EP2911586A1 - Procédés et appareils de détection de néoplasie du côlon à spectres raman de haute fréquence - Google Patents

Procédés et appareils de détection de néoplasie du côlon à spectres raman de haute fréquence

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Publication number
EP2911586A1
EP2911586A1 EP13849363.0A EP13849363A EP2911586A1 EP 2911586 A1 EP2911586 A1 EP 2911586A1 EP 13849363 A EP13849363 A EP 13849363A EP 2911586 A1 EP2911586 A1 EP 2911586A1
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European Patent Office
Prior art keywords
raman
optical
tissues
optical path
tissue
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EP13849363.0A
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German (de)
English (en)
Inventor
Michael Short
Isabella Tai
Haishan Zeng
Wenbo Wang
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British Columbia Cancer Agency BCCA
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British Columbia Cancer Agency BCCA
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0075—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by spectroscopy, i.e. measuring spectra, e.g. Raman spectroscopy, infrared absorption spectroscopy

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • LF low frequency range
  • 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.
  • 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.
  • 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.
  • tissue characterization based on Raman spectra which does not include the portion of the Raman spectrum at lower Raman shifts can provide good diagnostic sensitivity and specificity.
  • 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.
  • 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.
  • the probe may extend along an instrument channel of the endoscope.
  • the methods may be applied, for example to the identification of colonic neoplasias.
  • the present invention can be used to detect other neoplasias of the gastrointestinal tract.
  • the present invention can be used for the identification of preneoplastic lesion and the like which are at risk of progressing to neoplasia.
  • the endoscope lacks filters of the type often used to block light at a wavelength of exciting light from reaching a Raman spectrometer.
  • the apparatus comprises a data processor configured to process Raman spectrum data to indicate a characteristic of the tissues.
  • 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.
  • 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 ).
  • 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 ).
  • 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.
  • 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.
  • 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 .
  • 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.
  • the ratio of peaks at 1445 cm “1 and 1735 cm “1 is applied to distinguish normal from malignant tissue.
  • both peak ratios may be used in combination to distinguish normal from malignant tissue.
  • 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 .
  • 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).
  • multivariate analysis of the Raman spectra is used to characterize tissues (for example to distinguish normal from malignant tissues).
  • the present invention provides a device for obtaining HF
  • the optical fiber catheter has separate excitation and collection channels.
  • a multifiber probe is used to collect the HF Raman signal.
  • a dual-fiber probe with beveled fibers is used to collect the HF Raman signal.
  • 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.
  • pigtailed connections to an isolating wavelength division multiplexer are used to focus light in and out of the double clad fiber.
  • the catheter may be miniaturized for some medical applications.
  • 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 ).
  • 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.
  • 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.
  • the 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 5 shows results of the statistical analyses on the LF spectra
  • 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.
  • LDA linear discriminant analysis
  • 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.
  • 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.
  • Figure 8 shows schematically Raman spectrometry apparatus as used for in vivo collection of Raman spectrum data.
  • Figure 9 shows test spectra obtained using the apparatus of Figure 8 on palm skin.
  • Figure 10 shows test spectra taken with and without narrow band illumination light turned on.
  • Figure 11 shows in vivo Raman spectra of colon tissues in the range from 2800- 3050 cm "1 after fluorescence removal, and normalization.
  • Figure 12 shows in vivo Raman spectra of colon tissues with and without white light illumination (WLI).
  • 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.
  • 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.
  • 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.
  • 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.
  • Figures 17 and 18 illustrate the use of a multiplexer to couple excitation and collected light into and out of an optical fiber.
  • Figure 18A is a schematic illustration showing an example multiplexer.
  • Figure 19 illustrates a novel sheath for covering a Raman probe.
  • Figure 20 illustrates a Raman spectrum obtained using a Raman probe covered with a sheath as illustrated in Figure 19.
  • Embodiments of this invention use data from Raman spectroscopy to characterize tissues.
  • 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.
  • HF Raman Raman
  • 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.
  • HF Raman spectrum 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.
  • 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.
  • the optical paths may be provided by one or more optical fibers extending through or along an endoscope.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PCA principle component analysis
  • PCA involves generating a set of principle components which represent a given proportion of the variance in a set of training spectra.
  • 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.
  • 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).
  • PCs Principal components
  • the PCs generally provide a reduced number of orthogonal variables that account for most of the total variance in the original spectra.
  • 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
  • LDA Linear discriminant analysis
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • Examples 1 and 2 illustrate application of the invention to characterizing colon tissues.
  • the apparatus used in each case was similar.
  • 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.
  • CCD charge coupled device
  • 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.
  • 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
  • the 3.0 mm diameter, 2 m long, trifurcated probe contained a centre 200 ⁇ diameter fiber for excitation, surrounded by 31, 100 ⁇ diameter fibers, 28 of which were used for emission collection.
  • the remaining three 100 ⁇ 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.
  • 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.
  • This bundle consisted of 120, ultra pure, 50 ⁇ 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].
  • 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.
  • 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.
  • 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 mm 3 . 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.
  • 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.
  • 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.
  • Figures 6A and 6B 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.
  • FIGS 7A and B show receiver operator characteristics (ROCs) for the excised and biopsy tissue spectra measured in the LF and HF ranges respectively.
  • ROCs receiver operator characteristics
  • 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.
  • 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.
  • the malignant excised tissue shows increases in Raman peaks around 1340 (lipids), and 1450 (lipids), and 1650 cm “1 (amide I, and H 2 0) 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • WLI white light
  • NBI narrow band
  • 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.
  • 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.
  • 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.
  • probes that may be applied for Raman spectroscopy of colonic tissues and may additionally have other applications.
  • 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.
  • probes that are small are desirable.
  • probes for Raman spectroscopy 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.
  • FIG. 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
  • SPF short-pass filter
  • FC large core single fiber catheter
  • LI fused-silica lens
  • Backscattered radiation is filtered by a chevron-type high-pass filter (F1/F2) and focused (L2) into the detection fiber (DF).
  • F1/F2 chevron-type high-pass filter
  • L2 focused
  • DF detection fiber
  • 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.
  • 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
  • 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).
  • one of the optical fibers has a flat end and the other has a beveled end.
  • the collection optical fiber may have a flat end while the excitation optical fiber has a beveled end.
  • An example size for the smaller excitation fiber 151 is 50 ⁇ diameter.
  • An example size for the larger collection fiber 152 is 400 ⁇ - 1000 ⁇ 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.
  • a probe like probe 150 may be used to provide smaller-diameter probes which may be useful in applications other than HF Raman spectroscopy.
  • LF Raman spectroscopy filters may be provided.
  • 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;
  • 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.
  • FIGs 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.
  • 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).
  • M mirror
  • BP band pass filter
  • DM dichroic mirror
  • Double clad fibers with inner clad diameters up to 400 ⁇ are commercially available.
  • the FibercoreTM 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 ⁇ ).
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • collimating lenses 187 are provided to couple light into and out of the optical fibers.
  • a mirror 188 is wavelength selective.
  • 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).
  • excitation optical fiber 183 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • FIG 19 illustrates a sheath 190 that may be applied to a fiber optic probe for use in endoscopic applications.
  • the sheath may be disposable.
  • 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.
  • 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.
  • Sheath 190 comprises a tubular body 192 sealed to optical window 191.
  • 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.
  • PVDF polyvinylidene difluoride
  • 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).
  • 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.
  • the optical window at the distal end of sheath 190 facilitates spectra measurement and can result in improved data quality.
  • 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.
  • 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 .
  • colonoscope effect on efficiency and miss rates
  • Ramirez-Eli as, M.G., Alda, J., Gonzalez, F.J., "Noise and artefact characterization of in vivo Raman spectroscopy skin measurements", Applied Spectroscopy 2012;66:650-655. Movasaghi, Z., Rehman, S., Rehman, I.U., "Raman spectroscopy of biological tissues", Applied Spectroscopy Reviews 2007;42:493-541. Tominaga, Y., Fujiwara, A., Amo, Y., “Dynamical structure of water by Raman spectroscopy", Fluid Phase Equilibria 1998;144:323-330.
  • connection or coupling 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;
  • 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.
  • 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.
  • ASICs application-specific integrated circuits
  • LSIs large scale integrated circuits
  • VLSIs very large scale integrated circuits
  • 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”)).
  • PALs programmable array logic
  • PLAs programmable logic arrays
  • FPGAs field programmable gate arrays
  • 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.
  • DSPs digital signal processors
  • embedded processors embedded processors
  • graphics processors graphics processors
  • math co-processors general purpose computers
  • server computers cloud computers
  • mainframe computers mainframe computers
  • computer workstations and the like.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention may be implemented in software.
  • "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.
  • a component e.g. a software module, processor, assembly, device, circuit, etc.
  • reference to that component 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.

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Abstract

L'invention concerne l'application d'une spectroscopie de Raman avec des décalages Raman supérieurs, par exemple des décalages Raman dépassant 1 000 cm-1 pour caractériser des tissus du côlon. Cette caractérisation peut comprendre des comparaisons de pics dans le spectre de Raman et/ou dans une analyse à variables multiples. Il est possible de proposer des sondes Raman compactes, économiques et jetables. Selon un mode de réalisation, une sonde Raman comprend une seule paire de fibres optiques présentant des extrémités biseautées.
EP13849363.0A 2012-10-26 2013-10-25 Procédés et appareils de détection de néoplasie du côlon à spectres raman de haute fréquence Withdrawn EP2911586A1 (fr)

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GB201604331D0 (en) * 2016-03-14 2016-04-27 Univ Edinburgh Fibre-optic probe
CA3086931A1 (fr) 2018-01-25 2019-08-01 Provincial Health Services Authority Dispositif endoscopique de spectroscopie raman
WO2019157078A1 (fr) * 2018-02-06 2019-08-15 The Regents Of The University Of Michigan Systèmes et procédés d'analyse et d'interprétation distante d'images histologiques optiques
US20240225451A1 (en) * 2020-10-08 2024-07-11 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Raman spectroscopy system and methods of using the same
CN113324968A (zh) * 2021-04-01 2021-08-31 吉林大学 一种基于拉曼光谱的结直肠癌错配修复蛋白表达的检测方法

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