likely to result in additional measurement inconsistencies.

The application of the invention described in this patent to a device, system and/or methods development will both simplify and improve the collection and interpretation of optical emission data contributing to more accurate and repeatable spectral measurements.

SUMMARY OF THE INVENTION

The present invention overcomes the problems and disadvantages associated with current strategies and designs, and provides new devices, systems, and methods for efficiently optically coupling an area of tissue with a light source and spectroscope, probe, or any type of device used for the measurement of optical properties of tissue, e.g. skin.

Accordingly, the invention provides, in preferred embodiment, an optical coupling device for optically coupling tissue to components comprising a cardioid mirror; means for decreasing, increasing, or stabilizing the pressure inside a volume enclosed by the cardioid mirror and tissue; a first detector disposed at the tissue surface; a first mirror connected to the first detector; means for directing excitation light of a first wavelengths (or multiple wavelengths) into the volume to illuminate the tissue; and means for collecting light of a second wavelength (or multiple wavelengths), which may or may not be the same as the first wavelength, emitted from the tissue illuminated with the light having the first wavelength.

In another preferred embodiment, the invention provides, a spectroscopic system comprising an optical coupling device; a plurality of fiber optic cables, and a spectrometer, wherein the plurality of fiber optic cables connect the optical coupling device to the spectrometer and are provided to transmit light comprising excitation radiation from a radiation source to the tissue and radiation comprising fluorescence away from the tissue. A pump is provided to increase, decrease, or stabilize the pressure of the volume within the optical coupling device.

another preferred embodiment, the invention provides, a method comprising the steps of placing an optical coupling device in close proximity to a tissue; decreasing, increasing, or stabilizing the pressure within the optical coupling device; exposing tissue to radiation from a source; and collecting fluorescent light emitted by said tissue.

In another preferred embodiment, the invention provides, emission light at the excitation or absorption wavelength(s) is collected in addition to fluorescence light.

Therefore, this device can be also be used to make absorption measurements insofar as the light emitted is the same wavelength as the excitation light or absorption measurements in other regions. The instant invention provides distinct advantages over existing devices, systems and methods of collecting spectral data from tissue. One such advantage of the invention is that leakage of excitation light into detection optics is eliminated. Another advantage of this invention is that interference from outside electromagnetic radiation is minimized, if not entirely eliminated. Another advantage is that this invention provides accurate measurements of physiological properties by applying a consistent pressure to the skin over time. Another advantage of this invention is the ability to stretch or smooth the skin by changing the pressure applied to the skin, thereby minimizing air gaps that can cause scattering of excitation light. Another advantage of this invention is that it better integrates the emission and excitation light over a skin area large in comparison to typical spatial heterogeneities thereby reducing signal variance across sites.

Other embodiments and advantages of the invention are set forth, in part, in the following description including the figures, and, also in part, will be obvious from this description, or may be learned from the practice of the invention.

DESCRIPTION OF THE FIGURES

While the invention is described and disclosed here in connection with certain preferred embodiments, the description is not intended to limit the invention to the specific embodiments shown and described here, but rather the invention is intended to cover all alternative embodiments and modifications that fall within the spirit and scope of the invention as defined by the claims included herein as well as any equivalents of the disclosed and claimed invention.

Figure 1 An optical coupling device according to an embodiment of the invention.

Figure 2 An optical coupling device having three mirrors (Ml , M2, M3) and three detection options (Dl, D2, D3).

Figure 3 An optical coupling device having four detection options (D 1 , D2, D3 ,

D4), and two sources (SI, S2). Figure 4 An optical coupling device directed to exciting glucose and osmolyte sensors in near UV and NLR.

Figure 5 An optical coupling device have four detector options (Dl, D2, D3, D4), disposed at a diaphragm or skin surface.

Figure 6 An optical coupling device wherein a transparent membrane is eliminated.

DESCRIPTION OF THE INVENTION

As embodied and broadly described herein, the present invention comprises devices, systems and methods for optically coupling tissue to a spectroscope. In particular, the invention is directed to an optical coupler that can be used on skin and other tissues.

Conventional devices and methods for taking optical measurements of tissue do not take into account the nature of the tissue being measured. For example, discrepancies including tissue surface heterogeneity are often overlooked or ignored for the sake of simplicity, or simply because it is not possible to address the discrepancy. However, as is well known, tissue, and specifically skin, contains a vast amount of heterogeneity that includes pigmentation differences, the presence or absence of hair follicles, major and minor wrinkles, and hydration differences, to name a few. These and other variations can both complicate and create inaccuracies in optical measurements.

It has been surprisingly discovered that heterogeneity in the area being examined can be eliminated, completely or at least substantially, by applying certain physical stresses to the area being measured. These physical interactions can be created using an optical coupling device structured according to the strategies of the invention. Preferred embodiments of the invention are discussed in the context of efficiently coupling tissue (e.g. skin), with a spectral analyzer, hi these embodiments, the collected spectral data is useful for studying physiology of the tissue, particularly measuring glucose levels or other analytes. See U.S. Patent Application No. 09/785,547, entitled "Non-hivasive Tissue Glucose Level Monitoring," filed February 20, 2001, which is herein incorporated by reference in its entirety. However, the invention can be practiced in any type of spectroscopic technique where optical coupling is necessary between a sample and optical components.

A preferred embodiment of the invention is depicted in Figure 1, wherein an optical coupling device according to an embodiment of the invention is shown. Preferably, the device comprises a curved reflector having a cross-sectional shape of a cardioid. In other embodiments, alternative cross-sectional and three-dimensional shapes include, but are not limited to, an ellipse, circular structure, oval, sphere, triangle, rectangle, dome structure such as a geodesic dome, or other suitable shapes.

At the focus of this reflector is disposed a detector on top of a small curved mirror. This mirror is fixed on top of a transparent membrane sealed to the curved reflector.

The membrane is flexible and most preferably, elastic. The membrane, which may comprise a means for performing a calibration methodology, can be permanent or alternatively, inexpensively mass-produced and thereby, disposable. The curved reflector is rigid and has a fluid port disposed thereon.

Further, a second opening is provided to allow the transmission of excitation light from a source and emission light collected from the detector without compromising the seal of the fluid, e.g., air, inside the curved reflector, h a preferred embodiment, the second opening is at the top of the curved reflector, however, in other embodiments the opening maybe disposed at other locations.

The device is placed on top of a tissue, such as skin, and the pressure inside the reflector can be reduced causing the membrane to be drawn into the reflector, pulling the skin upwards. A pressure reduction causes the skin surface to convexly deform, as shown in Figure 1. Preferably, the pressure differential is not sufficient to induce discomfort, significant physiological sequellae, or intentional change. Alternatively, the pressure inside can be increased to concavely deform the skin surface. As noted above, this preferably occurs without discomfort, intentional change, or manifest physiological consequence.

The device may be positioned on any desired location of a patient. Preferably, the device may be positioned on skin such as on a forearm or leg, fingernails, earlobes, lips or internal tissues for example during an invasive surgical procedure. In another preferred embodiment, the device is placed on an arm or leg of a patient. The patient is generally a mammal such as a dog or cat, and preferably is a human. However, one will appreciate that the instant invention is applicable to testing other tissues in vitro.

When acquiring a measurement, the exposure to the excitation light is minimal. For example, the exposure time is less than a minute, preferably less than 15 seconds, and more preferably less than 5 seconds. In another embodiment of the invention, two or more tissue areas are examined with the device and emission data averaged, thereby reducing heterogeneity effects, to acquire a more accurate measurement. Further, sampling may be taken over a wide area for normalization purposes. In this manner, changes associated with convex and/or concave deformations can be minimized or completely eliminated. Anomalous measurements maybe removed prior to averaging using commonly known techniques (M.S. Srivastava Methods of Multivariate Statistics, Wiley, 2002).

Another preferred embodiment is shown in Figure 6, wherein the membrane is eliminated and the combination of detector and curved mirror is disposed in either direct contact or close proximity with the skin. Herein, the detector and curved mirror may be connected to curved reflector by a wire grid, which would permit free passage of radiation, or may be connected directly to the inside wall of the curved reflector.

Further, a pump may be provided to increase, decrease, or stabilize the pressure in between the skin and the and curved reflector, thereby allowing the skin to be deformed into a respective concave, convex, or flat configuration. The dashed line denotes changes in tissue deformation, secondary to pressure alterations or adjustments, hi another embodiment of the invention, measurements may be taken at multiple pressures thereby allowing for the evaluation of potential differences between said measurements.

Compositions useful as interface media for facilitating contact between a skin surface and the curved reflector may be used for optically coupling, standardizing, and/or improving contact between the skin surface and curved reflector with or without the membrane. Thus the surfaces of these locations may be prepared prior to excitation to enhance the study of the physiology of the sample. For example, lotions for hydrating tissue, optical gels, pharmaceuticals, desiccants, disinfectants, or blood vessel treatments may be used. Such an arrangement is particularly suited for collecting fluorescence, absorbance or other spectral information from the skin using the embodiments of the present invention. See also U.S. Patent Application No. 09/704,421, entitled "Interface Medium for Tissue Surface Probe," filed November 3, 2000, which is herein incorporated by reference in its entirety.

The source and the detector may be discrete devices or they may consist of optical fibers that may be independently connected to a light source and a spectrometer. The source excites tissue at a first wavelength or first range of wavelengths. Further, the source may be disposed at any location within the device. The detector collects light emitted from the tissue, due to fluorescence, absorption, or scatter of tissue components, at a second wavelength or second range of wavelengths either equal to, overlapping, or entirely different from the first wavelength or range of wavelengths. Particularly in fluorescence of biological molecules, emission wavelengths are longer than the excitation wavelength due to non-radiative transfer of energy to the molecular vibrational and rotational states.

hi other preferred embodiments, a plurality of sources and/or detectors may be employed. For example, multiple sources and detectors can be placed at different angles within the device to take advantage of various wavelengths of light via their reflection at varying angles. Moreover, one or more detectors may be used to acquire depth-resolved spectral information from the tissue.

The present invention effectively couples excitation radiation from a source onto a tissue surface, such as skin, and the emission light from said tissue surface, such as the skin, to a detector. The collection of said emissions is improved by virtue of the curved reflector, which focuses said emitted light onto a detector. Excitation is similarly improved insofar as the cardioid mirror facilitates uniform tissue illumination. The device also has the potential to be used with wavelength or temporally resolved spectral data.

In each case, uniform illumination of the tissue surfaces and collection of spectral data is achieved by careful matching of the optical properties of the fiber, the mirror, and the curvature of the reflector. Particularly, light emitted from the skin will impact the reflector surface, which will then focus said emissions onto a detector. If a fiber is used as the detector then the end can be terminated in a sphere. The sphere is placed at the focus of the reflector, and the light is collected over a large solid angle.

By varying the pressure within the device, tissue surface homogeneity can be increased, thereby improving the accuracy of the spectral measurements. For example, unperturbed skin is generally heterogeneous due to normal variations in contour resulting from wrinkles, appendages and air gaps. Decreasing or increasing pressure in the device, when placed on the skin, will stretch the skin and smooth out its superficial topography. Therefore, skin heterogeneity, a leading contributor to noise generation, can be easily reduced leading to more accurate optical measurements. In addition, the pressure within the device can be adjusted to a desired constant pressure to allow for temporal and spatial pressure normalization between patients, thereby, reducing inter- subject variation(s). Moreover, because measurements are taken over a fixed area of skin, i.e., the area of skin enclosed by the curved reflector, the device allows the use of area integration to normalize skin heterogeneity.

Referring to the preferred embodiment of Figure 2, another optical coupling device of the instant invention is shown. In this embodiment, cardioid mirror M₂ is connected to a transparent diaphragm and placed on a tissue surface, e.g., arm, either with or without a coupling substance interposed between the diaphragm and the skin. A coupling substance can enhance both the transfer of light and the collection of light such as fluorescent spectra, from the tissue to the probe. Usually a liquid, cement (adhesive), or gel, with an index of refraction that closely approximates that of an optical fiber, and is used to reduce specula reflection at the fiber end face, coupling substances herein may be used to match refractive indices of the skin surface and the diaphragm. Further, the substance would be preferably water soluble, non-toxic, and pH buffered. The substance may also contain known fiuorophores and/or chromophores that can support calibration efforts.

The volume enclosed by either the cardioid mirror and diaphragm, the diaphragm and tissue surface, or both, may be evacuated by a pump operating through an opening in the cardioid mirror. Said enclosed volume may hold air or a coupling substance, as described above. Excitation light enters a small hole in the cardioid mirror at point S, and is reflected by mirror M₁ illuminating the surface of the cardioid to produce rays, which enter the tissue at oblique angles and illuminate, by photon diffusion, object Z. Particularly, targets within the tissue, e.g. structural or matrix components such as mitochondria or collagen fibers, are excited by the excitation energy. In turn, the tissue absorbs, emits, and scatters light, some of which will reenter the cardioid by traversing a reverse course whereupon it will be focused on mirror M_\ and returned to the source position S. The longer wavelength fluorescence is readily separated from the incident excitation by mirror M₃, which may be dichroic or silvered, or any other means that reflects a particular band of spectral energy and transmits all others, and collected at detector D₃. Thus, the device fully exploits the inherent optical reciprocity of the cardioid system. A further advantage is conferred through the provision of multiple pathway illumination and secondarily collecting the spectral emissions for analysis. Scattered light may be acquired over the large solid angle created by the geometry of the device, thereby providing additional data for analysis.

Detector D₁ is disposed in contact with the diaphragm and between mirror Mi and the tissue surface. This detector collects radiation directly emitted from the skin. Alternatively, detector D₂ may be disposed inside mirror M_\ so that it may collect returning light reflected off the cardioid. Mirror Mi can be a half silver reflector or dichroic reflector. Excitation and detection wavelengths are determined based on the target within the tissue being investigated. For example as shown in Figure 2, the source wavelength may be 366 nm and the detected wavelength may be 460 nm. Nevertheless, the source wavelength and detected wavelength can be configured to any desired wavelength depending on the desired type of application. Another preferred embodiment of the instant invention is shown in Figure 3, wherein fluorescence targets, such as NADH, are the desired targets within skin. This embodiment is similar to that of Figure 2 with at least the exception that a second source and fourth detector are employed. The second source is positioned in the general location of the first source. A fourth detector, D₄, is added at the location of detector

D₃. In a particular example, the first source emits radiation at 366 nm and the second source emits radiation at 800 nm. Further, detectors D₃ and D₄ collect radiation at 460 and 800 nm, respectively.

One of ordinary skill in the art will recognize that the above source and detection wavelengths are exemplary only and can be changed to other desired wavelengths . For example, the excitation light can comprise one or more unique wavelengths or a continuous distribution of wavelengths in the range of 250-2000 nm, preferably 270- 1100 nm, and more preferably 285-500 nm. The fluorophores responsible for skin autofluorescence in the ultraviolet and blue regions of the spectrum include metabolic components and intermediates, plus proteins and collagen. This includes tryptophan, which fluoresces in the 295-350 nanometer (nm) region, keratin, which fluoresces in the 295-340 nm region, nicotinamide adenine dinucleotide ("NADH"), which fluoresces in the 460 nm region, flavin adenine dinucleotide ("FAD"), which fluoresces in the 525 nm region, and fluorophores associated with collagen cross-links, which fluoresce in a broad region from 420 to 490 nm. (J. Invest. Dennatol. 111:776-780, 1998, and references therein). The collagen cross-link fluorophores are thought to arise through a number of possible chemical transformations, including the Maillard reaction, into stable entities known as advanced glycosylation end products (AGE's). These AGE's form at a higher rate in people with diabetes presumably because of chronic exposure to elevated of tissue glucose levels.

Detected light can comprise one or more unique wavelengths or a continuous distribution of wavelengths in the range of 250-2,000 nm, preferably 320-510 nm, more preferably 315-600 nm, and even more preferably 335- 620 nm, and even more preferably 340-620 nm. In a preferred embodiment of the invention, skin is excited using ultraviolet-visible wavelengths such that the collected spectral information is indicative of a glucose level or diabetic condition of a patient.

Another preferred embodiment is shown in Figure 4, wherein an optical coupling device according to an alternative embodiment is directed to exciting glucose and osmolyte sensors. An osmolyte sensor, in this embodiment, determines osmotic strength on the basis of the numbers of particles suspended in a given solution. In this embodiment, source S₂, along with the distal ends of fiber optic cable, are connected to detectors D₃ and D , such that they are disposed along the diaphragm.

h alternative embodiments, the transparent diaphragm shown in Figures 2-4 can be eliminated allowing the detectors to be placed in direct contact, or indirect contact via an interface coupling substance, with the skin. As in earlier embodiments, the pressure inside the cardioid mirror can be changed to raise, lower, or stabilize (e.g. flatten the skin surface).

In another embodiment of the optical coupling device, shown in Figure 5, all detectors Di, D₂, D₃, and D₄ are disposed along the diaphragm or, in the absence if the diaphragm, on the skin surface. Sources Si and S are disposed at separate locations along the cardioid mirror.

Generally, illuminating a tissue with excitation comprises exciting a target within said tissue. The target may be a structural, cellular, matrix, or molecular species in a patient. Preferably, the target comprises pepsin- or collagenase-digestible collagen cross links, non-pepsin digestible collagen cross links, tryptophan, elastin, elastin cross-links, keratin, serum proteins, Glu-T proteins, NADH, NADPH, flavoproteins (e.g. FAD), melanin precursors, porphyrins (e.g. including hemoglobin, glycosylated hemoglobin Ale, or red blood cells), cytochromes, vitamin B complexes, carotenoid, salicylate (aspirin), lactate, pyruvate, ketones (e.g. acetoacetate and beta-hydroxybutyrate), free fatty acids, succinate, fumarate, dihydroxyacetone phosphate (DHAP), 3- phosphoglycerate, acetyl CoA, succinyl CoA, alpha-ketoglutarate, malate, citrate, isocitrate, bicarbonate, insulin, triglyceride, cholesterol, phosphorus, calcium, blood urea, electrolytes, bilirubin, creatinine, albumin, lactate dehydrogenase (LDH), or combinations thereof.

hi a preferred embodiment, the instant method and apparatus may be combined in a method for the in vivo measurement of at least one biological analyte through tissue exposure to radiation, followed by spectroscopic analysis, preferably selectively evaluating ultraviolet and/or visible light fluorescence, in combination with at least one adjunctive optical measurement selected from the group consisting of infrared, near infrared, and/or visible light absorbance. Alternatively, the spectroscopic measurement is combined with at least one adjunct physiological parameter measurement and/or at least one adjunct informational parameter measurement. These types of adjunct measurements can also be utilized to enhance the calibration of an analyte level quantitation device. An advantage of this preferred embodiment is that robustness and accuracy is added to the non-invasive measurement of the analyte (e.g. glucose), thereby reducing the error of the measurement. At the same time the technology has a clearly viable miniaturization and cost reduction strategy. See U.S. Patent Application entitled

"Adjunct Quantitative System and Method for Non-invasive Measurement of In Vivo Analytes, filed July 24, 2002, based on provisional Application No. 60/307,389 filed My 25, 2001.

hi a preferred embodiment, the invention takes into account contributions of diffuse reflectance (DR) of light that has diffusely reflected from tissue. The diffuse nature arises from scatter that invariably occurs in a medium that has an inhomogeneous distribution of refractive index. Examples of such include cellular tissue where blood, cell organelles, components of interstitial fluid, and variations in cell structure and size all give rise to spatial and/or temporal variations in refractive index. The character of the scatter and the information it contains obviously depends, among other things, on whether the scatter arises from temporal or spatial variations in refractive index.

A convenient way to describe the diffuse reflected spectrum (when more than one wavelength of light is used to interrogate the tissue) is to take the logarithm of the ratio the signal to the incident spectrum. In transmission absorption spectroscopy, this is routinely done to provide estimates of concentrations of absorbers in the medium when Beer-Lambert's law is know to apply. Although Beer-Lambert's law does not apply in tissue reflectance studies, it is none-the-less a useful transformation. The resulting "absorbance" spectrum contains features that may be useful in identifying constituent components.

In the preferred embodiment, the DR spectrum is an optical measurement of the same tissue region from which auto-fluorescence arises and hence provides information about the same tissue area that affects the fluorescence spectra used to measure glucose concentrations. This information can provide improvement in our ability to measure glucose. The sections below describe applications of the DR spectrum toward this end.

With respect to the practical (i.e. engineering) implementation of DR, the instant invention can incorporate components to do such a measurement, such as the instant light sources and spectral filters. The instant coupler's performance requirements may be easily altered between DR and fluorescence measurements in the following areas: (1) resolution of the spectrometer (depending on use of DR spectrum); (2) dynamic range of detector; (3) reference measurement; and (4) interface probe design.

Although only a few exemplary embodiments of the present invention have been described in detail in this disclosure, those skilled in the art who review this disclosure will readily appreciate that many modifications are possible in the exemplary embodiments (such as variations in sizes, structures, shapes and proportions of the various elements, values of parameters, or use of materials) without materially departing from the novel teachings and advantages of the invention. Accordingly, all such modifications are intended to be included within the scope of the invention as defined in the appended claims. Other substitutions, modifications, changes and omissions maybe made in the design, operating conditions and arrangement of the preferred embodiments without departing from the spirit of the invention as expressed in the appended claims.

As used herein and in the following claims, articles such as "the", "a" and "an" can com ote the singular or plural. It is intended that the specification and examples be considered exemplary only.

Claims

Claims:

1. An optical coupling device for optically coupling a tissue to optical components comprising: a rigid partial enclosure having an inside wall, wherein said inside wall reflects electromagnetic radiation; an opening on said rigid partial enclosure for passage of light; and one or more mirror and light detector pairs.

2. The optical coupling device of claim 1 wherein the opening does not allow for the passage of fluid.

3. The optical coupling device of claims 1-2 further comprising another opening disposed on said rigid partial enclosure for the passage of fluid.

4. The optical coupling device of claims 1-3 further comprising a membrane sealed to said rigid partial enclosure such that said rigid partial enclosure and said membrane completely encloses a first volume, wherein said mirror and light detector pairs are disposed on said membrane and in said first volume.

5. The optical coupling device of claim 4 wherein the membrane is elastic and transparent to light.

6. The optical coupling device of claims 4r5 wherein the membrane or interposed with a material that provides calibration information.

7. The optical coupling device of claims 1-6 wherein the light is selected from the group consisting of visible light, fluorescent radiation, ultraviolet radiation, infrared radiation, microwave, and combinations thereof.

8. The optical coupling device of claims 1-7 wherein the rigid partial enclosure has a shape selected from the group consisting of a portion of a cardioid, a portion of an ellipse; a portion of a geodesic dome; a portion of a rectangle, and a portion of a sphere.

9. A spectroscopic system comprising: said optical coupling device of claim 1; a plurality of fiber optic cables; and a spectrometer, wherein said plurality of fiber optic cables connect said optical coupling device to said spectrometer and are provided to transmit said light, wherein said light comprises excitation radiation and radiation collected from said tissue.

10. The spectroscopic system of claim 9 wherein the excitation radiation is visible, ultraviolet, infrared, fluorescence, or combinations thereof.

11. The spectroscopic system of claims 9-10 further comprising a fluid pump, wherein said fluid pump allows a pressure of said fluid inside said optical coupling device to be increased, decreased or stabilized.

12. A method for optically coupling tissue to optical components comprising: placing the optical coupling device of claim 1 in close proximity to, or in contact with said tissue; illuminating said tissue with excitation light; and collecting radiation emitted from said tissue.

13. The method of claim 12 wherein the excitation light is selected from the group consisting of one or more of visible light, fluorescent radiation, ultraviolet radiation, infrared radiation, microwaves, diffuse reflectance, and combinations thereof.

14. The method of claims 12-13 wherein the radiation emitted comprises infrared, fluorescence radiation, Raman, scattering, visible light, or combinations thereof.

15. The method of claims 12-14 further comprising the step of decreasing or increasing the pressure of said fluid in said optical coupling device to induce deformation of said tissue.

16. The optical coupling device of claims 12-15 further comprising measuring the pressure of said fluid in said optical coupling device.

17. An optical coupling device for optically coupling a tissue to optical components, comprising: a cardioid mirror; a first detector disposed substantially near said tissue surface; a first mirror connected to said first detector; means for directing excitation light of a first wavelength or a first range of wavelengths to illuminate said tissue; and means for collecting light of a second wavelength or a second range of wavelengths emitted from said tissue illuminated with said excitation light.

18. The optical coupling device of claim 17 further comprising a transparent diaphragm between said first detector and mirror, and said tissue.

19. The optical coupling device of claims 17-18 further comprising means for changing a pressure inside a volume enclosed by said cardioid mirror and said tissue.

20. The optical coupling device of claims 17-19 further comprising a means for changing a pressure inside a volume enclosed by said cardioid mirror and said diaphragm.

21. The optical coupling device of claims 17-20 further comprising a second detector disposed between said first mirror and said first detector, wherein said first mirror is a dichroic mirror.

22. The optical coupling device of claims 17-21 wherein the first wavelength is less than said second wavelength.

23. The optical coupling device of claims 17-22 wherein the first wavelength comprises 366 nm, and the second wavelength comprises 460 nm.

24. The optical coupling device of claims 17-23 wherein the first range of wavelengths overlaps said second range of wavelengths.

25. The optical coupling device of claims 17-24 further comprising means for directing excitation light of a third wavelength to illuminate said tissue, and means for collecting light of a fourth wavelength emitted from said tissue illuminated with said light of first or third wavelength.

26. The optical coupling device of claim 25 wherein the third and fourth wavelengths are from about 320 nm to 699 mn.

27. The optical coupling device of claims 25 -26 wherein the third and fourth wavelengths are from about 700 nm to 1200 nm.

28. The optical coupling device of claims 17-27 further comprising one or more additional detectors disposed in proximity to said tissue.

29. The optical coupling device of claims 17-28 wherein the first detector is disposed at a focus of said cardioid mirror.

30. A spectroscopic system comprising: said optical coupling device of claims 17-29; a plurality of fiber optic cables; and a spectrometer, wherein said plurality of fiber optic cables connect said optical coupling device to said spectrometer and are provided to transmit said light, wherein said light comprises excitation radiation and radiation emitted from said tissue.

31. The spectroscopic system of claim 30 further comprising a pump for increasing, decreasing, or stabilizing a pressure inside a volume enclosed by said cardioid mirror and said tissue.

32. A method of optically coupling tissue to optical components comprising: placing said optical coupling device of claims 17-29 in close proximity to said tissue; illuminating said tissue with excitation light; and collecting light emitted by said illuminated tissue.

33. The method of claim 32 further comprising increasing, decreasing, or stabilizing a pressure inside a volume enclosed by said cardioid mirror and said tissue.

34. The method of claims 32-33 further comprising increasing, decreasing or stabilizing a pressure inside a volume enclosed by said cardioid mirror and said diaphragm.

35. The method of claims 32-34 further comprising analyzing the light collected to obtain a measurement of an analyte level in said tissue.

36. The method of claim 35 wherein the analyte is glucose.

37. The method of claims 35-36 further comprising: repeating the illuminating tissue step and the collecting light step one or more times; analyzing the light collected from said one or more collecting steps to obtain one or more measurements of said analyte level in said tissue and determining the analyte level.

38. The method of claim 37 wherein analyzing comprises a statistical averaging of the one or more measurements.

39. The method of claim 38 wherein statistical averaging comprises error checking, outlier rejection, or both.

40. The method of claims 32-39 wherein the collected light is fluorescent light emitted by said tissue.

41. The method of claims 32-40 further comprising increasing, decreasing, or stabilizing a pressure within said optical coupling device.

42. The method of claims 32-41 wherein the wavelengths of the excitation light are equal to, overlapping or different than the collected light.

43. The method of claims 32-42 wherein said illuminating is performed with a plurality of sources.

44. The method of claims 32-43 wherein said collecting is preformed with a plurality of detectors.

45. The method of claims 32-44 wherein the tissue comprises skin, fingernails earlobes or lips.

46. The method of claims 32-45 wherein the tissue comprises skin from a leg, an arm, or an inter digital web space of a patient.

47. The method of claims 32-46 wherein illuminating said tissue comprises exciting a target within said tissue.

48. The method of claim 47 wherein the target is a structural, cellular, matrix, or molecular species in a patient.

49. The method of claims 47-48 wherein the target is selected from the group consisting of pepsin- or collagenase-digestible collagen cross links, non-pepsin digestible collagen cross links, tryptophan, elastin, elastin cross-links, keratin, serum proteins, Glu-T proteins, NADH, NADPH, flavoproteins, FAD, melanin precursors, po hyrins, hemoglobin, glycosylated hemoglobin Ale, red blood cells, cytochromes, vitamin B complexes, carotenoid, salicylate, lactate, pyruvate, ketones, acetoacetate, beta-hydroxybutyrate, free fatty acids, succinate, fumarate, dihydroxyacetone phosphate, 3-phosphoglycerate, acetyl CoA, succinyl CoA, alpha-ketoglutarate, malate, citrate, isocitrate, bicarbonate, insulin, triglyceride, cholesterol, phosphorus, calcium, blood urea, electrolytes, bilirabin, creatinine, albumin, lactate dehydrogenase, and combinations thereof.

50. The method of claims 32-49 further comprising measuring diffuse reflectance of light that has diffusely reflected from said tissue.