WO2014063257A1 - Methods and apparatus for colonic neoplasia detection with high frequency raman spectra - Google Patents

Abstract

Raman spectroscopy at higher Raman shifts, for example, Raman shifts exceeding 1000 cm^-1 is applied for characterizing tissues of the colon. Characterization may comprise comparisons of peaks in the Raman spectrum and/or multivariate analysis. Compact, cost effective and disposable Raman probes may be provided. In one embodiment a Raman probe comprises a single pair of optical fibers having beveled ends.

Description

METHODS AND APPARATUS FOR COLONIC NEOPLASIA DETECTION WITH HIGH FREQUENCY RAMAN SPECTRA

Related Applications

[0001] This application claims priority from United States Application No. 61/718830 filed 26 October 2012. For purposes of the United States, this application claims the benefit under 35 U.S.C. §119 of United States Application No. 61/718830 filed 26 October 2012 and entitled METHODS AND APPARATUS FOR COLONIC

NEOPLASIA DETECTION WITH HIGH FREQUENCY RAMAN SPECTRA which is hereby incorporated herein by reference for all purposes.

Technical Field

[0002] This invention relates to methods and apparatus for in vivo characterization of tissues by Raman spectroscopy. Embodiments have application to screening for cancer, particularly colon cancer.

Background

[0003] Like all cancers, the earlier colon cancers are found the better the prognosis for the patients. Several advances have taken place in endoscopic imaging technologies that improve the ability of clinicians to localize small or occult lesions. However there is some subjectivity on behalf of the clinician for selecting which sites to biopsy during a colonoscopy. Only a fraction of the suspicious sites identified during a colonoscopy are neoplastic lesions, which can lead to a significant number of biopsies which are negative for high grade dysplasia or worse, and potential early cancers at some sites are missed because they were considered benign and not requiring a biopsy by the clinician. To address these deficiencies, reliable, highly specific, optical techniques are required which will function as optical biopsy tools to objectively classify lesions in vivo.

[0004] Raman spectroscopy systems have tremendous potential as adjunct devices for endoscopes to improve the in vivo identification of early cancers. Raman spectroscopy studies the non-elastic scattering of light. Excitation light, for example from a laser is directed at an area of tissue. The light interacts with the tissue. Light that is non-elastically scattered undergoes a frequency shift. The spectrum of such frequency-shifted light can reveal information regarding the makeup of the tissues being studied.

[0005] Research on the use of Raman emissions in the low frequency range (0 to 1800 cm^-1) for differentiating between normal and neoplastic tissue in vivo, has significantly increased over the last decade.

[0006] Raman is a powerful analytical technique, but the inherently weak emission prevented its use as a fast medical diagnostic method until relatively recent advances in lasers, spectrometers, detectors and optical fibers made it possible. In endoscopic applications the technical challenges in collecting good quality Raman spectra are increased. One challenge with Raman spectroscopy is that the wavelengths in the Raman spectrum are close to that of the exciting light. Other sources of interference are background fluorescence from flourophores in the tissue and, where an endoscope is used, Raman scattering from optical components in a probe used with the endoscope. These problems are particularly acute in the case of low-frequency Raman scattering which has frequencies close to that of the excitation light.

[0007] The excitation wavelength of choice for clinical Raman systems is 785 nm since it penetrates deeper into tissue, stimulates less emission in the fiber optic catheter and less tissue autofluorescence than excitation with visible wavelengths. However, interference from tissue autofluorescence and emission in the fiber optic catheter remain problematic because both can be strong in the low frequency range (LF) which coincides with the Raman spectrum. Simply subtracting the contribution to the spectrum from the fiber is not a good solution since it is difficult to reliably remove leading to a decrease in diagnostic accuracy. Another approach is to reduce the fiber emission by elaborate and expensive optical filters placed at the distal end of the fiber optic catheter. The drawback is that optically filtered endoscopic catheters have to be reprocessed for reuse after each procedure multiple times to make them economically viable. Unfortunately the long, narrow and flexible catheters can be damaged during the subsequent reprocessing, significantly reducing the average number of reuse cycles. Furthermore this damage can subtly change the optical properties of the filters, which may lead to diagnostic inaccuracies if it goes undetected. Even if these expensive catheters could be made to survive multiple procedures, the high tissue autofluorescence in the LF range remains and cannot be optically filtered out, this results in a further reduction in the diagnostic accuracy.

[0008] It would be beneficial to provide diagnostic and screening methods and apparatus which can take advantage of Raman spectroscopy while alleviating some of the foregoing disadvantages. Of particular benefit would be methods and applications which can use Raman spectroscopy in endoscopic applications, such as making studies of the colon.

Summary

[0009] The present invention has a number of aspects which all relate to methods and apparatus for characterizing tissues based at least in part by analysis of Raman spectra for the tissues. Some aspects of the invention relate to ranges of Raman shift that are analyzed. Advantageously, such embodiments may use portions of the Raman spectrum at higher Raman shift and may ignore portions of the Raman spectrum at lower Raman shifts. The effectiveness of this range at discriminating between different colon tissue pathologies has not previously been demonstrated. Surprisingly, not least because the Raman spectrum at higher Raman shifts tends to have fewer features than the Raman spectrum at lower Raman shifts, tissue characterization based on Raman spectra which does not include the portion of the Raman spectrum at lower Raman shifts (low- frequency Raman) can provide good diagnostic sensitivity and specificity.

[0010] Some other aspects relate to the use of specific features (e.g. specific peaks) in the Raman spectrum for use in tissue characterization, particularly characterization of colonic tissues in some embodiments. Some peaks that have been found to be significant for tissue characterization are at higher Raman shifts and others are at lower Raman shifts.

[0011] One aspect provides methods for characterizing tissues by Raman spectroscopy. In some embodiments the tissues are colon tissues and the method involves carrying light to a Raman spectrometer by way of a probe comprising one or more optical fibers extending along an endoscope. For example, the probe may extend along an instrument channel of the endoscope. The methods may be applied, for example to the identification of colonic neoplasias. In alternative embodiments, the present invention can be used to detect other neoplasias of the gastrointestinal tract. In still further alternative embodiments, the present invention can be used for the identification of preneoplastic lesion and the like which are at risk of progressing to neoplasia. In some embodiments the endoscope lacks filters of the type often used to block light at a wavelength of exciting light from reaching a Raman spectrometer.

[0012] Another aspect of the invention provides apparatus useful for characterizing tissues. In various embodiments the apparatus comprises a data processor configured to process Raman spectrum data to indicate a characteristic of the tissues. In some embodiments the data processor is combined with a Raman spectrometer and a probe comprising one or more elongated optical fibers suitable for use with an endoscope. The data processor may be configured to characterize issues bases on Raman spectra lacking information regarding lower Raman shifts and/or to characterize tissues using specific Raman peaks as described herein.

[0013] Some aspects of the invention involves the use of high frequency Raman spectroscopy (spectroscopy of that part of the Raman spectrum having a Raman shift in excess of 1800 cm^"1). In some embodiments tissue characterization is performed using no part of the Raman spectrum below 1000 cm^"1 (in some embodiments below 1200 cm^"1 or 1500 cm^"1 or 1800 cm^"1). Advantageously, autofluorescence and Raman signals from the optical fiber in an endoscope probe affect Raman signals with higher Raman shifts less than signals with lower Raman shifts. Consequently, it can be unnecessary to provide filters to block excitation light from entering the return optical fiber in an endoscopic probe in cases where Raman signals at lower Raman shifts are not used. In some embodiments light is passed from tissues being studied to a Raman spectrometer along an optical path that passes wavelengths corresponding to that of the excitation light source from end to end. In some embodiments light is passed from tissues being studied to a Raman spectrometer along optical fibers leading through or along an endoscope and the optical fibers pass wavelengths corresponding to that of the excitation light source from end to end.

[0014] In one embodiment, the present invention provides a method of detecting colonic neoplasias comprising measurement of HF Raman Spectroscopy signal peaks at 2853 cm^" 2866 cm^"1 and 2930 cm^"1.

[0015] In another embodiment, present invention provides a method of detecting colonic neoplasias comprising measurement of peak ratios calculated from LF Raman spectra. The ratio of the 1340 and 1735 cm^"1 peaks may be applied to distinguish normal from malignant tissue. In some embodiments, the ratio of peaks at 1445 cm^"1 and 1735 cm^"1 is applied to distinguish normal from malignant tissue. In still further embodiments, both peak ratios may be used in combination to distinguish normal from malignant tissue.

[0016] One embodiment provides methods for detecting colonic neoplasias comprising measurement of LF Raman spectroscopy signal peaks at 590, 780, and 1030 cm^"1. Another embodiment provides methods for detecting colonic neoplasias comprising measurement of LF Raman spectroscopy signal peaks at 1340, 1450, 1650 and 1730 cm^"1. In further embodiments, one or more additional peaks selected from 1000, 1150, 1540 and 1550 cm^{" x}are also measured and applied to characterizing tissues (for example to evaluate the likelihood that the tissues comprise a colonic neoplasia).

[0017] In some embodiments multivariate analysis of the Raman spectra is used to characterize tissues (for example to distinguish normal from malignant tissues).

[0018] In one embodiment, the present invention provides a device for obtaining HF

Raman spectra utilizing optically unfiltered catheters . In one embodiment, the optical fiber catheter has separate excitation and collection channels. In one embodiment, a multifiber probe is used to collect the HF Raman signal. In an alternative embodiment, a dual-fiber probe with beveled fibers is used to collect the HF Raman signal.

[0019] In a further alternative embodiment of the present invention, a double clad fiber is used which delivers the laser light through the central core and collects backscattered radiation and Raman signals from tissue via the inner cladding. In a still further alternative embodiment, pigtailed connections to an isolating wavelength division multiplexer are used to focus light in and out of the double clad fiber. In one embodiment of the present invention, the catheter may be miniaturized for some medical applications.

[0020] Another aspect of the invention provides a non-transitory tangible computer- readable medium storing instructions for execution by at least one data-processor that, when executed by the at least one data-processor cause the at least one data processor to execute a method for characterizing tissue comprising the steps of processing at least one Raman spectrum of a colon tissue, characterizing the colon tissue in response to the Raman spectrum and generating an indication of the characterization of the colon tissue. Characterizing the colon tissue is based on one or more features of that part of the Raman spectrum having Raman shifts (relative wavenumbers) above 1000 cm^"1 (in some embodiments above 1200 cm^"1 or 1500 cm^"1 or 1800 cm^"1). In some embodiments, characterizing the colon tissue is performed using no part of the Raman spectrum below 1000 cm^"1 (in some embodiments below 1200 cm^"1 or 1500 cm^"1 or 1800 cm^"1). The tissue characterization may comprise determining ratios of peaks in the Raman spectrum and/or performing multivariate analysis of the Raman spectrum, for example using principle components analysis / linear discriminant analysis.

[0021] Another aspect of the invention provides a probe for use in in vivo Raman spectroscopy. The probe comprises a first optical fiber arranged to carry light from an excitation light source to illuminate a tissue and a second optical fiber arranged to provide an optical path to carry scattered light to a spectroscope. Ends of the first and/or second optical fibers are beveled. In some embodiments he first optical fiber has a smaller diameter than the second optical fiber. In some embodiments, distal ends of the fibers are not provided with filters and/or are transmissive at an excitation wavelength. Such probes may be used in the apparatus and methods of the aspects and embodiments described herein and/or used in other applications.

[0022] Another aspect of the invention provides a sheath for use in in vivo Raman spectrometry. The sheath comprises a tubular member having an inside diameter dimensioned to receive a Raman spectroscopy probe and an outside diameter dimensioned to fit within an instrument channel in an endoscope. An optical window is sealed to the tubular member at a distal end thereof. The sheath may be provided in combination with a probe received within the sheath and/or an endoscope having an instrument channel dimensioned to receive the sheath. Such sheaths may be used in the apparatus and methods of the aspects and embodiments described herein and/or used in other applications.

[0023] Further aspects and example embodiments are illustrated in the accompanying drawings and/or described in the following description.

Brief Description of the Drawings

[0024] The accompanying drawings illustrate non-limiting example embodiments of the invention.

[0025] Figures 1A to ID show LF and HF Raman emission spectra taken in vivo from the palm skin of a volunteer. Spectra were obtained with both optically filtered and unfiltered catheters. Figures 1 A and IB are the calibrated emission including autofluorescence, Raman and fibre emission. Figures 1C and ID are the spectra obtained after removal of the fluorescence by polynomial fits.

[0026] Figures 2A to 2D show spectra from excised (Figures 2A and 2C ) and biopsy

(Figures 2B and 2D) colon samples using only an optically filtered catheter. Figures 2A and 2B are the calibrated emission including autofluorescence, Raman and fibre emission.

Figures 2C and 2D are the corresponding spectra obtained after removal of the autofluorescence by a polynomial fit. Tissue Raman peaks and fibre catheter emissions are present in the spectra. Error bars shown are the standard errors on the mean.

[0027] Figures 3A to 3D show HF emission spectra from excised (Figures 3A and 3C) and biopsy (Figures 3B and 3D) samples. Spectra were obtained with both optically filtered and unfiltered catheters. Figures 3A and 3B are the calibrated emission including autofluorescence, and Raman. Figures 3C and 3D are the corresponding spectra obtained after removal of the autofluorescence by a polynomial fit. Only the main Raman emission range is shown. The spectra of Figures 3C and 3D contain predominantly tissue Raman peaks with very little fibre catheter emission. Error bars shown are the standard errors on the mean.

[0028] Figures 4A to 4D show peak ratios calculated from two peaks in both the LF and HF spectral ranges for excised and biopsy tissue using data collected with the optically filtered catheter only. Spectra were calibrated, with autofluorescence subtracted, and normalized as described herein. Figures 4A and 4C are for excised tissue in the LF and HF ranges respectively. Figures 4B and 4D are for biopsy tissue in the LF and HF ranges respectively. The ordinate axis title indicates the wavenumber location of each peak used in the ratio calculation and these values refer directly to the corresponding abscissa values in Figures 2C and 2D and 3C and 3D.

[0029] Figure 5 shows results of the statistical analyses on the LF spectra

(autofluorescence subtracted) from excised (Figures 5A and 5C) and biopsy (Figures 5B and 5D) samples using data collected with the optically filtered catheter only. Figures 5A and 5B are 2D scatter plots of the two principal component factors scores which are highly correlated (by t-test) with tissue pathology. The symbol "A" marks the average position of a group. Figures 5C and 5D are the posterior probability plots derived from validated linear discriminant analysis (LDA) that predicts the likelihood that a spectrum is from normal tissue, and the symbols indicate the actual pathology determined by histology.

[0030] Figures 6A to 6D show results of statistical analyses on the HF spectra (autofluorescence subtracted) from excised (Figures 6A and 6C) and biopsy (Figures 6B and 6D) samples using data collected with the optically filtered catheter only. Only the range 2800-3050 cm^"1 was used. Figures 6A and 6B are 2D scatter plots of the two principal component factors scores which are highly correlated (by t-test) with tissue pathology. The symbol "A" marks the average position of a group. Figures 6C and 6D are the posterior probability plots derived from validated LDA that predicts the likelihood that a spectrum is from normal tissue, and the symbols are the actual pathology determent by histology.

[0031] Figures 7A and 7B are ROC curves showing the sensitivity and specificity for predicted the tissue pathology from the LF (Figure A) and HF (Figure 7B) Raman spectra using data collected with the optically filtered catheter only. Data for excised tissue is represented by open diamond symbols and data for biopsy tissue is represented by solid square symbols.

[0032] Figure 8 shows schematically Raman spectrometry apparatus as used for in vivo collection of Raman spectrum data.

[0033] Figure 9 shows test spectra obtained using the apparatus of Figure 8 on palm skin.

[0034] Figure 10 shows test spectra taken with and without narrow band illumination light turned on.

[0035] Figure 11 shows in vivo Raman spectra of colon tissues in the range from 2800- 3050 cm^"1 after fluorescence removal, and normalization.

[0036] Figure 12 shows in vivo Raman spectra of colon tissues with and without white light illumination (WLI).

[0037] Figure 13 illustrates an alternative Raman spectrometry probe that uses a single optical fiber both to carry excitation light to a tissue of interest and to carry collected scattered light back to a spectrometer. [0038] Figure 14 illustrates an alternative Raman spectrometry probe that uses separate sets of optical fibers to carry excitation light to a tissue of interest and to carry collected scattered light back to a spectrometer.

[0039] Figure 15 illustrates an alternative Raman spectrometry probe that uses single beveled fibers to carry excitation light to a tissue of interest and to carry collected scattered light back to a spectrometer.

[0040] Figures 16A and 16B illustrate alternative Raman spectrometry apparatus using a double clad fiber to carry excitation light to a tissue of interest and to carry collected scattered light back to a spectrometer.

[0041] Figures 17 and 18 illustrate the use of a multiplexer to couple excitation and collected light into and out of an optical fiber.

[0042] Figure 18A is a schematic illustration showing an example multiplexer.

[0043] Figure 19 illustrates a novel sheath for covering a Raman probe.

[0044] Figure 20 illustrates a Raman spectrum obtained using a Raman probe covered with a sheath as illustrated in Figure 19.

Description

[0045] Throughout the following description specific details are set forth in order to provide a more thorough understanding to persons skilled in the art. However, well known elements may not have been shown or described in detail to avoid unnecessarily obscuring the disclosure. The following description of examples of the technology is not intended to be exhaustive or to limit the system to the precise forms of any example embodiment. Accordingly, the description and drawings are to be regarded in an illustrative, rather than a restrictive, sense.

[0046] Embodiments of this invention use data from Raman spectroscopy to characterize tissues. As an alternative to measuring that part of the Raman spectrum having Raman shifts of 0 to 1800 cm^-1 (LF Raman), one aspect of the invention uses primarily or entirely features of the Raman spectrum having Raman shifts of over 1800 cm^"1 (HF Raman) to characterize tissues. Features of the HF Raman spectrum may be used for tissue characterization alone or in combination with information retrieved from other modes of investigation of the tissues.

[0047] An advantage of the HF Raman spectrum is that there is substantially less interference from fiber optic noise and tissue autofluorescence in the HF Raman spectrum as compared to the LF Raman spectrum. However, the HF Raman spectrum tends to be less rich in features than the LF Raman spectrum. The inventors have compared LF and HF Raman emissions obtained from the same ex vivo colonic tissue sites to determine the sensitivity and specificity of each range at predicting the tissue pathology.

[0048] Raman spectroscopy systems are known to those of skill in the art. A Raman spectroscopy system includes a light source, generally a laser, an optical path arranged to carry light from the light source to the tissues to be studied, a Raman spectrometer, and an optical path arranged to carry light scattered from the tissues to the Raman spectrometer. For in-vivo spectrometry the optical paths may be provided by one or more optical fibers extending through or along an endoscope.

[0049] One issue with endoscopic Raman spectrometry is that light at the excitation wavelength may be scattered within optical fibers. The scattered light may have wavelengths that overlap with wavelengths in the LF Raman spectrum. Consequently, Raman systems typically include filters to block light at the excitation wavelength from entering the optical path that carries light back to the Raman spectrometer.

[0050] Example Raman spectroscopy systems are described in references [16] and [22] to [25] which are listed below.

[0051] Data representing a Raman spectrum may be processed in a data processor to yield one or more values characterizing the tissue to which the Raman spectrum corresponds. In embodiments of the present invention the one or more values are indicative of whether the tissues are normal, on one hand, or cancerous on the other. The one or more values may be binary values (e.g. tissue is indicated as being either 'normal' or 'diseased') or values in a range.

[0052] Tissue characterization values may be obtained by identifying and comparing features in the Raman spectrum (e.g. by comparing the ratios of different peaks in the Raman spectrum or, more generally, comparing, by ratios or otherwise, the values at one Raman shift or range of Raman shifts in the Raman spectrum to the values at one Raman shift or range of Raman shifts in the Raman spectrum. Another way to generate values characterizing tissues is by multivariate analysis. One example of multivariate analysis is principal components analysis followed by linear discriminant analysis.

[0053] A data processor connected to receive Raman spectrum data directly or indirectly from a Raman spectrometer may apply multivariate data analysis to classify tissues according to their Raman spectra. For example, a particular spectrum may be analyzed by performing a principle component analysis (PCA). PCA may be performed on part or all of the range of the acquired Raman spectra.

[0054] PCA involves generating a set of principle components which represent a given proportion of the variance in a set of training spectra. For example, each spectrum may be represented as a linear combination of a set of a few PCA variables. The PCA variables may be selected so that they account for at least a threshold amount (e.g. at least 70%) of the total variance of the set of training spectra. In the present case the training spectra may comprise Raman spectra of colon tissues having a range of pathologies (e.g. some normal tissues and some tissues that have been confirmed to be malignant). The training spectra (or at least the part of the training spectra used in the principle components analysis may consist of Raman spectra having Raman shifts exceeding 1000 cm^"1 (exceeding 1200 cm^"1 or 1500 cm^"1 or 1800 cm^"1 in some embodiments).

[0055] Principal components (PCs) may be derived by performing PCA on a standardized spectral data matrix to generate PCs. The PCs generally provide a reduced number of orthogonal variables that account for most of the total variance in the original spectra.

[0056] PCs may be used to assess a new Raman spectrum by computing a variable called the PC score, which represents the weight(s) of particular PC(s) in the Raman spectrum being analyzed. Linear discriminant analysis (LDA) can then be used to derive a function of the PC scores (a discriminate function) which indicates whether or not the tissue should be considered to be similar to one group of the training spectra (e.g. a 'normal' group) or another group of the training spectra (e.g. a 'malignant' or 'diseased' or 'unhealthy' group).

[0057] Leave-one-out cross validation procedures may be used in order to prevent over training. Leave-one-out cross validation involves removing one spectrum from the data set and repeating the entire algorithm, including PCA and LDA, using the remaining set of spectra. The resulting optimized algorithm is then used to classify the withheld spectrum. This process may be repeated until each spectrum has been individually classified.

[0058] The discriminate function may subsequently be applied to categorize an unknown tissue based on where a point corresponding to the PC scores for a Raman spectrum of the unknown tissue is relative to the discriminate function surface (e.g. a line in the case where two PCs are used).

[0059] Some embodiments of the invention comprise stored data representing PCS obtained for a training set comprising Raman spectra from normal colon tissues and Raman spectra from diseased colon tissues. The PCS may correspond to only to parts of the Raman spectra having relative wavenumbers exceeding 1000 cm^"1 (exceeding 1200 cm^"1 or 1500 cm^"1 or 1800 cm^"1 in some embodiments). The stored data may additionally characterize one or more linear discriminant functions for discriminating between different tissue pathologies using the stored PCs. Software instructions may be provided on a program data store accessible to a data processor that cause the data processor to process Raman spectra using the stored PCS to yield a PC score and to then perform a linear discriminant analysis using a discriminant function specified by the stored data to characterize tissues from which the Raman spectra were obtained.

[0060] Example approaches to tissue characterization which use empirically determined diagnostic algorithms based on the determined peak intensities, widths, and/or peak ratios of tissue spectra are described in the literature and may be applied, with suitable modification, in the context of the present invention. Some examples are Mahadevan- Jansen A, and Richards -Kortum R. Raman spectroscopy for the detection of cancers and precancers, J Biomed Opt 1996;1, 31-70; Mahadevan-Jansen A, et al. Near-infrared Raman spectroscopy for in vitro detection of cervical precancers Photochem Photobiol 1998;68: 123-132; and, Huang Z, et al., Near-infrared Raman spectroscopy for optical diagnosis of lung cancer, Int J Cancer, 2003; 107: 1047-1052. All of these references are hereby incorporated herein by reference for all purposes.

[0061] Example approaches to tissue characterization which use multivariate statistical techniques are described in the literature and may be applied, with suitable modification, in the context of the present invention. Some examples are: Bakker Schut TC et al. In vivo detection of dysplastic tissue by Raman spectroscopy Anal Chem 2000;72:6010-6018; Mahadevan-Jansen A, et al. Near-infrared Raman spectroscopy for in vitro detection of cervical precancers Photochem Photobiol 1998;68: 123-132; Stone N,et al. Near-infrared Raman spectroscopy for the classif ication of epithelial pre-cancers and cancers, J Raman Spectrosc 2002; 33: 564-573; Deinum G, et al., Histological classification of Raman spectra of human coronary artery atherosclerosis using principal component analysis, Appl Spectrosc 1999;53:938-942; and, Silveira L Jr et al., Correlation between near- infrared Raman spectroscopy and histopathological analysis of atherosclerosis in human coronary arteries, Lasers Surg Med 2002;30:290-7. All of these references are hereby incorporated herein by reference for all purposes.

[0062] Raman spectrometry methods as described herein may be combined with other fast, low specificity, optical modalities like white light, narrow band, or autofluorescence video imaging In such combinations a clinician may use one or more video imaging modalities to locate suspicious tissue sites (e.g. within the colon), and then collect point Raman spectra of these sites with a fiber optic probe or catheter passed down the instrument channel of an endoscope. These spectra can then be processed as described herein to predict the tissue pathology in real time.

[0063] Examples 1 and 2 illustrate application of the invention to characterizing colon tissues. The apparatus used in each case was similar.

[0064] For example 1, the Raman system used to take measurements used a 785 nm diode laser as an excitation light source. The maximum excitation power was 150 mW. Emission was analyzed with a spectrograph incorporating a manually tunable grating and a charge coupled device (CCD) detector. One of two detachable fiber optic catheters was used to deliver excitation light to the sample and collect emission from it. These catheters contained ultra low OH impurity fibers for carrying scattered light to the spectrograph and gold coated excitation fibers for carrying excitation light to the tissue samples. One catheter incorporated optical filters at the distal end to filter out laser noise, fiber emission, and to sharply attenuate all collected light with wavelengths < 820 nm (< 540 cm^-1 relative to 785 nm excitation). This catheter attached at its proximal end to a second set of optical filters with similar transmission characteristics to further reduce the unwanted emissions. The second catheter was identical to the first except with no filters at the distal end.

[0065] A fixed catheter to tissue distance of -7.5 mm was used which generates a tissue spot size -3.5 mm in diameter. Together the spectrograph, CCD and optical filters allow reliable spectra to be obtained from: 540 to 1800 cm^-1 (LF) and from 2050-3100 cm^-1 (HF) at a spectral resolution of -10 cm^"1. Custom designed software as described in references [26] and [27] was used to subtract the fluorescence background in real time using a modified 5^th order polynomial fit.

[0066] Figure 8 is a schematic illustration of the Raman system used to acquire the in vivo Raman spectra for the in vivo measurements of Example 2. This system was similar to the Raman system described above. The excitation light source was a 785 nm diode laser (model: BRM-785, B &W Tek, Newark, DE). Emission was analyzed with a

spectrograph/CCD (charge coupled device) combination both from Princeton Instruments (Trenton, NJ, models: LS-785, and PIXIS 1340x400BR respectively). The 3.0 mm diameter, 2 m long, trifurcated probe contained a centre 200 μπι diameter fiber for excitation, surrounded by 31, 100 μπι diameter fibers, 28 of which were used for emission collection. The remaining three 100 μπι diameter fibers were coupled to a 2 mW green (532 nm) guide laser (model: CORE, Wicked Lasers) to facilitate the accurate indication of the area being measured. No optical filters were incorporated into the probe.

[0067] At the proximal end of the probe the fibers were separated into excitation (E), collection (C) and guide (G) channels and coupled to collimating lenses and filters from SemRock (Rochester, NY, models: LL01-785, BLP01-785R and FF01-531/22 respectively). These filters reduced off-resonance laser noise and fiber emission, blocked all wavelengths < 790 nm from reaching the spectrometer, and ensured that the guide light contained only green emission.

[0068] A single 200 μπι fiber connected the laser to the filter module, and a second specially designed fiber bundle guided the filtered emission to the spectrometer. This bundle consisted of 120, ultra pure, 50 μπι diameter fibers packed in a round geometry at the filter end, but spread out into a parabolic arc at the spectrometer end to increase signal to noise ratio, spectral resolution and the throughput of the system as described in reference [22].

[0069] The system was wavelength calibrated using neon and mercury standard lamps (Newport Corporation, Stratford, CT), and intensity calibrated using a halogen standard lamp (RS-10, Gamma Scientific, San Diego, CA) . The spectral resolution was estimated to be ~ 8 cm^-1. The maximum excitation power at the tissue surface was 150 mW. A probe to tissue distance of between 5-10 mm was used which generates a tissue spot size between 2-5 mm in diameter. A TTL switch was incorporated into the laser and synchronized with the spectrometer data acquisition. This allowed an instantaneous on/off laser mode to be software controlled once the probe was focused on the point of interest. [0070] Custom designed software removed the CCD dark count, applied an 5 point spectral smoothing, and subtracted the autofluorescence background using a modified polynomial fitting routine all in real time. The complete system was mounted on a movable cart with an articulated arm.

Example 1 - Samples and Methods

[0071] A total of 47 colon tissue samples were collected from 18 patients. Excised tissue was collected from 8 patients during surgery to remove a previously identified malignant lesion. Samples were obtained from the lesion itself, and from the surrounding tissue visually free of disease. For some sites two tissue fragments were obtained, and these were treated separately giving a total of 11 lesion and 9 normal samples. The average volume of the samples was approximately 5 mm³. All excised lesion samples were classified by histology as adenocarcinomas, and those from the surrounding tissue were normal. The remaining samples were biopsies obtained from 10 polyps (with matched normal epithelium) during a routine colonoscopy. Eight polyps were classified by histological evaluation as adenomatous (with varying grades of dysplasia), and two were invasive adenocarcinomas. Two tissue fragments were obtained from four of the polyps and three of the normal epithelium sites and these were treated separately giving a total of 14 polyp and 13 normal samples. The average volume of a biopsy sample used for Raman measurements was approximately 1.5 mm³. All samples were immediately placed in saline (@ 4°C) after collection and the Raman spectra measured within 30 minutes. Each sample was placed on an aluminum foil covered glass microscope slide, placed under a fiber optic catheter fixed above the sample, and spectra obtained with a 1 second integration time. In addition to the colon samples, some preliminary in vivo tests of the Raman system were done on the palm skin of a volunteer.

[0072] The raw Raman spectra from the colon samples were standardized prior to analysis. This was accomplished with the following steps: removing the ambient background, calibrating, smoothing, fluorescence removal, and normalizing to reduce the effect of intensity variations from different tissue sites with the same pathology. The normalization was accomplished by summing the area under each curve and dividing each variable in the smoothed spectrum by this sum. Simple Raman peak ratios were calculated using two peaks from each range. These peaks were selected on the basis of a student's t- test which indicated which peaks were the most significantly different between normal and diseased tissue.

[0073] Commercial software was also used to perform multivariate statistical analyses (Statistica™ 10.0, StatSoft Inc. Tulsa, OK) where a spectral range is used rather than single Raman peaks (univariate). Principal components (PCs) for all the spectra in the dataset were computed to reduce the number of variables for the multivariate analyses. Student's t-tests were used on the PCs that accounted for 0.1 % or more of the variance to determine those most significant at separating spectral ranges into two pathology groups: normal and diseased tissue. A linear discrimination analysis (LDA) with leave-one-out cross validation was used on the most significant PCs. To avoid over fitting the data, the number of PCs used in the LDA was limited to one third of the total number of cases of the smallest subgroup. The excised tissue and the biopsy samples were analysed separately.

Example 1 - Results

[0074] The initial in vivo test spectra of palm skin in the LF taken with the optically filtered and unfiltered catheters are shown in Figures 1A and 1C. The difference spectrum was dominated by 3 broad peaks at 590, 780, and 1030 cm^"1. Below 500 cm^"1 the emission collected with the unfiltered catheter saturated the detector. In the HF range the palm skin spectra were very similar for both catheters apart from a small decrease in the emission intensity below 2400 cm^"1 and above 2850 cm^"1 with the optically filtered catheter (Figure IB). Removal of the background fluorescence results in nearly identical Raman spectra obtained with either catheter (Figure ID).

[0075] For the more reflective colon tissue, the LF emissions at 590, 780, and 1030 cm^"1 saturated the detector when using the optically unfiltered catheter (not shown). Even with the optically filtered catheter, these peaks were still clearly present in all excised tissue and biopsy samples (Figure 2), and very strong in a few. There was a substantial fluorescence contribution to the LF spectra for all colon samples (Figures 2A and 2B). Generally the Raman emission intensity from the biopsy samples was weaker but the fluorescence was stronger than from the excised tissue. Furthermore the fluorescence was stronger in diseased excised tissue than normal tissue, but the reverse of this for the biopsy samples.

[0076] After the fluorescence contribution was subtracted Raman peaks were clearly visible around 1340, 1450, 1650 and 1730 cm^"1 as well as a number of smaller peaks most notably around 1000, 1150 and 1550 cm^"1 (Fi gures 2C and D). There were also differences in the shape of the LF Raman spectra for excised and biopsy tissue.

[0077] For the HF range, a comparison of spectra taken of colon tissue with the optically filtered and unfiltered catheters (not shown) demonstrated similar differences below 2400 cm^"1 and at around 2850 cm^"1 as for the palm skin measurements (Figure IB and D).

Comparisons in the HF spectra obtained from diseased and normal tissues with the optically filtered catheter are shown in Figure 3: A and C for excised tissues and B and D for biopsy tissues. The HF tissue fluorescence intensity drops to less than half that of the LF range, and was stronger for the biopsy samples (Figure 3B) than the excised tissue as in the LF case. Furthermore the fluorescence was again stronger in diseased excised tissue than normal tissue, but the reverse of this for the biopsy samples. Raman emissions were observed in the raw data clustered around 2900 cm^"1, and low intensity broad peak centred at 2150 cm^"1. Between 2200 and 2800 cm^"1 of the emission range there were a number of weak peaks that did not seem to vary for different samples. Above 3000 cm^"1 the intensity rises steeply. , Figures 3C and D show the Raman range from 2825-3025 cm^"1 after fluorescence removal, differences in the shape of the spectra between excised and biopsy tissue are seen.

[0078] Due to saturation of most LF spectra obtained with the optically unfiltered catheter, statistical analyses were carried out on spectra obtained with the optically filtered catheter only. Even with this catheter, 4 LF spectra out of the 47 were rejected from the analyses because of partial detector saturation. The rejected spectra were from 3 excised tissue samples (2 malignant and one normal), and from one adenomatous polyp.

[0079] Simple Raman peak ratios for the remaining LF and all the HF spectra are shown in Figure 4. Figures 4A and C are for the excised tissue (LF and HF respectively), and Figures 4B and D are for the biopsy samples (LF and HF respectively). Note: different Raman peaks were used in the ratio calculation for the excised and biopsy samples. There were clearly various degrees of clustering in the Raman peak ratios. Ratios calculated from the peaks in the LF spectra of excised tissue produced the best cluster separation and those from the peaks in the HF spectra of biopsy tissue produced the worst.

[0080] The results of the multivariate analyses on the LF spectra are shown in Figure 5. Figures 5A and B are two dimensional (2D) scatter plots of the two principal components that are the most highly correlated with tissue pathology for the excised tissue and biopsy samples respectively. The pathology classes were clearly clustered in two groups. Figures 5C and D show the posterior probabilities derived from the leave-one-out LDA for the excised and biopsy tissue respectively.

[0081] The results of the multivariate analyses on the HF spectra are shown in Figures 6A and 6B which are 2D scatter plots of the two principal components that are the most highly correlated with tissue pathology for the excised tissue and biopsy samples respectively. The pathology classes were again clearly clustered in two groups, and this clustering appears better for the excised tissue samples. Figures 6C and D show the posterior probabilities derived from the leave-one-out LDA for the excised and biopsy tissue respectively.

[0082] Figures 7A and B show receiver operator characteristics (ROCs) for the excised and biopsy tissue spectra measured in the LF and HF ranges respectively. For the LF range 100% sensitivity and 87% specificity are obtained for the excised tissue and 100% sensitivity and 69% specificity for the biopsies. The area fractions under the ROCs were 0.986 and 0.944 for excised and biopsy tissue respectively. For the HF range 100% sensitivity and 89% specificity are obtained for the excised tissue and 100% sensitivity and 85% specificity for the biopsies. The area fractions under the ROCs were 0.929 and 0.934 for excised and biopsy tissue respectively.

Example 1 - Discussion

[0083] In the LF range the fibre emission was large with the optically unfiltered catheter despite the use of ultra low impurity materials. Catheters used to measure spectra in this range have to include expensive optical filters at their distal end to reduce fibre emission, avert detector saturation and increase the signal to noise ratio. In contrast, the HF Raman spectra showed only small differences in spectra obtained with the two different catheters. It is unclear what causes the divergence in the spectra below 2400 cm^"1 (Figure IB). The difference cannot be explained by greater fibre fluorescence tail since this is inconsistent with the LF spectra, although it could be caused by a broad fibre emission peak. In contrast the difference above 2850 cm^"1 was most likely due to a drop in transmission of the catheter filters in this range (from data provided by the filter manufacturer).

[0084] The LF Raman spectra from the colon samples included a significant fluorescence contribution to the spectra. Despite extensive optical filtering, fibre emission peaks were clearly evident as well. From this, it can be deduced that the measured fluorescence is in part coming from the fiber catheter in addition to autofluorescence from the tissue. The LF Raman spectra of biopsy tissues were generally less intense, and riding on a higher fluorescence background compared to the excised tissues.

[0085] The malignant excised tissue shows increases in Raman peaks around 1340 (lipids), and 1450 (lipids), and 1650 cm^"1 (amide I, and H₂0) and decreases at 1150 (proteins), 1540 (amino acids), and 1735 cm^"1 (lipids) compared to the normal excised tissue spectra. The changes in the spectra from the biopsy tissue seem to be largely the reverse of this apart from the 1735 cm^"1 peak. These differences are probably caused by variations in sample composition, where the excised tissue has more contribution from deeper tissue layers. It should be noted that in vivo spectra will likely be more similar to the excised tissue spectra shown here.

[0086] In the HF range the spectra were similar to those obtained from other organs. Fluorescence was still significant, but it was about half of that found in the LF range. It can be deduced from these data that only a small fraction of this fluorescence was coming from the delivery and collection fibres. The biopsy Raman spectra were again generally less intense, and riding on a higher fluorescence background compared to the excised tissue. The dominant Raman emissions near 2900 cm^"1 were mainly due to a combination of lipid (C-H) peaks (2853+2866 cm^"1) and generic protein vibrations (2930 cm^"1). The low intensity broad peak centred at 2150 cm^"1 and the steep intensity increase above 3000 cm^"1 may correspond to water molecule vibrational modes. The weak peaks occurring between 2200 and 2800 cm^"1 may be due to small CCD etaloning effects.

[0087] For the malignant excised tissue the intensity of the lipid peaks at 2853 and 2866 cm^"1 dropped, but the intensity of the generic protein peak at 2930 cm^"1 increased compared to normal samples. In contrast it is hard to see really significant changes in the HF spectra from the biopsy samples. These differences probably occur because of variations in sample composition as seen in the LF spectra.

[0088] The peak ratios calculated from the spectra were surprisingly good at separating the tissue into two pathology groups. For the LF excised tissue, one can clearly see the 1340 and 1735 cm^"1 peaks change significantly in the average spectra shown in Figure 2C, and the ratio of these peaks clearly separates normal and malignant tissue (Figure 4A). For the LF biopsy tissue, the 1340 cm^"1 peak does not show significant change between normal and diseased tissue (Figure 2D), instead the peak at 1445 cm^_1was used with the 1735 cm^"1 peak to calculate the ratio (Figure 4B). However in this case the two pathology groups are not as well separated.

[0089] For the HF spectra using a simple peak ratio to predict the pathology was not so reliable (Figures 4C and 4D) generating lower diagnostic accuracies. This is not too surprising since the relative changes in peak heights between normal and diseased tissue as indicated by the HF average spectra shown in Figures 3C and 3D were less than those found in the LF Raman spectra.

[0090] Multivariate analyses produced much better diagnostic statistics for separating normal and diseased tissue compared to the simple peak ratios. Furthermore the HF Raman spectra were better at predicting the pathology than the LF Raman spectra despite there being fewer Raman peaks in the HF measurement range. This result was not so surprising for the HF spectra from excised tissue, since one can clearly see changes in the shape of the average spectra for different pathologies. However the HF Raman spectra from the biopsy samples were also good at predicting the pathology, although in this case the average spectra of diseased and normal tissue were quite similar in appearance. This highlights the power of multivariate analyses where multiple small changes in a spectral range can be diagnostically very significant.

[0091] This example shows that HF Raman spectra can be used as a diagnostic tool to discriminate between different colon tissue pathologies. The Raman peak ratios calculated from the HF spectra were good at predicting the pathology but worse than ratios obtained from the LF spectra. In contrast, multivariate analyses indicated that HF spectra were very good at predicting the pathology and slightly better than the LF spectra. Multivariate statistical analyses predicted the pathology with 100% sensitivity and a specificity of >88% for both the low and high frequency data sets.

[0092] Although the spectra analysed in this study were obtained with catheters incorporating expensive optical filtering at the distal end such filtering can be omitted for HF spectroscopy with the right choice of fibre. Utilizing optically unfiltered catheters to obtain HF Raman spectra has several key advantages mainly around the design, reliability, cost and longevity of the catheters. The evidence points to HF Raman spectroscopy as having great potential for improving the in vivo detection of early neoplastic lesions of the colon. Example 2 - In vivo results

[0093] 60 spectra were obtained from normal tissue and polyps under video surveillance during a routine colonoscopy. The polyps were subsequently biopsied, and the pathology determined. The in vivo Raman spectra from different tissue pathologies were distinct and correlated well with the results presented in Example 1 for ex vivo tissues.

[0094] Two patients were enrolled for the test as part of their routine colonoscopies. Before each use the probes were cleaned with an enzymatic detergent, and then soaked in a high level disinfection solution following a reprocessing protocol similar to that used for the colonoscopes. Some initial in vivo tests of the system were conducted on the palm skin of a volunteer. Then the system was tried on the two patients: 73 year old female (patient #1), and a 64 year old male (patient #2) who consented to the tests as part of their routine colonoscopy procedure.

[0095] Polyps were located during the procedures using either white light (WLI) or narrow band (NBI) video imaging (Evis Exera II platform, Olympus America Inc., Center Valley, PA). The Raman probe was inserted into the 3.7 mm diameter instrument channel and positioned using the green guide light under video surveillance. A one second Raman spectrum was then obtained. Several spectra of each polyp and adjacent normal tissue were obtained. High quality Raman spectra were obtained with a Is integration time, which only marginally extended the colonoscopy procedure time. Very little interference was observed from inherent fibre emission, or from the video imaging light. Biopsies were obtained of the polyps and their pathology determined.

[0096] The initial in vivo test spectra of palm skin showed very clear HF Raman peaks centred at 2900 cm^"1 superimposed on background fluorescence (Figure 9). The test was done with the probe placed down the colonoscope and the image light on. As can be seen from Figure 9, there was very little interference of the Raman spectra by the imaging light apart from a small broad peak near 2220 cm^"1 with NBI (see Figure 10). The results of the in vivo tests on the colon tissue were similar to those of palm skin with Raman emissions clustered around 2900 cm^"1, and small broad peak near 2220 cm^"1 with NBI.

[0097] Figure 11 shows the Raman range from 2800-3050 cm^"1 after fluorescence removal, and normalization. Clear differences in the shape of the spectra for different sites are seen. The pathology of the polyp from patient #1 was determined to be consistent with hyperplasia, and for patient #2, a tubular adenoma.

[0098] There was very little interference from the surveilling imaging light in the Raman range, although this may not be the case for other colonoscope systems (see Figure 12). The observed Raman emissions are thought to be mainly due to a combination of lipid (C- H) peaks (2853+2866 cm^"1) and generic protein vibrations (2930 cm^"1). For the diseased sites the intensity of the lipid peaks dropped, but the intensity of the generic protein peak increased compared to normal samples. This is consistent with the findings of both ex vivo measurements on colon tissue and in measurements on tissue from other organs. These spectral changes have been shown to accurately predict pathology either by a peak ratio method or by multivariate statistical analyses. It is encouraging to see the hyperplasic polyp showed spectral differences from normal tissue even though it was at a very early disease stage.

[0099] This study shows that a Raman system utilizing optically unfiltered probes can obtain clear in vivo HF Raman spectra, and that these spectra show clear changes with tissue pathology. This detection modality may also be applied for discriminating ulcerative colitis and Crohn's disease and for identifying dysplasia in inflammatory bowel disease and flat or polypoid lesions in vivo.

Probe Systems

[0100] The following describes some alternative example probe systems that may be applied for Raman spectroscopy of colonic tissues and may additionally have other applications. In general it would be desirable to provide probes that are either or both inexpensive and reusable. For a probe to be reusable the probe should be able to withstand sterilization procedures. For application to characterization of colon tissues a probe must also have optical characteristics good enough to permit obtaining Raman spectra at least within a range of Raman shifts of suitable quality to use for tissue discrimination as described above. For some applications probes that are small (especially in cross section) are desirable.

[0101] As noted above, where tissue characterization is done using Raman spectra at higher Raman shifts a probe may be made simpler and less expensive because it is not necessary to provide an expensive optical filter at the distal end of the probe to block light at the excitation wavelength. Accordingly probes for Raman spectroscopy according to some embodiments include a return optical pathway that does not include an optical filter that blocks light of the excitation wavelength at its distal end. This pathway may, for example, be provided by one or more optical fibers. Filtering is also not required on fiber(s) carrying excitation light.

[0102] Since it is not required to block light at the excitation wavelength from the return optical path, the possibility exists for using an single optical fiber or group of optical fibers both to transmit excitation light and to carry light back to a Raman spectrometer. Some embodiments provide a single optical pathway that both carries excitation light to tissues and caries light scattered from the tissues to a spectrometer. Such embodiments can be advantageous because a fiber optic probe that illuminates and collects through the same fiber can offer an improved efficiency for collecting scattered light. On the other hand, care must be taken (by suitable optical design and selection of materials) to avoid fluorescence and other light emissions stimulated within the fiber by the relatively intense excitation light travelling to the tissues being studied in the wavelength range of interest from interfering with obtaining a satisfactory Raman spectrum.

[0103] Other embodiments provide arrangements where excitation light is carried to the tissues in one channel and light scattered from the tissues is carried back to the spectrometer in a second separate channel. Each channel may, for example, comprise one or more optical fibers. [0104] Figure 13 is a schematic diagram illustrating an example setup for Raman spectrometry using a single optical fiber. The Figure 13 setup is similar to that described in Reference [20]. Laser light at an excitation wavelength (e.g. 785 nm) passes through a short-pass filter (SPF) and is focused into a large core single fiber catheter (FC) by a fused-silica lens (LI). Backscattered radiation is filtered by a chevron-type high-pass filter (F1/F2) and focused (L2) into the detection fiber (DF). Large cores up to 1 mm 0 are available which is equivalent in area to 100 x 100 μπι fibers

[(1000umxl000um)/(100umxl00um)=100]. There is a tradeoff between using fewer individual optical fibers to achieve improved light collection efficiency and general simplicity at the cost of some flexibility (bendability) and using more optical fibers to provide a channel for carrying light to and from the tissues of interest.

[0105] Figure 14 shows an example set up for collection of HF Raman Spectra with an optical fiber catheter having separate excitation and collection channels. This example provides the advantage that any fluorescence or other emissions of light in the wavelength range of interest that occur in the fiber(s) making up the excitation channel are much less effectively collected by the collection fibers. Maximizing the number collection fibers increases efficiency and maintains probe flexibility. However fiber probes with large numbers of fibers are expensive and have to be reprocessed (too expensive to be disposable). The collection channel in the embodiment of Figure 14 does not require a filter coating on its distal end.

[0106] Figure 15 shows a probe 150 according to another embodiment in which one optical fiber 151provides a channel for excitation light and a second optical fiber 152 provides a separate channel for collected scattered light. Ends 151A and 152A of the optical fibers may be flat or may be beveled, as shown. Beveled fibers can provide improved light collection efficiency as compared to a dual fiber with a flat tip

configuration. The angled ends of the fibers steer light. The fiber ends are angled such that light collected by the collection fiber 152 overlaps with the light beam emitted by the excitation fiber 151. The ends of the fibers may be angled in the range of 0 degrees to 60 degrees (with 0 degrees corresponding to the case where the fiber ends are flat and perpendicular to the fibers). In some embodiments, one of the optical fibers has a flat end and the other has a beveled end. For example, the collection optical fiber may have a flat end while the excitation optical fiber has a beveled end.

[0107] An example size for the smaller excitation fiber 151 is 50 μπι diameter. An example size for the larger collection fiber 152 is 400 μπι - 1000 μπι diameter. Probe 150 may be made to be conveniently small and inexpensive enough to be disposable. No filter coatings are needed on the distal ends of the optical fibers for use in HF Raman spectroscopy.

[0108] A probe like probe 150 may be used to provide smaller-diameter probes which may be useful in applications other than HF Raman spectroscopy. For LF Raman spectroscopy filters may be provided. For example: o The distal end of the excitation fiber may be coated to provide a SP filter that passes laser wavelengths but blocks longer wavelength fiber fluorescence and Raman noises; o The distal end of the collection fiber may be coated with a LP filter that blocks laser wavelengths, but passes longer wavelength tissue Raman signals.

[0109] Figures 16A and 16B illustrate alternative Raman spectrometry apparatus using a double clad fiber to carry excitation light to a tissue of interest and to carry collected scattered light back to a spectrometer. Here 785 nm laser excitation light is reflected by a mirror (M) and passes band pass filter (BP) then through a dichroic mirror (DM) which transmits 785 nm laser light and reflects longer wavelength Raman signals. The excitation light is focused into the centre core of a double clad fiber catheter by a lens (LI).

Backscattered radiation and Raman signals from tissue is collected by inner cladding and collimated by LI and reflected by DM into a spectrometer. [0110] Double clad fibers with inner clad diameters up to 400 μπι are commercially available. For example, the Fibercore™ model F-SMM900 is an example double core fiber. The laser needs to be well aligned with the center core, thus a larger center core is advantageous (say 50 μπι). With a large center core, a SMA or FC connector may be used to align the fiber with the laser beam through LI. This will enable disposable use of the fiber catheter. A long pass coating maybe applied onto the inner cladding surface at the proximal end of the fiber to prevent laser light from getting into the inner cladding. This will prevent fluorescence noise being generated in the inner cladding and get collected by the spectrometer. Antireflection coating may be applied at both end of the fiber to increasing efficiency of the probe.

[0111] A probe using a double clad fiber may be miniaturized for use in a wide range of applications. If the probe will be used for LF Raman spectroscopy the probe To facilitate LF Raman measurements, the following filtering may be provided:

• Coating the fiber distal end center core with a SP filter that passes excitation laser wavelengths and blocks longer wavelength fiber fluorescence and Raman noises.

• Coating the fiber distal end of the inner cladding with a LP filter that blocks excitation laser wavelengths, but passes longer wavelength tissue Raman signals.

[0112] As shown in Figures 17 and 18, an alternative to using lenses and mirrors to focus light in and out of double clad fiber is to use pigtailed connections to an isolating wavelength division multiplexer.

[0113] Figure 18A shows an example multiplexer 180 which optically connects a probe optical fiber 182 to both an excitation light source (not shown) by way of an optical fiber 183 and a spectrometer by way of a spectrometer optical fiber 184. A support point (e.g. an eye bolt) 181 is provided to facilitate supporting multiplexer 180.

[0114] Spectrometer optical fiber 184 comprises a number of individual fibers that are arranged in a bundle at the end of spectrometer optical fiber 184 that couples to multiplexer 180 and are arranged in a parabolic configuration at the end of spectrometer optical fiber 184 from which light is coupled to spectrometer 185. Optical fibers 182, 183 and 184 are coupled to multiplexer 180 by couplings 186 which may be SMA type couplings.

[0115] In an example embodiment, optical fiber 183 comprises a 100 μπι core fibre NA=0.22, with a SMA termination. In an example embodiment, probe optical fiber 182 comprises a medical MM 600/660 μπι fiber here, with NA=0.3, and a SMA connector. In an example embodiment, spectrometer optical fiber 184 comprises an 836 μπι active area fibre bundle NA=0.22, with SMA termination.

[0116] Collimating lenses 187 are provided to couple light into and out of the optical fibers. A mirror 188 is wavelength selective. In an example embodiment, mirror 188 comprises a dichroic mirror that reflects light at an excitation wavelength (e.g. 785 nm) and transmits light at longer wavelengths (e.g. 800-1200 nm). Light from excitation optical fiber 183 is reflected by mirror 188 into probe optical fiber 184. Scattered light collected by probe optical fiber 184 is directed onto mirror 188 which transmits reflects light near the excitation wavelength and transmits longer wavelength light to spectrometer optical fiber 184.

[0117] Filter housings 189A and 189B are provided. Filters may be inserted into filter housings 189 A and/or 189B to improve the quality of the light. For example filter holder 189 A may hold a bandpass filter that passes only wavelengths close to the desired excitation wavelength. Filter holder 189B may hold a filter that blocks light at one or more wavelengths outside of a desired range of wavelengths (where the desired range includes Raman shifts of interest). In an example embodiment, each filter holder is configured to hold a 12.5 mm (0.5 inch) diameter filter and has a filter locking ring to accept 3.5 mm thick filters.

[0118] In addition to use for HF Raman spectroscopy of colon tissue, probes as described herein may be applied for HF Raman spectroscopy of other tissues of the body. Such probes (modified with filters as described above) may also be used for LF Raman spectroscopy of colon tissues or other tissues.

[0119] Figure 19 illustrates a sheath 190 that may be applied to a fiber optic probe for use in endoscopic applications. The sheath may be disposable. As shown in Figure 19, disposable sheath 190 can be fit over a Raman catheter 193 (which may have a construction like that of any of the probes described herein for example). An optical window 191 fitted at the distal end of sheath 190 allows even illumination of target tissue by excitation light and efficient collection of scattered light containing Raman signals.

[0120] Window 191 may provide improved coupling of light both to and from tissues of interest than would be provided by an air gap (as in the embodiment of Figure 8) due to improved matching of index of refraction between the optical fibers of the probe, the window and the tissues. Window 191 may be coated with anti-reflection coatings on one or both of its surfaces.

[0121] Sheath 190 comprises a tubular body 192 sealed to optical window 191. In a prototype embodiment body 192 was made of medical grade polyvinylidene difluoride (PVDF) tubing having an inner diameter of 3.00 mm and a wall thickness of 0.15mm. A custom made fused silica window was fitted at the end of the PVDF tubing and bonded to the inner surface of the tubing using medical epoxy. The sheath assembly was sanitized using an electron beam at 25 kGy and successfully passed a sterility test (USP 71).

[0122] Sheath 190 protects the Raman catheter against possible contamination by viruses and germs inside the gastrointestinal tract and protects the patient from contact with the Raman probe. For Raman spectra measurement, the optical window at the distal end of sheath 190 facilitates spectra measurement and can result in improved data quality.

[0123] A sterile sheath 190 may be packaged in sterile packaging for use as a single-use disposable sheath. The sheath may be disposed of after use.

[0124] Raman spectra of both normal colonic tissue and adenoma of the same patient are shown in Figure 20. The spectrum of normal colonic tissue was taken at a location 5cm away from the adenoma polyp. The Raman spectra clearly show differences between normal and precancerous growth at relative wavenumbers 1680 cm^"1 and between 2800 and 3000 cm^"1.

References

The following references describe various medical Rama spectroscopy systems and applications. These references are all hereby incorporated herein by reference for all purposes.

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Advances in endoscopic imaging of colorectal neoplasia", Biophotonics 2011 ; 4:482-489. Wang, T.D., Van Dam, J., "Optical biopsy: a new frontier in endoscopic detection and diagnosis", Clinical Gastroenterology Hepatology 2004; 2:744-753. Hanlon, E.B., Manoharan, R., Koo, T.-W. et al., "Prospects for in vivo Raman spectroscopy", Physics in Medicine and Biology 2000; 45:Rl-59. Molckovsky, A., Wong, L.-M., Shim, M.G. et al., "Diagnostic potential of near- infrared Raman spectroscopy in the colon: differentiating adenomatous from hyperplastic polyps", Gastrointenstinal Endoscopy 2003; 57:396-402. Crow, P., Uff, J.S., Farmer, J.A., Wright, M.P., Stone, N., "The use of Raman spectroscopy to identify and characterize transitional cell carcinoma in vitro", British Journal of Urology International 2004; 93: 1232-1236. Lau, D.P., Huang, Z., Lui, H., Anderson, D.W., Berean, K., Morrison, M.D. et al., "Raman spectroscopy for optical diagnosis in the larynx: Preliminary findings", Lasers in Surgery and Medicine 2005; 37: 192-200. Malini, R., Venkatakrishna, K., Kurien, J. et al., "Discrimination of normal, inflammatory, premalignant, and malignant oral tissue: a Raman spectroscopy study", Biopolymers 2006; 81 : 179-193. Robichaux-Viehoever, A., Kanter, E. et al., "Characterization of Raman spectra measured in vivo for the detection of cervical dysplasia", Appl. Spectrosc. 2007; 61 :986-997. Teh, S.K., Zheng, W., Ho, K.Y., Teh, M., Yeoh, K.G., Huang, Z., "Diagnostic potential of near infrared Raman spectroscopy in the stomach: differentiating dysplasia from normal tissue", British Journal of Cancer 2008; 98:457-465. Magee, N.D., Villaumie, J.S., Marple, E.T., Ennis, M., Elborn, J.S., McGarvey, J., "Ex Vivo diagnosis of lung cancer using a Raman miniprobe", Journal of Physical Chemistry B 2009; 113:8137-8141. Almond, L.M., Hutchings, J., Shepherd, N., Barr, H., Stone, N., Kendall, C, "Raman spectroscopy: a potential tool for early objective diagnosis of neoplasia in the oesophagus", Biophotonics 2011 ; 4:685-695 Lui, H., Zhao, J., Mclean, D.I., Zeng, H., "Real-time Raman spectroscopy for in vivo skin cancer diagnosis", Cancer Research 2012 (In press, doi: 10.1158/0008- 5472.CAN-11-4061). Zeng, H., Fawzy, Y.S., Short, M.A. et al., "Combining field imaging endoscopy with point analysis spectroscopy for improving early lung cancer detection", Proc. 30th Annual International IEEE Engineering in Medicine and Biology Society Conference 2008; 1849-1850. Shim, M.G., Wilson, B.C., Marple, E.C. et al., "Study of fiber optic probes for in vivo medical Raman spectroscopy", Applied Spectroscopy 1999; 53: 619-627. Santos, L.F., Wolthuis, R., Koljenovic, S. et al., "Fiberoptic probes for in vivo Raman spectroscopy in the high-wavenumber region", Anal. Chem. 2005;

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33. http://www.wickedlasers.com/core

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Interpretation of Terms

[0125] Unless the context clearly requires otherwise, throughout the description and the claims:

• "comprise", "comprising", and the like are to be construed in an inclusive sense, as opposed to an exclusive or exhaustive sense; that is to say, in the sense of

"including, but not limited to";

• "connected", "coupled", or any variant thereof, means any connection or coupling, either direct or indirect, between two or more elements; the coupling or connection between the elements can be physical, logical, or a combination thereof;

• "herein", "above", "below", and words of similar import, when used to describe this specification, shall refer to this specification as a whole, and not to any particular portions of this specification;

• "or", in reference to a list of two or more items, covers all of the following

interpretations of the word: any of the items in the list, all of the items in the list, and any combination of the items in the list;

• the singular forms "a", "an", and "the" also include the meaning of any appropriate plural forms. [0126] Words that indicate directions such as "vertical", "transverse", "horizontal", "upward", "downward", "forward", "backward", "inward", "outward", "vertical", "transverse", "left", "right", "front", "back", "top", "bottom", "below", "above", "under", and the like, used in this description and any accompanying claims (where present), depend on the specific orientation of the apparatus described and illustrated. The subject matter described herein may assume various alternative orientations. Accordingly, these directional terms are not strictly defined and should not be interpreted narrowly.

[0127] Embodiments of the invention may be implemented using specifically designed hardware, configurable hardware, programmable data processors configured by the provision of software (which may optionally comprise "firmware") capable of executing on the data processors, special purpose computers or data processors that are specifically programmed, configured, or constructed to perform one or more steps in a method as explained in detail herein and/or combinations of two or more of these. Examples of specifically designed hardware are: logic circuits, application-specific integrated circuits ("ASICs"), large scale integrated circuits ("LSIs"), very large scale integrated circuits ("VLSIs"), and the like. Examples of configurable hardware are: one or more

programmable logic devices such as programmable array logic ("PALs"), programmable logic arrays ("PLAs"), and field programmable gate arrays ("FPGAs")). Examples of programmable data processors are: microprocessors, digital signal processors ("DSPs"), embedded processors, graphics processors, math co-processors, general purpose computers, server computers, cloud computers, mainframe computers, computer workstations, and the like. For example, one or more data processors in a control circuit for a device may implement methods as described herein by executing software instructions in a program memory accessible to the processors.

[0128] Processing may be centralized or distributed. Where processing is distributed, information including software and/or data may be kept centrally or distributed. Such information may be exchanged between different functional units by way of a communications network, such as a Local Area Network (LAN), Wide Area Network (WAN), or the Internet, wired or wireless data links, electromagnetic signals, or other data communication channel.

[0129] Software and other modules may reside on servers, workstations, personal computers, tablet computers, spectrometers, customized medical instruments and other devices suitable for the purposes described herein.

[0130] The invention may also be provided in the form of a program product. The program product may comprise any non-transitory medium which carries a set of computer-readable instructions which, when executed by a data processor, cause the data processor to execute a method for characterizing tissue, specifically colon tissues in some embodiments, according to the invention. Program products according to the invention may be in any of a wide variety of forms. The program product may comprise, for example, non-transitory media such as magnetic data storage media including floppy diskettes, hard disk drives, optical data storage media including CD ROMs, DVDs, electronic data storage media including ROMs, flash RAM, EPROMs, hardwired or preprogrammed chips (e.g., EEPROM semiconductor chips), nanotechnology memory, or the like. The computer-readable signals on the program product may optionally be compressed or encrypted.

[0131] In some embodiments, the invention may be implemented in software. For greater clarity, "software" includes any instructions executed on a processor, and may include (but is not limited to) firmware, resident software, microcode, and the like. Both processing hardware and software may be centralized or distributed (or a combination thereof), in whole or in part, as known to those skilled in the art. For example, software and other modules may be accessible via local memory, via a network, via a browser or other application in a distributed computing context, or via other means suitable for the purposes described above.

[0132] Where a component (e.g. a software module, processor, assembly, device, circuit, etc.) is referred to above, unless otherwise indicated, reference to that component (including a reference to a "means") should be interpreted as including as equivalents of that component any component which performs the function of the described component (i.e., that is functionally equivalent), including components which are not structurally equivalent to the disclosed structure which performs the function in the illustrated exemplary embodiments of the invention.

[0133] Specific examples of systems, methods and apparatus have been described herein for purposes of illustration. These are only examples. The technology provided herein can be applied to systems other than the example systems described above. Many alterations, modifications, additions, omissions, and permutations are possible within the practice of this invention. This invention includes variations on described embodiments that would be apparent to the skilled addressee, including variations obtained by: replacing features, elements and/or acts with equivalent features, elements and/or acts; mixing and matching of features, elements and/or acts from different embodiments; combining features, elements and/or acts from embodiments as described herein with features, elements and/or acts of other technology; and/or omitting combining features, elements and/or acts from described embodiments.

[0134] It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions, omissions, and sub-combinations as may reasonably be inferred. The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

WHAT IS CLAIMED IS:

1. A method for characterizing colon tissues, the method comprising:

obtaining Raman spectrum data representing a Raman spectrum of the colon tissues; and

processing the Raman spectrum data to yield data characterizing the tissues;

wherein the data characterizing the colon tissues is not based on any part of the Raman spectrum having a Raman shift of less than 1000 cm^"1.

2. A method according to claim 1 wherein processing the Raman spectrum data comprises performing a multivariate analysis on a part of the Raman spectrum having a Raman shift exceeding 1800 cm^"1.

3. A method according to any one of claims 1 and 2 wherein processing the Raman spectrum data comprises detecting and measuring peaks in the Raman spectrum at Raman shifts of two or more of 2853 cm^"1, 2866 cm^"1 and 2930 cm^"1.

4. A method according to any one of claims 1 to 3 wherein the tissues are in vivo.

5. A method according to any one of claims 1 to 4 wherein obtaining the Raman spectrum data comprises illuminating the tissues with excitation light at an excitation wavelength, collecting scattered light that has been scattered by the tissues, and carrying the scattered light to a Raman spectrometer via an optical path that carries scattered light at the excitation wavelength.

6. A method according to claim 5 wherein the excitation wavelength is 785 nm.

7. A method according to any one of claims 5 and 6 comprising carrying the scattered light to the Raman spectrometer by way of a return optical path extending along an endoscope.

8. A method according to claim 7 wherein the return optical path comprises a single optical waveguide.

9. A method according to claim 7 wherein the return optical path comprises a group of optical waveguides.

10. A method according to claim 7 comprising carrying the excitation light to the tissues by way of a forward optical path extending along the endoscope.

11. A method according to claim 10 wherein the forward optical path comprises a single optical waveguide.

12. A method according to claim 10 wherein the forward optical path comprises a group of optical waveguides.

13. A method according to claim 10 wherein the forward optical path and the return optical path are provided in the same optical waveguide.

14. A method according to claim 13 wherein the forward and return optical paths are provided by a single-use fiber probe extending along the endoscope.

15. A method according to any one of claims 7 to 13 wherein the return optical path is provided by a structure and the method comprises inserting the structure into a tubular sheath.

16. A method according to claim 15 wherein the sheath comprises an optical window and the method comprises illuminating the tissues by the excitation light and collection of the scattered light through the optical window.

17. A method according to claim 16 wherein the sheath comprises a tubular plastic body sealed to the optical window.

18. A method according to any one of claims 15 to 17 wherein the sheath is a single- use disposable sheath and the method comprises disposing of the sheath after use.

19. Apparatus for characterizing colon tissues by in vivo Raman spectroscopy, the apparatus comprising a data processor configured by software to:

obtain Raman spectrum data representing a Raman spectrum of the tissues; and

process the Raman spectrum data to yield data characterizing the tissues; wherein the data characterizing the tissues is not based on any part of the Raman spectrum having a Raman shift of less than 1000 cm^"1.

20. Apparatus according to claim 19 wherein the software comprises instructions that, when executed by the data processor causes the data processor to process the Raman spectrum data by performing a multivariate analysis on a part of the Raman spectrum having a Raman shift exceeding 1000 cm^"1.

21. Apparatus according to any one of claims 19 to 20 comprising a data store storing stored data representing principal components obtained for a training set comprising Raman spectra from normal colon tissues and Raman spectra from diseased colon tissues.

22. Apparatus according to claim 21 wherein the principal components correspond only to parts of the Raman spectra having relative wavenumbers exceeding 1000 cm^"1.

23. Apparatus according to claim 21 wherein the principal components correspond only to parts of the Raman spectra having relative wavenumbers exceeding 1500 cm^"1.

24. Apparatus according to any one of claims 21 to 23 wherein the stored data

additionally characterizes one or more linear discriminant functions for discriminating between different tissue pathologies using the stored principal components.

25. Apparatus according to claim 24 wherein the software instructions are configured to cause the data processor to process the Raman spectrum data using the stored principal components to yield a principal component score and to then perform a linear discriminant analysis using a discriminant function specified by the stored data to characterize tissues from which the Raman spectrum data was obtained.

26. Apparatus according to any one of claims 19 to 25 wherein the software comprises instructions that, when executed by the data processor cause the data processor to process the Raman spectrum data by detecting and measuring peaks in the Raman spectrum at Raman shifts of two or more of 2853 cm^"1, 2866 cm^"1 and 2930 cm^"1.

27. Apparatus according to claim 26 wherein the software instructions are configured to cause the data processor to compute one or more ratios of the peaks in the Raman spectrum and to characterize the colon tissues based on the ratios.

28. Apparatus according to any one of claims 19 to 27 comprising a probe, the probe comprising a first optical fiber arranged to carry light from an excitation light source to a tissue and a second optical fiber arrange to provide a return optical path.

29. Apparatus according to claim 28 wherein ends of the first and second optical fibers are beveled.

30. Apparatus according to claim 29 wherein ends of the first and second optical fibers are beveled at angles in the range of 20 degrees to 60 degrees..

31. Apparatus according to any one of claims 19 to 30 comprising:

an excitation light source arranged to illuminate tissues of interest with excitation light having an excitation wavelength;

a Raman spectrometer; and

a return optical path arranged to carry light scattered from the tissues of interest to the Raman spectrometer;

wherein the return optical path is transmissive to light at the excitation wavelength.

32. Apparatus according to claim 31 wherein the excitation wavelength is 785 nm.

33. Apparatus according to any one of claims 31 and 32 wherein the return optical path extends along an endoscope.

34. Apparatus according to claim 33 wherein the return optical path comprises a single optical waveguide.

35. Apparatus according to claim 33 wherein the return optical path comprises a group of optical waveguides.

36. Apparatus according to claim 33 wherein the excitation light source is coupled to a forward optical path that extends along the endoscope.

37. Apparatus according to claim 36 wherein the forward optical path comprises a single optical waveguide.

38. Apparatus according to claim 36 where the forward optical path comprises a group of optical waveguides.

39. Apparatus according to claim 36 wherein the forward optical path and the return optical path are provided in the same optical waveguide.

40. Apparatus according to claim 36 wherein the forward and return optical paths are provided by a single use fiber probe.

41. Apparatus according to claim 36 wherein the forward and return optical paths are provided by separate sets of optical fibers to carry excitation light to a tissue of interest and to carry collected scattered light back to a spectrometer.

42. Apparatus according to any one of claims 31, 32, 33 or 41 comprising a

multiplexer arranged to couple excitation and collected light into and out of an optical fiber.

43. Apparatus according to claim 36 wherein the forward and return optical paths are respectively provided by a core and outer layer of a double clad optical fiber.

44. Apparatus according to any one of claims 31 to 40 wherein the endoscope

comprises an instrument channel, the return optical path extends along the instrument channel, and the apparatus comprises a sheath extending around the return optical path, the sheath comprising a tubular member extending along the instrument channel and an optical window sealed to the tubular member at a distal end of the return optical path.

45. A method according to any one of claims 39 to 41 wherein the sheath comprises a single-use disposable sheath.

46. A probe for use in in vivo Raman spectroscopy, the probe comprising a first optical fiber arranged to carry light from an excitation light source to illuminate a tissue and a second optical fiber arrange to provide an optical path to carry scattered light to a spectroscope wherein ends of the first and/or second optical fibers are beveled.

47. A probe according to claim 46 wherein the first optical fiber has a smaller diameter than the second optical fiber.

48. A probe according to any one of claim 46 and 47 wherein a distal end of the

second optical fiber is optically transmissive to light having a wavelength of 785 nm.

49. A sheath for use in in vivo Raman spectrometry, the sheath comprising a tubular member having an inside diameter dimensioned to receive a Raman spectroscopy probe and an outside diameter dimensioned to fit within an instrument channel in an endoscope and an optical window sealed to the tubular member at a distal end thereof.

50. The sheath of claim 49 comprising an anti-reflective coating on the optical

window.

51. The sheath of claim 49 or 50 packaged in a sterile packaging wherein the sheath comprises a single-use disposable sheath. Apparatus having any new and inventive feature, combination of features, or subcombination of features as described herein.

Methods having any new and inventive steps, acts, combination of steps and/or acts or sub-combination of steps and/or acts as described herein.