WO2010144081A1

WO2010144081A1 - Apparatus for raman spectroscopy having an optical fiber probe

Info

Publication number: WO2010144081A1
Application number: PCT/US2009/046823
Authority: WO
Inventors: Robert W. Mcclane; Brandon G. Bentz
Original assignee: University Of Utah Research Foundation
Priority date: 2009-06-10
Filing date: 2009-06-10
Publication date: 2010-12-16

Abstract

Disclosed are apparatuses for detecting Raman scattered light from a sample, including in vivo biological tissue samples. The apparatuses include a sample probe comprising an optical fiber, the optical fiber adapted to deliver excitation light along the fiber to the sample and to collect the Raman scattered light from the sample along the same fiber; an optical module coupled to the sample probe, the optical module adapted to direct the excitation light and the Raman scattered light and to separate the excitation light from the Raman scattered light; and a detector module coupled to the optical module, the detector module adapted to detect the Raman scattered light from the sample over a particular spectral region, including about 2000 cm^-1or less. Also disclosed are methods of using the apparatuses.

Description

APPARATUS FOR RAMAN SPECTROSCOPY HAVING AN OPTICAL

FIBER PROBE

BACKGROUND

[0001] Raman spectroscopy is a particularly powerful analytical technique for identifying and quantifying specific molecular species in complex matrices, including human tissues. When monochromatic light illuminates a molecule of interest, a minute fraction of the incident light can be scattered inelastically, with one or more discrete shifts in wavelength. These light wavelength shifts, termed Stokes shifts, correspond exactly to the various vibrational energy levels of the light scattering molecules. This process, termed Raman scattering, and can provide a highly specific optical fingerprint of the molecule of interest. See, e.g., Bernstein, P. S., et al., Invest Ophthalmol Vis Sci 1998, 39, 2003-2011 and Hata, T. R., et al., Invest Dermatol 2000, 115, 441-448. Moreover, Raman scattering intensity can be correlated to the concentration of the scattering molecules. In resonance Raman spectroscopy, incident illumination having a frequency which overlaps with the absorption spectrum of the molecule of interest is used. This has the effect of strongly enhancing the Raman output signal without using a higher intensity input signal. These resonance Raman signals have much higher intensity than off-resonance Raman signals, which otherwise may be undetectable in some cases.

[0002] Fiber optic probes are often used to analyze biological tissues via Raman spectroscopy. However, the design of suitable fiber optic probes, especially for clinical use and in vivo biological tissues, is particularly challenging. First, the intensity of the Raman signals from the molecules of interest tend to be very weak since only a small proportion of scattered light is Raman shifted. Second, the significant background light from a variety of sources (e.g., broad-band tissue fluorescence, Rayleigh scattered illumination light, fiber optic fluorescence, and Raman signals from the fiber optic) may overwhelm the Raman signals of interest, especially when using shorter wavelength incident light. Finally, accessing in vivo biological tissues with minimal invasion demands that the fiber optic probes be small and flexible. These and other challenges have been thoroughly discussed in at least the following references: Santos et al.,

Analytical Chemistry, Vol. 77, No. 20, Oct. 15, 2005, 6747; Koljenovic et al., Journal of Biomedical Optics, 10(3), 031116 (May/June 2005); Koljenovic et al, Analytical Chemistry, Vol. 79, No. 2, Jan. 15, 2007 , 557; Nijssen et al., Journal ofBiophotonics, 2, No. 1-2, 29-36 (2009); and U.S. Pat. No. 7,499,153.

[0003] Current fiber optic probes have met with limited success in reconciling each of the challenges described above. For example, in order to minimize background light and maximize the intensity of the Raman signal of interest, many conventional fiber optic probes use multiple fiber optics with separate fiber optics for delivering the illumination light and for collecting the Raman signal of interest. These fiber optic probes may also use filters at the ends of the illumination fiber optics to block certain background light from the fiber optic itself. However, due to the number of fiber optics and additional optical components, these probes are bulky, complex, expensive, and not compatible with standard endoscopes. In addition, it can be difficult to maintain the alignment of the illumination and collection fiber optics, further contributing to decreased collection efficiency.

[0004] Another conventional approach uses a single fiber optic to deliver the illumination light and to collect the Raman signal of interest. However, these probes are restricted to detecting Raman signals over a limited spectral region — from about 2500- 3700 cm^"1 — and require the use of specific fiber optic materials which do not exhibit background light within this spectral region. Unfortunately, much useful spectroscopic information is found within lower wavenumber regions, e.g., within the well-known "finger print" region of about 400-2000 cm^"1. Moreover, the restrictions on the types of fiber optic materials that may be used further limits the practicality of these probes.

[0005] Finally, both types of fiber optic designs are most often used with illumination light at relatively long wavelengths (i.e., greater than 600 nm, including about 720 nm or about 785 nm) in order to minimize background tissue and fiber optic fluorescence. However, use of longer wavelengths precludes the use of resonance Raman spectroscopy at lower wavelengths of light (i.e., less than 600 nm, including about 450 nm to about 550 nm) in which numerous molecules of interest exist. SUMMARY

[0006] Provided herein are apparatuses for detecting molecules within a sample using Raman spectroscopy. The disclosed apparatuses include a sample probe having an optical fiber; an optical module; and a detector module. The optical fiber of the sample probe is adapted to both deliver excitation light along the fiber to the sample and collect the Raman scattered light from the sample along the same fiber. Also provided are methods of using the apparatuses in a variety of applications, including medical applications.

[0007] The disclosed apparatuses exhibit a number of advantages over conventional apparatuses using fiber optic sample probes. First, the sample probes of the disclosed apparatuses are considerably less complex, less bulky, and cheaper than those of conventional apparatuses. Second, the disclosed sample probes also exhibit greater collection efficiency and eliminate any need to align separate excitation and collection optical paths. Third, the disclosed sample probes are also compatible with a variety of standard endoscopes, avoiding the need for costly custom-made endoscopes. Although the disclosed apparatuses may be used to detect Raman scattered light from a sample over a broad range of spectral regions, they are particularly suitable for detecting Raman scattered light below about 2000 cm^"1, including within the finger print region. Finally, although the disclosed apparatuses may be used with a variety of excitation wavelengths, they are particularly suitable for use with short wavelengths, enabling further enhancement of the Raman signal due to the wavelength dependency of the Raman cross-section and by facilitating the use of resonance Raman spectroscopy. These and other advantages are further discussed in greater detail below.

[0008] The disclosed apparatuses include a sample probe having an optical fiber; an optical module; and a detector module. The optical fiber of the sample probe is adapted to both deliver excitation light along the fiber to the sample and collect the Raman scattered light from the sample along the same fiber. The optical fiber of sample probe may include one or more such optical fibers. In some embodiments, the sample probe includes multiple optical fibers, each of which is adapted to both deliver excitation along the fiber and collect the Raman scattered light along the same fiber. In other embodiments of the disclosed apparatuses, the sample probe includes no more than a single optical fiber, the single optical fiber adapted to both deliver excitation light along the fiber to the sample and collect the Raman scattered light from the sample along the same fiber. The composition and dimensions of the optical fiber may vary. Exemplary compositions and dimensions are provided below.

[0009] The apparatuses also comprise an optical module coupled to the sample probe. The optical module is adapted to separate light passing through the optical module, including the excitation light used to illuminate the sample and the Raman scattered light generated from the sample. The optical module may serve several other functions, including, but not limited to directing the excitation light to the sample probe and sample and directing the Raman scattered light to the detector module. The optical module may include a variety of optical components configured in a variety of ways to achieve these and other functions. Exemplary optical components and configurations are described below. In some embodiments, the optical module comprises one or more of a wavelength selective device, a collimator, or a lens. In some such embodiments, the wavelength selective device is a holographic optical element.

[0010] The apparatuses also comprise a detector module coupled to the optical module. The detector module is adapted to detect the Raman scattered light from the sample — specifically, from molecules of interest within the sample. The detector module may serve several other functions, such as further separating the excitation light from the sample's Raman scattered light, measuring the intensity of the Raman scattered light, processing the Raman scattered light, and displaying the Raman scattered light. The detector module may include a variety of components configured in a variety of ways to achieve these functions. Exemplary components and configurations are described below. In some embodiments, the detector module comprises one or more of a wavelength selective device, a light measuring device, or a computer. Exemplary wavelength selective devices and light measuring devices are described below. In some embodiments, the wavelength selective device is a spectrometer and the light measuring device is a CCD camera. In some embodiments, the detector module comprises a computer having software adapted to identify and remove background light signals from the Raman scattered light. [0011] The apparatuses having the disclosed detector modules are capable of detecting Raman scattered light from a sample over a variety of spectral regions. In some embodiments, the apparatuses are adapted to detect Raman scattered light from the sample over a spectral region ranging from about 400 cm^"1 to about 4000 cm^"1; or a spectral region of about 2000 cm^"1 or less; or a spectral region ranging from about 1200 cm^"1 to about 1900 cm^"1. However, other spectral regions are possible.

[0012] The apparatuses are compatible with a variety of wavelengths of excitation light. In some embodiments, the wavelength of the excitation light ranges from about 450 nm to about 550 nm, although other wavelengths are possible. Similarly, the power of the excitation light may vary.

[0013] The apparatuses may include a variety of other components, including, for example, a light source. The apparatuses may also include a tubular housing coupled to the sample probe. In some embodiments, the apparatus includes an endoscope coupled to the sample probe. In other embodiments, the apparatus includes a needle coupled to the sample probe.

[0014] The apparatuses may be used to detect a broad range of molecules in a broad range of samples. Regarding the types of molecules, the disclosed apparatuses may be used to detect any molecule capable of generating a sufficiently intense Raman signal. In some embodiments, the types of molecules that may be detected are molecules comprising C=C, C - C₅ C - H bonds, or combinations thereof, wherein the Raman scattered light is generated from the vibrations of one or more of these bonds. In other embodiments, the types of molecules that may be detected are molecules having an optical absorption spectrum that overlaps with the wavelength of the excitation light, thereby providing resonance enhanced Raman scattered light from the sample. However, the apparatuses are not limited to detecting molecules under resonant enhanced conditions. Examples of molecules that may be detected by the disclosed apparatuses include, but are not limited to carotenoids, polymers, drugs, cooking oils, and glycerol. Examples of samples that may be analyzed by the disclosed apparatuses include, but are not limited to consumer products, food products, drug products, and agricultural products. In some embodiments, the sample is a biological tissue sample. The tissue sample may be an in vivo or in vitro tissue sample. [0015] Also disclosed are methods for using any of the disclosed apparatuses to detect molecules within a sample via Raman spectroscopy. These methods find use in a variety of applications, including, but not limited to medical applications, food processing applications, process control applications, pharmaceutical applications, and homeland security applications. These methods and applications are further described below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 shows a schematic illustration of an exemplary embodiment of the disclosed Raman apparatus, including a sample probe, optical module, and detector module. The sample probe includes a single optical fiber used to deliver excitation light to the sample and to collect Raman scattered light from the sample.

[0017] FIG. 2 shows a more detailed schematic illustration of the apparatus of FIG. 1.

[0018] FIG. 3 shows the co-oxidation of β-carotene (BC) in the presence of LOX- 1/LA using UV spectroscopy. These results have been correlated with the decrease in BC due to oxidation observed with the disclosed Raman apparatus.

[0019] FIGs. 4A and B show the appearance over time of β-carotene breakdown products in the presence of LOX/LA as measured using HPLC. These results have been correlated with the decrease in BC due to oxidation observed with the disclosed Raman apparatus.

[0020] FIGs. 5A-5B show the Raman spectra obtained from a variety of samples using the disclosed Raman apparatus, including human soft palate (A) and raw carrot (B).

[0021] FIGs. 6A-6G show the Raman spectra obtained from a variety of samples using the disclosed Raman apparatus, including aspirin (A), soybean oil (B), polycaprolactone (C), glycerol (D), sudafed (E), polycarbonate (F), and ibuprofen (G). DETAILED DESCRIPTION

[0022] Provided are apparatuses for detecting molecules within a sample using Raman spectroscopy and methods of using the apparatuses.

Apparatus

[0023] The disclosed apparatuses include a sample probe having an optical fiber; an optical module; and a detector module. An embodiment of such an apparatus 100 is shown in FIG. 1, including the sample probe 104 having an optical fiber 108, the optical module 112, and the detector module 116. Each of these components is further described below.

Sample Probe

[0024] The optical fiber of the sample probe is adapted to both deliver excitation light along the fiber to the sample and collect the Raman scattered light from the sample along the same fiber. In other words, the excitation light and the Raman scattered light propagate along the same optical fiber in the disclosed sample probes. These sample probes are distinguished from conventional sample probes which generally use separate optical fibers for delivery of excitation light and for collection of Raman scattered light in order to minimize background light generated in the excitation optical fiber. See, e.g., Santos et al, Analytical Chemistry, Vol. 77, No. 20, Oct. 15, 2005, 6747; Koljenovic et al., Journal of Biomedical Optics, 10(3), 031116 (May/June 2005); Koljenovic et al., Analytical Chemistry, Vol. 79, No. 2, Jan. 15, 2007 , 557; and U.S. Pat. No. 7,499,153. Because the disclosed sample probes use the same optical fiber for both excitation and collection, they are considerably less complex, less bulky, and cheaper than conventional sample probes. This feature of the disclosed sample probes also increases collection efficiency and eliminates any need to align separate excitation and collection fibers, since the excitation and collection light cones of the disclosed sample probes inherently overlap.

[0025] The sample probe may include multiple optical fibers in a "fiber bundle" in which one, more than one, or each of the optical fibers are adapted to both deliver excitation light along the fiber to the sample and collect the Raman scattered light from the sample along the same fiber. In some embodiments, the sample probe includes multiple optical fibers, each of which is adapted to both deliver excitation along the fiber and collect the Raman scattered light along the same fiber. In other embodiments of the disclosed apparatuses, the sample probe includes no more than a single optical fiber, the single optical fiber adapted to both deliver excitation light along the fiber to the sample and collect the Raman scattered light from the sample along the same fiber. By "no more than a single optical fiber," it is meant that the sample probe does not include more than one such optical fiber, although the sample probe could include other components.

[0026] The composition and dimensions of the optical fiber may vary. The composition of the optical fibers is not particularly limited and a wide variety of commercially available optical fibers, including standard laser surgery fibers may be used. By way of example only, those optical fibers exhibiting the least background light (e.g., fluorescence and/or Raman scattering from the optical fiber itself) at the excitation wavelength of interest may be chosen. Any of the optical fibers described in U.S. Pat. No. 7,499,153, the entirety of which is hereby incorporated by reference, may be used. However, even those optical fibers exhibiting background light from about 2500-3700 cm^"1 may be used. Similarly, the dimensions of the optical fibers is not particularly limited. By way of example only, those optical fibers having dimensions compatible with standard endoscopes (as opposed to custom-made endoscopes) may be used. Optical fibers having diameters of no more than 1 mm, 600 μm, 400 μm, 100 μm, or even 50 μm may be used. In some embodiments, the optical fiber is a 200 - 600 μm polymer-clad silica fiber with a Tefzel jacket. This and other suitable optical fibers are commercially available.

[0027] Finally, it is noted that the disclosed optical fibers are solid optical fibers. Such optical fibers are distinguished from hollow optical waveguides.

Optical Module

[0028] As noted above, the disclosed apparatuses also comprise an optical module coupled to the sample probe. By "coupled" it is meant that the sample probe and the optical module are in optical communication. The sample probe and the optical module may also be physically connected by a variety of means, examples of which are further described below. The optical module is adapted to separate light passing through the optical module, including the excitation light used to illuminate the sample and the Raman scattered light generated from the sample. The optical module may serve several other functions, such as receiving the excitation light from a light source, directing the excitation light to the sample probe and sample, receiving the collected Raman scattered light from the sample probe and sample, and directing the Raman scattered light to the detector module. The optical module may include a variety of optical components configured in a variety of ways to achieve these functions. By way of example only, the optical module may include a wavelength selective device for separating the excitation light from the Raman scattered light. Wavelength selective devices include, but are not limited to holographic optical elements (HOEs), dielectric filters, prisms, and the like. In some embodiments, the wavelength selective device is a HOE. Similarly, by way of example only, the optical module may include collimators, mirrors, lenses, and the like for directing the excitation light to the sample and the Raman scattered light from the sample.

[0029] In some embodiments, the optical module includes one or more of a holographic optical element, a collimator, or a lens. A non-limiting exemplary embodiment of an optical module 208 is shown in FIG. 2. As shown in FIG. 2, the optical module 208 includes a holographic optical element (HOE) 220 adapted to separate the excitation light 216 from the Raman scattered light 232. The HOE can be selected to reflect a large percentage of the excitation light 216 and transmit a large percentage of the Raman scattered light 232. The optical module 208 also includes a collimator 212 adapted to direct the excitation light 216 to the HOE 220. The HOE 220 is at an angle sufficient to direct the excitation light 216 towards the sample probe 228. The optical module 208 further includes two other lenses 224 and 236. Both lenses 224 and 236 direct the Raman scattered light 232 towards the detector module 238. Lens 224 also focuses the excitation light 216 into the sample probe 228. The optical module 208 of FIG. 2 is further described in the Examples below.

Detector Module

[0030] As noted above, the disclosed apparatuses also comprise a detector module coupled to the optical module. In other words, the detector module and the optical module are in optical communication, although the modules may be physically connected by a variety of means, examples of which are further described below. The detector module is adapted to detect the Raman scattered light from the sample — specifically, from molecules of interest within the sample. By "detect" it is meant that the module is capable of identifying and distinguishing this Raman scattered light above background light (e.g., broad-band sample fluorescence, optical fiber fluorescence, Raman signals from the optical fiber and optical module components, and residual Rayleigh scattered excitation light). The detector module may serve several other functions, such as further separating the excitation light from the sample's Raman scattered light, measuring the intensity of the Raman scattered light, processing the Raman scattered light, and displaying the Raman scattered light. The detector module may include a variety of components configured in a variety of ways to achieve these functions. By way of example only, the detector module may also include a wavelength selective device for further separating the excitation light from the Raman scattered light from the sample. Such wavelength selective devices include, but are not limited to a spectrograph (also known as a spectrometer) for resolving the Raman scattered light from the sample into its frequency components; filters; gratings; prisms; acousto-optic tunable filters; and the like. The detector module may also include a light measuring device for measuring the intensity of the Raman scattered light. Light measuring devices include, but are not limited to a CCD (charge-coupled device) camera, a CMOS (complimentary metal-oxide semiconductor) camera, a Linear Diode Array, a photomultiplier tube, a photodiode, or the like. The detector module may also include a computer for processing and displaying the Raman scattered light. The computer may include software adapted to identify and remove certain background light from the Raman scattered light. By "identify and "remove" it is meant that the software is adapted to mathematically characterize the background light and to subtract this component from the composite spectrum as further described in the Examples below.

[0031] In some embodiments, the detector module includes one or more of a spectrometer, a CCD camera, or a computer. A non-limiting exemplary embodiment of a detector module 238 is shown in FIG. 2, including a spectrometer 244 coupled to the optical module 208, a CCD camera 248 coupled to the spectrometer, and a computer 252 coupled to the CCD camera. In such an embodiment, the computer 252 can include software adapted to identify and remove background light from the Raman scattered light. This background light may include broad-band sample fluorescence, fluorescence from the optical fiber of the sample probe, Raman signals from the optical fiber of the sample probe and optical module components, and residual Rayleigh scattered excitation light. The software may be adapted to identify one or more of these components and subtract it from the spectrum obtained from the CCD camera, leaving the signal of interest — the Raman scattered light from the sample — for further analysis. The computer and software may also be used to control other elements of the apparatus (e.g., components within the optical module 208). The detector module 238 of FIG. 2 is further described in the Examples below.

[0032] It is emphasized that the functions of the optical module and the functions of the detector module may be combined into a single unit. In such embodiments, although there may be a single physical unit, certain components within the unit can be associated with the optical module and the detector module as those modules are defined above.

[0033] The detector module and thus, the disclosed apparatuses, are capable of detecting Raman scattered light from the sample over a variety of spectral regions. The range of spectral regions is not particularly limited, but rather depends upon the Raman spectrum of molecule of interest in the sample to be analyzed. In some embodiments, the apparatuses are adapted to detect Raman scattered light from the sample over a spectral region ranging from about 400 cm^"1 to about 4000 cm^"1. In other embodiments, the apparatuses are adapted to detect the Raman scattered light from the sample over a spectral region of about 2000 cm^"1 or less. In further embodiments, the spectral region is about 1900 cm^"1 or less. In yet other embodiments, the spectral region is from about 1200 cm^"1 to about 1900 cm^"1, including from about 1300 cm^"1 to about 1800 cm^"1, from about 1400 cm^"1 to about 1700 cm^"1, or from about 1500 cm^"1 to about 1600 cm^"1 _. Many of these regions fall within the so-called "fingerprint region," a particularly useful spectral region from about 400 cm^"1 to about 2000 cm^"1 rich in information for identifying a variety of molecules, including biomolecules. The detection of Raman scattered light (i.e., identifying and distinguishing this light over background light) below about 2000 cm^"1 is not possible with conventional single optical fiber sample probes (i.e., those sample probes having a single optical fiber for excitation and collection) because these apparatuses have not been able to solve the problem of the significant background light and fluorescence generated in the sample probe itself. See, e.g., Santos et al, Analytical Chemistry, Vol. 77, No. 20, Oct. 15, 2005, 6747; Koljenovic et al., Journal of Biomedical Optics, 10(3), 031116 (May/June 2005); Koljenovic et al., Analytical Chemistry, Vol. 79, No. 2, Jan. 15, 2007 , 557; and U.S. Pat. No. 7,499,153. As shown in the Examples below, the disclosed apparatuses have solved these problems.

Excitation Light

[0034] The wavelength of the excitation light delivered by the sample probe is not particularly limited, but rather depends upon the available light sources (further described below) and the molecule of interest in the sample to be analyzed. In some embodiments, the wavelength of the excitation light ranges from about 450 nm to about 550 nm. This includes embodiments in which the wavelength of the excitation light is about 473 nm, about 488 nm, about 514 nm, or about 532 nm. However, other wavelengths are possible, including near infrared and infrared light. Use of any of the shorter wavelengths (i.e., less than about 600 nm) would not be possible, or would be avoided with conventional sample probes (both sample probes with multiple, separate excitation and collection optical fibers and sample probes with a single excitation and collection fiber) due to the significant background light generated at these short wavelengths. See, e.g., Huang, et al., Photochemistry and Photobiology, 2005, 81 : 1219 and U.S. Pat. No. 7,499,153.

[0035] The power of the excitation light may vary and depends upon considerations such as generating large enough Raman signals from the sample to be detected and minimizing damage to the sample, especially when the sample is an in vivo biological tissue. In some embodiments, the power of the excitation light is no more than about 10 mW, no more than about 5 mW, no more than about 2 mW, or no more than about 1 mW.

Other Components

[0036] The disclosed apparatuses may include a variety of other components. By way of example only, the apparatuses may include a light source coupled to the optical module. A variety of light sources may be used, depending upon the desired wavelength of the excitation light. Non-limiting examples of light sources include a lamp, a laser, or a LED (light emitting diode). Lamps, lasers, and LEDs capable of providing any of the disclosed excitation wavelengths are known and are commercially available. The apparatuses may further include any components necessary for coupling the various elements of the apparatus. By way of example only, optical fiber cables may be used to couple the light source to the optical module and the optical module to the detector module. Such cables are known and are commercially available.

[0037] The disclosed apparatuses may also include a tubular housing coupled to the sample probe. This tubular housing may be flexible or rigid and may serve to protect the optical fiber of the sample probe as well as facilitate insertion of the sample probe into the sample to be analyzed. In some embodiments, the disclosed apparatuses include an endoscope coupled to the sample probe. The sample probe may be adapted to fit within the working channel of the endoscope or a Raman-capable endoscope may be constructed with an optical fiber permanently incorporated into its design. Any endoscope may be used. As noted above, certain embodiments of the disclosed sample probes have the advantage of being compatible with a variety of standard endoscopes, avoiding the need for a costly custom-made endoscope, which may be bulky and invasive. Standard endoscopes are known and are commercially available.

[0038] In another embodiment, the disclosed apparatuses include a needle, such as those used to biopsy human tissue, coupled to the sample probe. Because very small diameter optical fibers may be used for the disclosed sample probes, the sample probes can easily fit within the lumen of a needle. The sample probe may be glued to the inside of the needle. Sample access is particularly easy when the sample probe is coupled to a needle.

Samples

[0039] The disclosed apparatuses may be used to detect a broad range of molecules in a broad range of samples. Regarding the types of molecules, the disclosed apparatuses may be used to detect any molecule capable of generating a sufficiently intense Raman signal. By "sufficiently intense" it is meant that the molecule generates a Raman signal that is intense enough to be identified and distinguished above background light. Whether the molecule is capable of generating a sufficiently intense Raman signal depends upon a number of factors such as the nature of the molecule, the nature of the sample, the wavelength and intensity of the excitation light, and the composition of the optical components and other elements in the apparatus. In some embodiments, the types of molecules that may be detected are molecules comprising C=C, C - C₅ C - H bonds, or combinations thereof, wherein the Raman scattered light is generated from the vibrations of one or more of these bonds. In other embodiments, the types of molecules that may be detected are molecules having an optical absorption spectrum that overlaps with the wavelength of the excitation light, thereby providing resonance enhanced Raman scattered light from the sample. Resonance enhanced Raman spectroscopy has been described above.

[0040] In some embodiments, the molecules are carotenoids or polymers. Non- limiting examples of polymers include polyethylene and polystyrene. Non- limiting examples of carotenoids include α-carotene, β -carotene, lycopene, zeaxanthin, and lutein. Any of the carotenoids described in the following references, the entirety of each of which is hereby incorporated by reference, may be used: U.S. Pat. Nos. 5,873,831, 6,205,354, 7,039,452, and 7,215,420. Carotenoids, a subclass of phytochemicals, are described in further detail below. Other possible molecules include, but are not limited to drugs, cooking oils, and glycerol. The Examples below show Raman spectra of such types of molecules obtained using an exemplary embodiment of the apparatus described herein.

[0041] Similar to the types of molecules that may be analyzed with the disclosed apparatuses, the types of samples that may be analyzed are not particularly limited. In general, a suitable sample is any sample that may include a molecule of interest to be detected. Non-limiting examples include consumer products, food products, drug products, and agricultural products. In some embodiments, the sample is a biological sample, such as a tissue sample. The term "tissue" refers to tissue of a human, animal, or plant origin, but in some embodiments, the sample is human or animal tissue. Non- limiting exemplary tissues include skin, tissues of the mouth (e.g., tongue, buccal mucosa, floor of mouth, soft palate), trachea, cervix, colon, and lungs. However, the sample may be any tissue that may be accessed with an endoscope or a needle (which may be coupled to the disclosed sample probes as described above). Both in vitro and in vivo tissue samples may be analyzed with the disclosed apparatuses. As discussed above, conventional sample probes (both sample probes with multiple, separate excitation and collection optical fibers and sample probes with a single excitation and collection fiber) have significant difficulties analyzing both types of tissues, especially using shorter wavelength excitation light (e.g., 450 nm - 550 nm) and detecting Raman signal at lower wavenumbers (e.g., less than 2000 cm -^"U ).

Carotenoids

[0042] Phytochemicals are bioactive non-nutrient plant compounds within fruits, vegetables, and whole grains thought to have great potential as chemopreventive agents. One of the most widely studied subclasses of phytochemicals is the carotenoids¹. Carotenoids are fat soluble pigments comprised of a 40-carbon skeleton characterized generally by a centrally located all-trans polyene conjugated double bond backbone of eight isoprenoid units. This conjugated polyene backbone is very effective at trapping singlet oxygen or nitrogen radicals, thus reducing oxidative stress (See Heber, D.; Lu, Q. Y. Exp Biol Med (Maywood) 2002, 227, 920-923.) and consequently reducing the risk of cancer or cardiovascular disease. In addition, carotenoids seem to offer a variety of other mechanisms of cancer protection (See Heber, D.; Lu, Q. Y. Exp Biol Med (Maywood) 2002, 227, 920-923.), contributing to their overall cancer benefit. Based upon prevailing epidemiologic and in vitro data, clinical trials have explored the use of carotenoids as cancer chemopreventive agents.

[0043] Despite scientific optimism that a specific phytochemical such as carotenoids may represent the "magic bullet" to cancer prevention, the true impact of carotenoids on the incidence of cancer has been brought into question as a result of several conflicting clinical trials. See Greenberg, E. R., et al, N EnglJ Med, 1990, 323, 789-795.; N EnglJ Med 1994, 330, 1029-1035; Hennekens, C. H., et al., N EnglJ Med 1996, 334, 1145- 1149; and Omenn, G. S., et al., N EnglJ Med, 1996, 334, 1150-1155. Initial trials demonstrated a protective effect, such as the Linxian, China trial (See Blot, W. J.,et al., J Natl Cancer Inst, 1993, 85, 1483-1492.) that found that the patients receiving β- carotene (BC)/vitamin E/selenium had a lower rate of cancer development (RR=O.87, 95% CI =0.75-1.00) and specifically a lower risk of stomach cancer (RR=0.79, 95% CI=O.64-0.99). Other carotenoid trials demonstrated no effect upon the risk of cancer development, such as the Physician's Health Study group. See Hennekens, C. H., et al., N Engl J Med, 1996, 334, 1145-1149. More surprisingly, two large trials (the ATBC and CARET trials) have found that supplementation with BC, α-tocopherol, and/or vitamin A resulted in a statistically significant increase in the risk of developing lung cancer and an increased overall mortality in actively smoking subjects, while the non- smoking cohort actually benefited from a slight protective effect. See N Engl J Med, 1994, 330, 1029-1035 and Omenn, G. S., et al, N EnglJ Med, 1996, 334, 1150-1155. A better understanding the nuances of carotenoid biology may allow for tailoring of chemopreventive interventions based upon carotenoid's modes of action under various oxidative conditions.

[0044] Several hypotheses have been proposed for these surprising results, including carotenoids: 1) being a potential promoter of pre-existing latent lung cancer in smokers (See Elsayed, N. M., et al, Nutr Res, 2001, 21, 551-567.); 2) decreasing the absorption of better antioxidant carotenoids (See Elsayed, N. M., et al., Nutr Res, 2001, 21, 551- 567.); 3) enhancing smoker's lung function, thus permitting tobacco associated carcinogens to reach deeper into smoker's lungs (See Grievink, L., et al., Eur J Clin Nutr, 1999, 53, 813-817.); and 4) inducing the formation of metabolites diminishing retinoic acid signaling (See Wang, X. D., et al., J Natl Cancer Inst, 1999, 91, 60-66.). More recently, the possibility has been posed that carotenoids may act as antioxidants in certain circumstances (See Palozza, P., et al., Methods Enzymol, 1992, 213, 403-420.), while being potent and long-acting pro-oxidants in other circumstances. See Palozza, P., et al., Free Radic Biol Med, 2001, 30, 1000-1007 and Palozza, P., et al., Int J Cancer, 2002, 97, 593-600. Under conditions of high oxidative stress, such as during ongoing smoking, smoke-induced oxidative stress may tilt the balance of carotenoid's effect toward pro-oxidant characteristics. See Palozza, P., Nutr Rev, 1998, 56, 257-265 and Palozza, P., et al., Carcinogenesis, 2004, 25, 1315-1325. Cigarette smoke contains an abundance of free radical species (See Pryor, W. A., Environ Health Perspect, 1997 ', 105 Suppl 4, 875-882 and Pryor, W. A., et al., Chem Res Toxicol, 1998, 11, 441-448.), and evidence demonstrates that exposure of plasma to cigarette smoke leads to the destruction of carotenoids and α-tocopherol. See Grievink, L., et al., Eur J Clin Nutr, 1999, 53, 813-817 and Handelman, G. J., et al., Am JCHn Nutr, 1996, 63, 559-565. Furthermore, the oxidation of BC by smoke has been reported to generate various oxidation products, including 4-nitro-β -carotene, β-apocarotenals, and BC epoxides (See Wang, X. D., et al., J Natl Cancer Inst, 1999, 91, 60-66; Baker, D. L., et al., Chem Res Toxicol, 1999, 12, 535-543; and Arora, A., et al., Carcinogenesis, 2001, 22, 1173- 1178.), some of which are unstable under conditions of high oxidative stress potentially contributing to further oxidation. Moreover, the pro-oxidant character of BC may be enhanced by high carotenoid concentrations, such as with supplementation during the ATBC and CARET trials (3.0 and 2.1 mg/1 respectively) when compared to those reported for the average US population (0.05-0.5 mg/1). See Mayne, S. T., Faseb J, 1996, 10, 690-701. Overproduction of free radical species by BC itself has been demonstrated in vitro as a consequence of high concentrations (See Palozza, P., et al., Free Radic Biol, Med 2001, 30, 1000-1007.), which may occur through different mechanisms such as changes in P450 enzymes (See Paolini, M., et al., Carcinogenesis, 2001, 22, 1483-1495.) and/or iron levels (See Garcia-Casal, M., et al., JNutr, 1998, 128, 646-650.). Finally, BC has been found to synergize with and exacerbate tobacco smoke condensate's ability to cause DNA oxidative damage and alter p53 -related pathways of cellular proliferation and apoptosis. See Palozza, P., et al., Carcinogenesis, 2004, 25, 1315-1325. Therefore, these data suggest that carotenoids may be either protective or detrimental relative to the oxidative environment in which they exist.

[0045] Carotenoids are an excellent example of an area of nutrition where relatively little understanding of variation in bioavailability exists. Previous chemopreventive trials have estimated carotenoid bioavailability by measuring dietary intake or serum concentrations. Estimations of carotenoid bioavailability by intake or serum measures are fraught with many shortcomings. See Faulks, R. M., et al., Biochim Biophys Acta, 2005, 1740, 95-100. Because carotenoids are hydrophobic, and predominantly associated with lipid domains of foods and tissues, lipid-containing foods that are ingested with the carotenoid source can significantly impact absorption. The measurement of plasma concentrations must take into account several dynamic processes including gut absorption, chylomicron response, conversion to other interrelated carotenoids, isomerization, and tissue absorption. Therefore, present studies evaluating only carotenoid intake or serum concentrations are only very rough estimates of bioavailability in at-risk tissues. Therefore, methods to measure carotenoids concentrations within specific tissues at risk of malignant degeneration have great potential for further defining carotenoid biology and pharmacokinetics.

[0046] Today, carotenoid levels within tissues are measured using high-pressure liquid chromatography (HPLC). This technique is difficult to perform in epithelial tissue because it requires relatively large volumes of tissue. The biopsy process itself induces a huge oxidative insult within the tissues under study, confounding results obtained. Nevertheless, the scientific basis of carotenoid function in the human body has been extensively studied for over 30 years using HPLC methodology.

[0047] Fluorescence in biological tissues often masks weak Raman signals, especially when shorter wavelength visible excitation light is used. Carotenoids have a long conjugated carbon double-bond backbone structure which when resonantly excited in their absorption bands in the visible (blue-green) wavelength range of 450 to 550 nm exhibit strong resonant enhancement (~ 5 orders of magnitude) of the vibrational energy levels of these bonds to be selectively detected with Raman spectroscopy. The resonance enhancement of carotenoid' s Raman signal allows measurements to be performed even in the presence of intense tissue fluorescence. However, it is emphasized that the disclosed apparatuses may be used to detect carotenoids and other molecules even when the Raman spectra of such molecules in not resonantly enhanced.

Methods and Applications

[0048] Also disclosed are methods for using any of the disclosed apparatuses to detect molecules within a sample via Raman spectroscopy. The methods involve generating Raman scattered light from the sample using any of the apparatuses described herein and detecting the Raman scattered light with the apparatuses. The step of generating the Raman scattered light from the sample involves bringing the sample probe of the apparatus, e.g., the tip of the sample probe, in proximity to the sample to be analyzed. Excitation light from the sample probe illuminates the sample (as well as the molecules of interest within the sample), thereby generating Raman scattered light from the sample. By "proximity," it is meant that the sample probe may directly contact the sample to be analyzed or the sample probe may be brought to within a small distance of the sample. By way of example only, the sample probe may be brought to within 5 cm, 2 cm, 1 cm, 5 mm, 2 mm, 1 mm, or even less of the sample. However, other distances are possible. The sample may be illuminated with excitation light from the sample probe for various periods of time. The period of time may be long enough to obtain a sufficiently intense Raman signal from the sample, but short enough to minimize any damage to the sample from the excitation light. By way of example only, the sample may be illuminated with excitation light for about 1 minute, 30 seconds, 20 seconds, 10 seconds, 5 seconds, or even less. [0049] The step of detecting the Raman scattered light involves directing the Raman scattered light from the sample probe towards the detector module of the apparatus. As described above with reference to the disclosed detector modules, detecting the Raman scattered light can involve processing the Raman scattered light (e.g., to remove background light) and displaying the Raman scattered light. The existence of a Raman signal within a particular spectral region can be used to determine the existence and identity of a molecule within the sample. The intensity of the Raman signal can be correlated to the concentration of the molecule within the sample.

[0050] The ability to identify and quantify certain molecules in certain samples using Raman spectroscopy has a broad range of applications, including, but not limited to, medical applications, food processing applications, process control applications, pharmaceutical applications, and homeland security applications. Raman spectroscopy is a form of rapid and non-destructive identification of chemical compounds. No sample preparation is needed. As noted above, the disclosed apparatuses for Raman spectroscopy provide access to samples not possible with conventional apparatuses and ensure inherent optical alignment between the excitation light and the sample volume to be measured. In addition, the disclosed sample probes are sufficiently low cost that they may be simply disposed if contaminated during a measurement. Finally, the sample probes, which may be of very small diameter, allow even the interior of a variety of samples to be measured in a minimally invasive way.

[0051] In a medical application, any of the disclosed apparatuses may be used in a method for detecting molecules in the tissues of a subject having a disease or disorder, or at risk for a disease or disorder, via Raman spectroscopy. The term "tissue" has been defined above. By "subject" it is meant any animal, including mammals, e.g., a human, a primate, a dog, a cat, a horse, a cow, a pig, or a rodent, e.g., a rat or mouse. The ability to identify and quantify certain molecules in the tissues of such subjects is important at least for determining whether the subject is at risk for the disease or disorder; for assessing the progression of the disease or disorder; and for assessing the effect of a treatment of the disease or disorder. The disclosed apparatuses may also be used to detect the margin of a tumor for subsequent surgical removal. [0052] By way of example only, carotenoids are a class of molecules that may provide a degree of biologic protection against the formation of malignancies in various tissues. Carotenoids have been shown in animal models to prevent carcinoma formation is tissues such as skin, salivary gland, mammary gland, liver, and colon. In addition, low levels of carotenoids and related substances such as retinoids have been assessed as high risk factors for malignant lesions. Having low levels of the carotenoid lycopene has been associated with prostate and cervical cancer; the carotenoids lutein, zeaxanthin, α-carotene, and β-carotene with lung cancer; and β-carotene with oral cancer. Thus, identifying and quantifying these and other carotenoids in the tissues of subjects can be used to provide an indicator of the subjects' risk of developing the cancer, the presence and extent of the cancer, and/or the efficacy of any kind of cancer treatment. See also, e.g., U.S. Pat. Nos. 5,873,831, 6,205,354, and 7,039,452, each of which is hereby incorporated by reference in its entirety.

[0053] As noted above with respect to the description of carotenoids, in other diseases or disorders, possibly in conjunction with other environmental situations (e.g., tobacco or alcohol use), the relationship between carotenoids, the underlying disease or disorder, and the environmental situation is less well understood. In such cases, identifying and quantifying carotenoids, especially β-carotene, can be used to provide a wealth of information about the relationship between carotenoids, the underlying disease or disorder, and the environmental situation.

[0054] In a homeland security application, the disclosed apparatuses may be used to detect a variety of explosives, including, but not limited to RDX (a C4 explosive) or TNT. Similarly, bio-weapon material, such as anthrax, may be detected with the apparatuses. As noted above, the disclosed sample probes may be used to analyze samples that may contain such hazardous materials in a minimally invasive way, even through containers.

[0055] In a food processing or agriculture application, the disclosed apparatuses may be used to analyze the interior of a variety of food or agriculture products for contaminants or quality control. By way of example only, the apparatuses may be used to detect the carotenoid levels in such products, which provides an indication of oxidative deterioration. See, e.g., U.S. Pat. No. 7,215,420, which is hereby incorporated by reference in its entirety.

[0056] Additional description is provided with use of the following non- limiting examples.

EXAMPLES

[0057] Chemical and Biological Materials: For the cell line work, ERC293 cells were maintained in DMEM in a humidified incubator at 37⁰C with 5% CO₂. The media was supplemented with 10% fetal bovine serum, 2mM L-glutamine, ImM sodium pyruvate, non-essential amino acids, and 50units/ml penicillin and streptomycin (GIBCO/Invitrogen, Carlsbad, CA). Ponasterone A (A.G. Scientific, Inc., San Diego, CA), β-carotene (BC) (Sigma-Aldrich, St. Louis, MO), linoleic acid (LA) (Cayman Chemical, Ann Arbor, MI), arachidonic acid (AA) (Nu-Chek Prep, Inc., Elysian, MN), methyl-β-cyclodextrin (MβCD) and soybean lipoxygenase- 1 type IB (15-LOX-l) (Sigma-Aldrich, St. Louis, MO) were all used in these cell experiments.

[0058] Single Optical Fiber Probe for Raman Spectroscopy: An apparatus for Raman spectroscopy was designed with a single optical fiber compatible with standard endoscopes. Based upon the absorption spectrum of BC, which exhibits a maximum at about 460 nm, an excitation laser (Spectra-Physics model 163) at 488 nm was chosen for these experiments. The apparatus 200 is shown in FIG. 2. The excitation light was launched into a multi-mode optical fiber patch cable 204 (SMA-connectorized 200 μm polymer-clad silica fiber; 3M). This cable 204 was then coupled to a custom made optical module 208 via a collimator 212 and gimbal mount. The collimated excitation light 216 was directed onto a custom made holographic optical element 220 (HOE: Ralcon Development Lab, Paradise, UT) at an angle such that it was diffracted toward a lens 224 focusing the light into the sample probe 228, which included a single optical fiber probe, a SMA-connectorized endoscopy probe fiber (200 - 600 μm polymer-clad silica fiber; 0.22-0.48 numerical aperture; with Tefzel jacket). The distal end of this single optical fiber probe was flat-cut and polished flush with the jacket making it compatible with most endoscope channels. The single optical fiber probe served as both an excitation fiber and a Raman scattered light collection fiber, directing the back- scattered light toward the optical module 208 where it was collimated and passed through the HOE 220. The considerable Rayleigh scattered light from the tissue was diffracted out of the collimated beam by the HOE 220, while Raman scattered light (and tissue fluorescence, etc.) 232 was selectively passed through the HOE to another lens 236 which then focused the Raman scattered light into a SMA-connectorized round-to- linear fiber bundle 240 coupled to the spectrometer 244 (Spectra-Physics model 77400, with fiber bundle input accessory). A high dynamic range CCD camera (SBIG ST- 9XEI) 248 was used in the spectrograph 244. The CCD camera 248 was coupled a laptop computer 252 running customized Lab View software (written in Lab View, National Instruments, Inc.) which controlled certain elements of the apparatus (e.g., the optical module, 208) and provided data acquisition and signal analysis. The optical module 208 also contained a software controlled shutter and laser light intensity monitor (not shown) which was used for signal correction.

[0059] The sample probe 228 was 4 m long and could be easily positioned in contact with tissue anywhere there is endoscopic access. The sample probe was momentarily ( about 5-20 seconds) held in contact with the tissue under study during a measurement. Laser light illuminated the tissue, and Raman back-scattered light was collected by the sample probe 228 and routed back to the optical module 208 and then to the spectrograph 244.

[0060] Light entering the spectrograph 244 was a mixture of background signal (broad-band tissue fluorescence, single optical fiber probe fluorescence, Raman signals from the probe and optical module components, and residual Rayleigh scattered excitation light), and the carotenoid's characteristic Raman signal, the carbon double bond "fingerprint signature" at 1525 cm^"1. The apparatus 200 was designed to specifically collect Raman signals only from the 1200 - 1900 wavenumber region, and block other interfering signals since the BC signal at 1525 cm^"1 is the peak of interest. The software characterized the relatively intense broad-band background fluorescence within this wavenumber region using a polynomial fit and subtracted this component from the composite spectrum, leaving only narrow-band Raman signals for further analysis. Confounding Raman signals arising from the optical fiber probe usually preclude using a single fiber for Raman endoscopy in this wavenumber range. However, in the disclosed apparatus, these obstructing signals are minimized by selecting the optical components to be out of resonance and/or out of the analysis wavenumber range, thereby causing no interference. The isolated 1525 cm^"1 C=C resonance Raman carotenoid peak was further characterized using a Lorentzian line shape, and the peak attributes are reported. The Lorentzian peak height at 1525 cm^"1 is linearly proportional to the tissue carotenoid concentration. See Hata, T. R., et al., J Invest Dermatol, 2000, 115, 441-448 and Bernstein, P. S., et al., Arch Biochem Biophys, 2004, 430, 163-169. The apparatus was calibrated for wavenumber and intensity stability using the polymer (a synthetic polyester film) Raman signature from the track pad of the laptop computer before any measurements are made. In this configuration and with this calibration procedure, this apparatus gave relative standard deviations of less than 10% on all tissue samples.

[0061] Confirmation of the ability of the apparatus to detect BC before and after oxidative stress in the Examples described below was confirmed by comparison to UV- vis spectrophotometry and HPLC measurements (not shown).

[0062] The apparatus was also used to analyze the tracheal mucosa of a human subject. A sample probe having a single 600 μM optical fiber was inserted through a ventilating bronchoscope. The probe was brought into contact with the tracheal mucosa just superior to the carina and mainstem bronchi. The mucosa was illuminated with the blue/green laser light and Raman measurements were made through the same fiber optic. A photograph of the sample probe contacting and illuminating the traceheal mucosa was taken (not shown).

Example 1: Measurement of β-carotene (BC) in Intact Human Cells in vitro.

[0063] Methods: To determine the utility of the sample probe with the single optical fiber to detect carotenoids in live cells, ERC293 cells engineered to conditionally express stable \5-lipoxygenase-l (15-LOX-l), a dioxygenase enzyme that imposes oxidative stress in the presence of AA or LA (See Cordray, P., et al., J Biol Chem, 2007, 282, 32623-32629.) were studied. Additionally, ERC293 cells were engineered to express the corresponding point mutations of these LOX genes which are catalytically inactive as controls.

[0064] 15-LOX-l engineered ERC293 cells under the control of the ponasterone- responsive promoter were grown to 80% confluence in media with or without lOμM ponasterone A as previously described. See Yu, M. K., et al., J Biol Chem, 2004, 279, 28028-28035. These cells were then exposed overnight to 0.16% (max) methyl-β- cyclodextrin (MβCD) alone or in combination with 0.2 or 1.0 μM BC. See Pfitzner, L, et al., Biochim Biophys Acta, 2000, 1474, 163-168. Cells were incubated for 10 minutes at 37⁰C with vehicle or 60 μM AA to generate 15-hydroperoxy-5,8,l l-cis-13-trans- eicosatetraenoic acid, and oxidant stress. See Cordray, P., et al., J Biol Chem, 2007, 282, 32623-32629. At the times indicated, cells were harvested by scraping, washed twice, resuspended in cold PBS, and pelleted by centrifugation (40Og, 5 minutes). Three sequential Raman measurements of the cell pellet were obtained and averaged. The cell pellet was subsequently solubilized by the method of Chichili et al. (See Chichili, G. R., et al., BrJNutr, 2006, 96, 643-649.), and BC was extracted with ethanol/hexane and quantified by absorption spectrophotometry at 456nm. Results were correlated with Raman measurements of BC in pelleted cells.

[0065] BC was also extracted from the cell pellets for HPLC by freeze/thawing the pellet in liquid nitrogen, solubilizing with ethanol, and extracting BC with three hexane extractions. Hexane was evaporated over nitrogen in a Rotovap. These samples were fractionated by HPLC on a Phenomenex Gemini Cl 8 analytical column (Torrance, CA) eluted with mobile phase consisting of 65/25/10 v/v/v of Acetonitrile, Methylene Chloride, and Methanol, with lg/L Butylated Hydroxytoluene and lOOμL/L Diisoproplyethylamine at lml/min. For each experimental run, BC standard was used to determine the retention time for unoxidized BC. One hundred μl's of the standard or cell sample was injected onto the column and compounds were separated by monitoring the absorbance of the column effluent at a wavelength of ~450nm. These experiments determine if an increase in Raman signal detectable after loading was attributable to BC. Moreover, HPLC was used to determine if a decrement in Raman signal after oxidative stress correlated with a decrease in the spectrophotometric signal and HPLC peak area.

[0066] Results: Detecting β-carotene in BC Loaded ERC293 Cells. ERC293 cells exposed to 0.3% MβCD alone, or in combination with 0.2 μM or 1 μM BC for 24 hours showed that Raman measurements of intracellular carotenoids correlated with the dose of BC/M CD, see Table 1. These Raman measurements also correlated with the UV spectrophotometric measurements of BC after solubilization of the cells. These data show that BC loading of cells using MβCD is accurately detected by the disclosed Raman apparatus.

Table 1: Raman Measurements of Loaded ERC293 Cells after Oxidative Stress

|: Significant difference between BC + AA + Pon vs. BC Alone (p<0.002), vs. BC + AA (p<0.006), and vs. BC + Pon (p<0.02) by Holm-Sidak One-way ANOVA

[0067] Based upon prior published results (see Wu, Z., et al, J Agric Food Chem, 1999, 47, 4899-4906) demonstrating the co-oxidation of BC in the presence of linoleic acid (LA) and soybean lipoxygenase- 1 type IB (15-LOX-l), the disclosed Raman apparatus indicated oxidation of BC as a decrement in the Raman signal. As shown in FIG. 3, a decrease in UV absorbance measurement over time at 456 nm was also observed. Solubilized BC (ImM) was incubated in the presence of Soybean lipoxygenase (LOX)-I and linoleic acid (LA) either separately or together and photobleaching was measured as the percent absorbance at 456nm. Control exposure to inactive LOX-I (Dead LOX-I) was used to confirm LOX specific activity. The co- oxidation of BC in the presence of another oxidative stress, peroxide, was also measured in comparison. As seen, a decrease in the percent absorbance was noted only in the presence of LOX-I /LA together, but not with either LOX-I or LA alone or in the presence of dead LOX-I /LA. Peroxide was used as an alternate source of oxidative stress to compare with the generation of endogenous oxidative stress by LOX. No appreciable decrement in absorbance was noted for IM peroxide. These data confirmed and expand upon prior published data that demonstrates co-oxidation of BC under these conditions. See Yu, M. K., et al, J Biol Chem, 2004, 279, 28028-28035.

[0068] As shown in FIG. 4, HPLC confirmed a decrement of the BC peak area over time and identified the appearance of alternate peaks with shorter retention times, suggesting the appearance of BC oxidative metabolites, β -carotene was exposed to the same reaction mixture as in FIG. 2 for 24 hours. Reactions were extracted 3 times with an equal volume of diethyl ether, and 0.5 g sodium sulfate to remove all trace of water. The ether fraction was decanted, dried under N₂, and resuspended in acetonitrile: Chloroform (3:2) and injected onto HPLC for analysis. 200 μL of the oxidize compound was injected on to a Phenomenex Gemini reversed phase Cl 8 column (250x4.6mm). Compounds were eluted from the column using a gradient method run at lmL/min: 0-5 min MeOH/H2O (9:1); 5-20 min to MeCN (100%); 20-35 min to MeCN:Ethyl Acetate (85:15); 35-85 min at MeCN:Ethyl Acetate (85:15); return to MeOH:H2O over 5 min; Equilibrate 30 min. Using a Beckman System Gold HPLC with Photodiode Array detector compounds were detected by measuring the absorbance from 250-50OnM. As shown in FIG. 4A, using the Beckman 32Karat Software it was possible to visualize the absorbance spectra and determine the lambda max for each compound. As seen, the β-carotene peak (tR=71 minutes and a λmax=450nm) decreased over time from t=0 minutes (top trace) through t=l minute (middle trace) to t=5 minutes (bottom trace). As shown in FIG. 4B, alternative peaks with lower retention times appeared over the same time frame. One candidate peak was chosen, and found to have a £_R= 18.45 minutes and a λmax=402 similar to 5,6-epoxy-b-apo-12'- or -lO'-carotenal, a published BC breakdown product (the absorption spectrum is not shown). See Wu, Z., et al., J Agric Food Chem, 1999, 47, 4899-4906 These results suggest that oxidation can cause a breakdown of BC to shorter chain molecular species.

[0069] Finally, tests were performed to determine if the disclosed Raman apparatus could detect the decrease in parent carotenoids in cells after exposure to a defined oxidative stress. As shown in Table 1, using the pronasterone promoter to express 15- LOX-I in the absence or presence of LA, experiments demonstrated that the Raman apparatus was able to detect the loading of BC in ERC293 cells and that 15-L0X-1 mediated co-oxidation of BC was detected as a decrement in Raman signal at 1525 cm^"1. These data demonstrate that the Raman apparatus can detect the oxidative breakdown of BC in loaded cells as a decrease in BCs Raman signal.

Example 2: Application of the Raman Apparatus to a Patient Cohort Study

[0070] Methods: Utilizing Raman Spectroscopy, carotenoid levels were measured at four subsites in the upper aerodigestive tract (UADT) (the buccal mucosa, anterior floor of mouth, oral tongue, and soft palate) by the techniques of Hata, et al. See Hata, T. R., et al., J Invest Dermatol, 2000, 115, 441-448. Each participant was administered a short validated clinical questionnaire, the AUDIT questionnaire, with supplemental dietary questions. See Am J Public Health, 1996, 86, 948-955. Answers to these questions were correlated with Raman measured UADT carotenoid concentrations. The sample probe of the Raman apparatus of FIG. 2 was brought into contact with each subsite (oral tongue, buccal mucosa, floor of mouth, and soft palate) for the requisite time (12 seconds for these tissues) necessary to obtain a characteristic waveform at 1525 cm^"1 wavelength, and the peak amplitude was recorded. In order to control for variations in the optical properties of regions within a subsite, three independent measurements within a given subsite of the UADT were measured and the average of three measurements reported. Variations in the optical properties within a subsite were minimized by consistently identifying the following anatomic landmarks with which to make measurements (1 cm right of the midline raphe of the tip of the oral tongue, 1 cm posterior to the right vermilion commissure on the buccal mucosa, 1 cm posterior of the right Wharton's duct on the floor of mouth, and 1 cm right of midline just posterior to the junction of the hard and soft palate). These mucosal sites were visually inspected for any gross pathologic processes that may effect the Raman measurements (i.e. tumor or inflammatory processes). If these areas were noted to have gross mucosal changes, the subject was eliminated from the study.

[0071] Fifty-one subjects (30 males, mean age of 47.3 ± 18.9 years, range 19 to 83 years of age) were used in the pilot study. Sixteen subjects within the study (31.4%) had FINSCCa (head and neck squamous cell carcinoma), while the rest (68.6%) were non-tumor bearing controls. Eight subjects currently used tobacco, while another eighteen admitted to former tobacco use, and the rest denied any prior tobacco intake. The mean pack/year smoking history (pack/years = number of packs per day x the number of years smoking in total) for smokers was 27.8 ± 33.5 pack/years. Twenty-two subjects admitted to active alcohol intake, five were former alcohol users, whereas the rest of the study cohort had never consumed alcohol.

[0072] HNSCCa offers an excellent model of tobacco-related cancer development with which to study tobacco and carotenoid's interrelationship. Despite accounting for a small percentage of the overall cancer incidence in the United States, HNSCCa is a huge global disease problem. HNSCCa is the sixth most common cancer worldwide, and in some areas of the world HNSCCa accounts for up to 50% of all new cancer cases presenting to an oncology clinic. Moreover, this global problem is most likely going to increase as tobacco companies target developing nation's large markets, and as other more treatable disease processes, such as infectious diseases, become less common. Therefore, studies of HNSCCa development may have a huge global impact on societal health. Evidence supports the fact that HNSCCa exist within an area of high oxidative stress (See Seven, A., et al., Clin Biochem, 1999, 32, 369-373.) with tobacco and alcohol consumption (See Epperlein, M. M., et al., IntJExp Pathol, 1996, 77, 197-200.), viral infection, lichen planus, and chronic gastroesophageal reflux all inducing chronic inflammation. See Brandsma, J. L., et al., Arch Otolaryngol Head Neck Surg, 1989, 115, 621-625 and Marshall, J. R., et al., Eur J Cancer B Oral Oncol, 1992, 28B, 9-15. Furthermore, decreased antioxidant mechanisms are found in head and neck cancer patients (See Subapriya, R., et al., Clin Biochem, 2002, 35, 489-493.) potentially further increasing the levels of oxidative stress. These characteristics in conjunction with the ease of clinical and pathologic assessment for both the primary and metastatic foci make HNSCCa a unique and powerful tool with which to unravel the nuances of oxidative stress induced carcinogenesis as a representative model for the wide variety of tobacco- induced cancers.

[0073] Statistical analysis: Statistical analysis of the relationship of categorical clinical variables to Raman measurements was undertaken by a dedicated Biostatistician. p-values were determined using Statistica 6.0 statistical analysis package (StatSoft Inc., Tulsa, OK) and a two-sided Fisher's Exact tests for binary variables or a Pearson chi-square test with variables with three or more categories. Statistical significance was established with a p<0.05, and values are expressed + standard error of the mean unless otherwise specified.

[0074] Results: No significant differences were found between HNSCCa subjects and non-tumor bearing controls with respect to tobacco or alcohol consumption (p=0.3 and p=0.2 by Chi-squared test). These investigations discovered the provocative findings that subjects consuming alcohol were found to have significantly higher Raman readings when compared to non-drinking subjects (p<0.05). Moreover, increased age also significantly increased the tissue Raman measurements (p<0.05). Interestingly, when controlling for tobacco use, lower Raman measurements were found in HNSCCa subjects (p<0.05) compared to non-tumor bearing subjects.

[0075] These data demonstrate the feasibility of using the disclosed Raman apparatus to analyze in vivo biological tissue samples and also raise some interesting points about carotenoid pharmacology and oxidative biology in cancerous tissues and in tissues at risk of cancer development. Without wishing to be bound to any particular theory, given the results of the ATBC and CARET clinical trials described above in conjunction with the data presented herein, it is hypothesized that carotenoids have a duality of function of being antioxidant under low oxidative stress conditions but becoming potent and long-acting pro-oxidants under a tobacco-induced high oxidative environment. If so, the synergistic effects of alcohol consumption with tobacco in HNSCCa development may in part be explained by a concentration of carotenoids as a result of alcohol use within this tobacco-related oxidative environment. Oxidative breakdown of carotenoids could remove carotenoid-related species, thus decreasing Raman signal. Moreover, these oxidative metabolites of carotenoids may potentiate cancer development through an enhancement of oxidative stress and/or influencing key cancer-related cellular pathways. Thus, the disclosed Raman apparatus could find use as a screening tool with which to measure unoxidized carotenoid concentrations in tissues at-risk of cancer development during future carotenoid chemoprevention trials.

Example 3: Analysis of Various Samples Using the Raman Apparatus

[0076] The Raman apparatus of FIG. 2 was used to analyze a variety of samples. For each sample, the sample probe of the apparatus was brought into contact with the sample under the conditions described below. Only the spectra in FIG. 5 were resonantly enhanced. For each of the other samples shown in FIG. 6, spectra were collected under non-resonant Raman conditions.

[0077] FIG. 5A shows an in vivo single fiber resonance Raman measurement in a human soft palate. The Raman peak at -1525 cm^"1 arises from carotenoid C=C bonds (Conditions: 488 nm excitation, <1 mW laser power, 9 sec exposure, 600 μm fiber diameter, 3 meter fiber length). FIG. 5B shows a single fiber resonance Raman measurement in a raw carrot. The Raman peak at -1525 cm^"1 arises from carotenoid C=C bonds (Conditions: 488 nm excitation, 2 mW laser power, 3 sec exposure, 400 μm fiber diameter, 4 meter fiber length). FIG. 6A shows a single fiber off-resonance Raman spectrum of uncoated aspirin from 1200 to 1900 cm^"1 (Conditions: 532 nm; 3 sec; 2.5 mW; 4 meter fiber in contact with surface). FIG. 6B shows a single fiber off-resonance Raman spectrum of soybean oil from 1300 to 1900 cm^"1 (Conditions: 532 nm; 12 sec; 3 mW; 4 meter fiber submerged in oil). FIG. 6C shows a single fiber off-resonance Raman spectrum of ploycaprolactone (PCL) polymer from 1200 to 1900 cm^"1 (Conditions: 532 nm;10 sec; 2 mW; 4 meter fiber in contact with surface). FIG. 6D shows a single fiber off-resonance Raman spectrum of gylcerol from 1200 to 1850 cm^"1 (Conditions: 532 nm; 15 sec; 3 mW; 3 meter fiber submerged in glycerol). FIG. 6E shows a single fiber off-resonance Raman spectrum of pseudoephedrine from 1200 to 1900 cm^"1 (Conditions: 532 nm;12 sec; 2.5 mW; 3 meter fiber in contact with uncoated inside surface). FIG. 6F shows a single fiber off-resonance Raman spectrum of Polycarbonate from 1200 to 1850 cm^"1 (Conditions: 532 nm; 5 sec; 2 mW; 4 meter fiber in contact with surface). FIG. 6G shows a single fiber off-resonance Raman spectrum of Ibuprofen from 1200 to 1900 cm^"1 (Conditions: 532 nm; 8 sec; 2 mW; 3 meter fiber in contact with uncoated inside surface). The identity of the molecules giving rise to the spectra in FIGs. 6A-6G may be readily determined by comparing the spectra to known libraries of Raman spectra.

[0078] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as "up to," "at least," "greater than," "less than," and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above.

[0079] All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document were specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

[0080] For the purposes of this disclosure and unless otherwise specified, "a" or an means one or more.

Claims

WHAT IS CLAIMED IS:

1. An apparatus for detecting Raman scattered light from a sample, the apparatus comprising: a sample probe comprising an optical fiber, the optical fiber adapted to deliver excitation light along the fiber to the sample and to collect the Raman scattered light from the sample along the same fiber; an optical module coupled to the sample probe, the optical module adapted to direct the excitation light and the Raman scattered light and to separate the excitation light from the Raman scattered light; and a detector module coupled to the optical module, the detector module adapted to detect the Raman scattered light from the sample over a spectral region of about 2000 cm^"1 or less.

2. The apparatus of claim 1, wherein the detector module is adapted to detect the Raman scattered light from the sample over a spectral region from about 1200 cm^"1 to about 1900 cm^"1.

3. The apparatus of claim 1, wherein the wavelength of the excitation light ranges from about 450 nm to about 550 nm.

4. The apparatus of claim 1 , wherein the optical module comprises one or more of a wavelength selective device, a collimator, or a lens.

5. The apparatus of claim 1, wherein the optical module comprises a wavelength selective device adapted to separate the excitation light from the Raman scattered light; a collimator adapted to direct the excitation light to the wavelength selective device; and a lens adapted to direct the Raman scattered light to the detector module.

6. The apparatus of claim 4 or 5, wherein the wavelength selective device is a holographic optical element.

7. The apparatus of claim 1 , wherein the detector module comprises one or more of a wavelength selective device, a light measuring device, or a computer.

8. The apparatus of claim 7, wherein the computer comprises software adapted to identify and remove background light signals from the Raman scattered light.

9. The apparatus of claim 1, wherein the detector module comprises a wavelength selective device coupled to the optical module, a light measuring device coupled to the wavelength selective device, and a computer coupled to the light measuring device, wherein the computer comprises software adapted to identify and remove background light signals from the Raman scattered light.

10. The apparatus of claim 7 or 9, wherein the wavelength selective device is a spectrometer and the light measuring device is a CCD camera.

11. The apparatus of claim 1 , further comprising a light source coupled to the optical module, the light source providing the excitation light.

12. The apparatus of claim 1, further comprising a tubular housing coupled to the sample probe.

13. The apparatus of claim 12, wherein the tubular housing is an endoscope or a needle.

14. The apparatus of claim 1, wherein the sample comprises a carotenoid or a polymer.

15. The apparatus of claim 1, wherein the sample comprises a carotenoid.

16. The apparatus of claim 1, wherein the sample comprises a molecule having an optical absorption spectrum that overlaps with the wavelength of the excitation light, thereby providing resonance enhanced Raman scattered light from the sample.

17. The apparatus of claim 1, wherein the sample is a biological tissue sample.

18. The apparatus of claim 1, wherein the sample is an in vivo biological tissue sample.

19. An apparatus for detecting Raman scattered light from a sample, the apparatus comprising: a sample probe comprising an optical fiber, the optical fiber adapted to deliver excitation light along the fiber to the sample and to collect the Raman scattered light from the sample along the same fiber; an optical module coupled to the sample probe, the optical module comprising a first wavelength selective device adapted to separate the excitation light from the Raman scattered light, a collimator adapted to direct the excitation light to the first wavelength selective device, and a lens adapted to direct the Raman scattered light to the detector module; and a detector module coupled to the optical module, the detector module adapted to detect the Raman scattered light from the sample over a spectral region of about 2000 cm^"1 or less wherein the detector module comprises a second wavelength selective device coupled to the optical module, a light measuring device coupled to the second wavelength selective device, and a computer coupled to the light measuring device, wherein the computer comprises software adapted to identify and remove background light signals from the Raman scattered light.

20. The apparatus of claim 19, wherein the first wavelength selective device is a holographic optical element, the second wavelength selective device is a spectrometer, and the light measuring device is a CCD camera.

21. A method for detecting Raman scattered light from a sample, the method comprising: generating the Raman scattered light from the sample using an apparatus comprising a sample probe comprising an optical fiber, the optical fiber adapted to deliver excitation light along the fiber to the sample and to collect the Raman scattered light from the sample along the same fiber; an optical module coupled to the sample probe, the optical module adapted to direct the excitation light and the Raman scattered light and to separate the excitation light from the Raman scattered light; and a detector module coupled to the optical module, the detector module adapted to detect the Raman scattered light from the sample over a spectral region of about

2000 cm^"1 or less, and detecting the Raman scattered light.

22. The method of claim 21 , wherein the detector module is adapted to detect the Raman scattered light from the sample over a spectral region from about 1200 cm^"1 to about 1900 cm^"1.

23. The method of claim 21 , wherein the wavelength of the excitation light ranges from about 450 nm to about 550 nm.

24. The method of claim 21, wherein the sample is an in vivo biological tissue sample and the sample comprises a carotenoid.