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EP0466828A1 - Diagnostic spectral des tissus malades - Google Patents

Diagnostic spectral des tissus malades

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Publication number
EP0466828A1
EP0466828A1 EP19900906721 EP90906721A EP0466828A1 EP 0466828 A1 EP0466828 A1 EP 0466828A1 EP 19900906721 EP19900906721 EP 19900906721 EP 90906721 A EP90906721 A EP 90906721A EP 0466828 A1 EP0466828 A1 EP 0466828A1
Authority
EP
European Patent Office
Prior art keywords
tissue
fluorescence
emission
spectrum
normal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP19900906721
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German (de)
English (en)
Inventor
Rebecca Richards-Kortum
Lucene Tong
Michael S. Feld
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Massachusetts Institute of Technology
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Massachusetts Institute of Technology
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Publication date
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Publication of EP0466828A1 publication Critical patent/EP0466828A1/fr
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/42—Detecting, measuring or recording for evaluating the gastrointestinal, the endocrine or the exocrine systems
    • 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
    • A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
    • 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
    • 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
    • A61B5/0082—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence adapted for particular medical purposes
    • A61B5/0084—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence adapted for particular medical purposes for introduction into the body, e.g. by catheters

Definitions

  • the present invention relates to the field of diagnosis of bodily tissue, and more particularly, to the differentiation of normal from abnormal tissue using laser- induced fluorescence to provide diagnostic
  • tissue including
  • gastrointestinal and colonic abnormalities for example.
  • An appraisal of the pathologic condition of many lesions or abnormalities can be made by endoscopic observation alone, but there remains a margin for error that can be substantial for certain types of lesions.
  • Microscopic assessment of biopsy specimens is often considered necessary for many lesions discovered during endoscopy.
  • certain abnormalities of a microscopic nature such as dysplasia in chronic ulcerative colitis or Barrett's esophagus, for example, are usually
  • a polyp of the colon can be defined as any lesion that protrudes above the surface of the
  • Adenomatous polyps are true neoplasms and sometimes harbor areas of carcinoma. Adenomatous polyps can be subdivided into three classes: tubular ( ⁇ 75%),
  • tubulovillous ( ⁇ 5-15%), and villous ( ⁇ 10-15%).
  • type of polyp There is a general correlation between the type of polyp, and its size and potential for harboring cancer. Hyperplastic polyps are the smallest, tubular adenomas are next in size, and villous adenomas are largest. Hyperplastic polyps are almost always benign. Overall, the incidence of carcinomas in tubular adenomas is about 3-5%.
  • hyperplastic polyps are composed of well-formed glands and crypts lined by non-neoplastic epithelial cells, most of which are well differentiated.
  • Tubular adenomas have slender stalks and rounded heads. They are composed of a central core of fibro-vascular tissue and are covered by an epithelium of elongated tubules and glands in which cells are not well
  • Villous adenomas are composed of fingerlike pappillae covered by polypoid epithelium. Each papilla is composed of a fibro-vascular core covered by
  • tubular adenomas having between 20-50% villous growth are referred to as tubulo-villous (See. Robbins, supra., at pp. 863-869).
  • Laser catheter systems have been developed for the purpose of inserting a light transmitting device into the human body to provide endoscopic examination of the tissue located in front of the catheter.
  • fluorescence can be transmitted along the catheter and analyzed at the proximal end of the catheter to produce an emission spectrum for the tissue being illuminated.
  • the present invention relates to the use of
  • gastrointestinal tissue removed from patients with familial multiple polyposis have been examined using methods of fluorescence spectroscopy.
  • Patients having colonic adenomas, hyperplastic polyps, or normal mucosa were examined during endoscopy using the present methods of fluorescence spectroscopy. Specific ranges of
  • excitation and emission wavelengths have been found to accurately diagnose the presence of abnormal tissue both in vivo and in vitro using " autofluorescence", that is, the fluorescence of tissue without the use of
  • tissue spectroscopy can be utilized to differentiate normal colonic mucosa and hyperplastic polyps from adenomatous polyps without the use of fluorescing agents such as dyes or stains.
  • abnormal gastrointestinal tissue as well as the tissue examined in vivo, have been irradiated with laser
  • the emission wavelengths longer than 400nm are used to differentiate normal and abnormal tissue. More specifically, differences between the emission spectra and reference spectra obtained from measurements of normal tissue are analyzed to determine functions which discriminate between normal and abnormal tissue. The standard deviation of emission spectra were calculated and used to assist in the determination of diagnos tically significant wavelengths for gastrointestinal tissue.
  • the methods of spectral diagnosis described herein can be used in vivo with a laser catheter system that can diagnose and treat the specific tissue of interest.
  • emission spectra The distinctive features identified in emission spectra as being diagnostically significant correspond to these chemical constituents and are used to measure the concentration of these constituents in the tissue.
  • the spectra can be deconvolved to isolate the different components of the tissue and their relative
  • Figure 1 illustrates a system for collecting spectra that are used to diagnose tissue condition.
  • Figure 2 illustrates excitation spectra of (a) normal and (b) polyp tissues at an emission wavelength of 500nm.
  • Figure 2c illustrates emission spectra of normal and
  • Figure 3 shows values of intensity ratios at (a) 335nm/ 365nm, (b) 335nm/440nm, (c) 440nm/390nm and (d) 415nm/ 425nm.
  • the solid lines indicate a criterion for determining tissue type from the value of the ratio. In (a) and (b), 94% accuracy is achieved with this criterion; in (c) and (d), the accuracy is 100%.
  • Figure 4 illustrates emission spectra of (a) normal and (b) polyp tissues. Excitation wavelength was 350nm.
  • Figure 5 shows values of intensity ratios at (a) 387/365, (b) 387/427, (c) 415/425, (d) 440/457,
  • Figure 6 Typical emission spectra of (a) normal and (b) polyp tissues. Excitation wavelength was 370nm.
  • the solid lines indicate a criterion for determining tissue type from the value o f the r at i o .
  • e ach c as e 100 % ac cur acy i s achi eve d .
  • Figures 8a and 8b illustrate average normalized fluorescence spectra of no rmal and adenomatous tissues at 330nm excitation.
  • Figures 9a and 9b illustrate the spectra of Figures 8a and 8b, respectively, with positive and negative standard deviation spectra superimposed thereon.
  • Figure 10 illustrates an average difference spectrum at 330nm excitation .
  • Figure 11 illustrates the discriminant function at the 330nm excitation.
  • Figure 12 graphically illustrates a combination diagnostic/algorithm based upon emission wavelengths at 438 and 384nm.
  • Figures 13a and 13b illustrate average fluorescence spectra at 350nm excitation.
  • Figures 14a and 14b illustrate the spectra of
  • Figure 15 illustrates an average difference spectrum at the 350nm excitation.
  • Figure 16 illustrates the discriminant function at the 350nm excitation.
  • Figure 17 graphically illustrates a combination diagnostic/algorithm based upon emission wavelengths at 436 and 556nm.
  • Figure 18 graphically illustrates a combination diagnostic/algorithm based upon emission wavelengths at 470 and 556nm.
  • Figures 19a and 19b illustrate fluorescence spectra for normal and adenomatous tissue at 370nm excitation.
  • Figures 20a and 20b show the spectra of Figures 19a and 19b, respectively, with positive and negative
  • Figure 21 illustrates an average difference spectrum at the 370nm excitation.
  • Figure 22 illustrates the discriminant function at the 370nm excitation.
  • Figure 23 graphically illustrates a combination diagnostic/algorithm bassed upon emission wavelengths at 442 and 558nm.
  • Figures 24a and 24b illustrate average normalized fluorescence spectra of normal and adenomatous tissues at 476nm excitation.
  • Figures 25a and 25b show the spectra of Figures 24a and 24b, respectively, with positive and negative
  • Figure 26 illustrates an average difference spectrum at the 476nm excitation.
  • Figure 27 illustrates the discriminant function at the 476nm excitation.
  • Figure 28 illustrates the use of a laser catheter device for the in vivo diagnosis and treatment of tissue in accordance with the invention.
  • Figures 29a and b are average fluorescence spectra of normal colon and adenomatous colon tissue at an excitation wavelength of 369.9nm.
  • Figures 30a and b are graphical illustrations of the ratio and difference between the averaged and normal tissue spectra of Figure 29, respectively.
  • Figures 31a, b and c illustrate diagnostic methods used in distinguishing normal or adenoma based upon excitation at 369.9nm.
  • Figures 32a and b are averaged, normalized spectra of normal and adenomatous colon, respectively obtained at 319.9nm excitation.
  • Figures 33a and b are averaged, normalized spectra of normal and adenomatous colon, respectively, obtained at 435.7nm excitation.
  • Figure 37 illustrates an average spectrum for adenoma superimposed on an average spectrum for normal colon.
  • Figure 38 illustrates the calculated ratio of the average adenoma spectrum to the normal spectrum.
  • hyperplastic polyps relative to those for normal and adenoma.
  • Figure 40 is a scatter plot of the average
  • Figure 41 illustrates an excitation emission matrix of averaged normal human colon.
  • Figure 42 illustrates an excitation emission matrix of averaged adenomatous colon.
  • Figure 43 illustrates average total reflectance spectra of normal colon and colonic adenoma.
  • Figures 44a-f shows emission spectra of selected chromophore.
  • Figures 45a-e show average spectra for selected morpholophores.
  • Low power laser illumination can induce endogenous tissue fluorescence (autofluorescence) with spectral characteristics that depend upon physicochemical
  • Fluorescence emission and attenuation can be measured and provide the basis of a diagnostic system adapted to endoscopy.
  • Autofluorescence can be used to differentiate adenoma from normal mucosa for in vitro measurements for tubular adenomas found in patients with familial adenomatous polyposis.
  • the following demonstrates that the excitation wavelength 370nm was optimal for in vitro discrimination of adenomas from normal tissue, however the excitation wavelengths 330nm and 430nm have also be used to effectively distinguish normal from abnormal tissue. Thus a range of wavelengths has been identified that provides an effective diagnostic procedure.
  • the colonic adenoma provides a procedure that reveals definite endoscopic and pathologic differences between normal and abnormal (adenomatous) tissue.
  • Laser induced fluorescence (LIF) spectroscopy has clinical significance in relation to the problem of the
  • adenoma-carcinoma sequence that is known to underlie the development of colon cancer. It is well established that endoscopic differentiation of adenomatous from nonadenomatous polyps is not possible in the case of small lesions. Management of these polyps may be
  • a biopsy is necessary for accurate diagnosis, but should a lesion prove to be an adenoma it may be difficult to locate it a second time for definitive treatment.
  • the immediate treatment of diminutive polyps upon discovery means that patients with nonadenomatous lesions incur an unnecessary, albeit small, risk of a complication as well as the additional cost of treatment.
  • LIF spectroscopy improves the management of small colon polyps, the method has additional applications with respect to the diagnosis of other mucosal disorders that are more difficult to recognize endoscopically.
  • the benign adenoma is similar to dvsplastic epithelium found in other conditions so that information derived from LIF spectroscopy of
  • adenomatous tissue can be applied to the recognition of dysplastic mucosa in disorders such as Barrett's
  • FIG. 1 A spectrofluorometry system employed to collect mucosal fluorescence spectra in vivo is illustrated in Figure 1.
  • An optical fiber fluorescence probe 10 was constructed that could be passed through the accessory channel of a standard colonoscope. The probe 10 delivers monochromatic light at 370nm produced by a nitrogen laser 40-pumped dye laser 50 through a centrally placed
  • excitation optical fiber 30 This light forms a 1 mm diameter excitation spot at the distal tip 25 of a 1 mm diameter transparent quartz outer shield 15.
  • This system can be used to deliver excitation light spanning the spectral region 360nm - 1.0um.
  • the laser furnishes an average power of 270uW at the distal tip, delivered in 3 nanosecond pulses at 20Hz.
  • Nine smaller peripherally placed optical fibers 20 surround the central excitation fiber. These collect the emitted tissue fluorescence only from the surface area directly illuminated by the excitation light.
  • the system has a well-defined excitation and collection geometry so that light that is scattered to the tissue surrounding the illuminated area is not collected. By substantially reducing the amount of light collected from the
  • the proximal ends of the nine collection fibers 20 were imaged at the entrance slit of an imaging
  • a 399nm long pass, low fluorescence filter 110 was used to block scattered excitation light from the detector.
  • Coupling lens 90, 100 directed the filtered light to the spectrograph 70 input.
  • a 1.0 microsecond collection gate pulser 150 synchronized by clock 130 to the laser pulse effectively eliminated the effects of the colonoscope's white illumination light during collection of the weaker tissue fluorescence.
  • differentiate gastrointestinal tissues as normal or adenomatous is based upon excitation in a range of wavelengths between 200 and 450 nanometers with the 370nm excitation providing the highest correlation.
  • the procedure followed for the first group of s amp le s included a total reflectance spectrum that was measured using a integrating sphere absorption
  • spectrophotometer A spectofluorimeter was used to measure fluorescence excitation and emission spectra of each sample. Excitation spectra were measured at
  • emission wavelengths varying from 350 to 600nm in 50nm steps. Peaks in the excitation spectra were noted, and emission spectra were measured at corresponding
  • the first group was analyzed using ratios of emission intensities at wavelengths that produced a high degree of correlation between the actual condition of the tissue and the spectrally determined condition.
  • the second group of in vitro samples used the same procedure for measuring the emission spectra, but a different method of analyzing the spectra.
  • the gratings in an emission monochromator were changed from 500nm blaze to 250nm blaze for those samples in the second group. This allowed for greater efficiency at UV wavelengths, and thus, for a greater signal-to- noise ratio in this region of the spectrum.
  • Fluorescence emission spectra were recorded at 290, 330, 350, 370 and 476nm excitation.
  • Figure 2(a) is a plot of fluorescence emission intensity versus excitation wavelength in nanometers for normal tissue, at 500nm emission.
  • a broad excitation peak is present from 300-400nm, with several sub-maxima at 310, 330, 350 and 370nm.
  • a second excitation peak is found at 460nm.
  • About half of the polyp samples showed a similar excitation spectrum, however, the other half showed an excitation spectrum typical of that in Figure 2(b). This shows a narrower excitation peak at 380nm with a shoulder at 400nm. A smaller excitation peak at 460nm is also present.
  • Emission spectra were collected at all peaks of the various excitation spectra; however, emission spectra obtained with 290, 350, and 370nm excitation are particularly suited for differentiating normal and polyp samples. Results obtained with these wavelengths will be described here.
  • Figure 2(c) shows typical emission spectra at 290nm excitation for normal tissues. About half of the polyp tissues showed similar emission spectra. The other half (marked with an asterisk in Table 1) had spectra typical of those shown in Figure 2(d). This unique emission profile always correlated with the unique excitation profile shown in Figure 2(b). Several differences between Figures 2(c) and (d) are immediately obvious.
  • the polyp spectrum shows a peak near 340nm and a
  • Figures 3(a) and 3(b) show that the ratios 335/365 and 335/440 can be used to diagnose tissues as normal or polyp correctly in 94% of the cases studied. 100% accuracy is achieved with the ratios 440/390, 415/425 and 440/457 as indicated in Figures 3(c-e).
  • the ratio 415/425 indicates little variation in its value for samples of a given tissue type and there is a large separation between the average values of normal andpolyps tissues.
  • Figure 4(a) shows fluorescence emission spectrum characteristic of normal tissue.
  • Figure 4(b) shows a spectrum characteristic of polyp tissues. Other polyp spectra were similar to normal tissue spectra and polyp tissues respectively with 350nm excitation. The normal spectrum exhibits a peak at 387nm, and a peak at 470nm. Subsequent maxima in this peak are created by
  • the polyp spectrum shows a three peak structure with maxima at 415, 440 and 460nm.
  • ratios of fluorescence intensities at the following wavelengths 387/365, 387/427, 415/425, 440/457, 495/440 and 475/440. Although many combinations of wavelengths were tried, these were also found to indicate separation of samples according to sample type. The value of these ratios versus tissue type are shown in Figure 5(a-f). The following table lists the average values and standard deviations for each ratio vs. tissue type.
  • Figures 5(a-c) show that the first three empirically defined ratios can be used to diagnose tissue type correctly in 91% of all cases studied.
  • Figures 5(d-f) illustrate that 100% accuracy can be achieved using the ratios 440/457, 495/440 and 475/440. With the last ratio there is a large separation between the value of the ratio for all normal and polyp samples.
  • Figure 6(a) shows a typical emission spectrum of normal tissue with 370nm excitation. Certain polyp samples showed emission spectra similar to this.
  • Figure 6(b) illustrates the emission spectrum characteristic for polyp samples. Normal samples showed a small peak at 420 and a large peak at 480nm with subsidiary maxima produced by hemoglobin reabsorption. Polyp samples again showed the three peaked structure at 415, 440 and 460nm.
  • Figures 7(a-c) illustrate that each of these three ratios was able to achieve separation of tissue by type accurately in 100% of cases studied.
  • the ratio 440/390 provided the largest degree of separation between normal and polyp tissues.
  • F Ad ( ⁇ ) were calculated from this normalized data.
  • standard deviation spectra were calculated for the normal ⁇ N ( ⁇ ) and adenomatous ⁇ Ad ( ⁇ ) tissue spectra.
  • Figure 10 shows the average difference spectrum. Two major differences are apparent. On average, normal tissue displays relatively more intense fluorescence at 380nm, while adenomatous tissue exhibit relatively more intense fluorescence at 440nm. In addition, smaller differences are present near 550nm, with adenomatous tissues exhibiting relatively more fluorescence in this region of the spectrum.
  • Figure 11 illustrates the discriminant function, D( ⁇ ), at this excitation wavelength. This Figure shows the same characteristic differences as the average difference spectrum; however, it shows that the most statistically consistent difference is that at 440nm.
  • fluorescence spectrum is that at 600nm for both types of tissue.
  • Figure 15 shows the average difference spectrum at 350nm excitation.
  • the major difference is the greater relative fluorescence intensity at 440nm for adenomatous tissues. Again, a smaller difference is present at 550nm, with adenomatous tissues having higher relative fluorescence intensity at this wavelength. Normal tissues, on the other hand, exhibit relatively more fluorescence at 470nm.
  • D( ⁇ ) is shown in Figure 16, and is discriminant spectrum at the 350nm excitation. The peaks of D( ⁇ ) were used in choosing wavelengths to define empirical
  • Figures 19(a) and (b) show average 370nm excited fluorescence spectra for no rmal and adenomatous tissues. Both tissue types exhibit emission peaks at 470nm, and hemoglobin absorption valleys at 420, 540 and 580nm.
  • Figure 21 the average difference spectrum, indicates greater relative fluorescence intensity at 440nm in adenomatous tissue. Again, small differences exist at 485 and 560nm.
  • the discriminant spectrum ( Figure 22), shows that the 440nm peak is statistically most
  • Figure 23 shows one possible combination utilizing Information at 442 and 558nm.
  • Figures 24(a) and (b) show average normalized fluorescence spectra of normal and adenomatous tissues with 476nm excitation. These spectra are strikingly similar to typical arterial fluorescence spectra at this excitation wavelength. A fluorescence peak is present at 520nm, with subsequent maxima at 550 and 600nm produced by hemoglobin reabsorption. Average spectra ⁇ standard deviation spectra, shown in Figures 25(a) and (b)
  • Figure 28 illustrates the use of a laser catheter 10 used for the diagnosis and or removal of selected tissue 334.
  • One or more fibers 20 may be positioned within a catheter tube 16 wherein the movement of the distal end of the catheter can be controlled by guidewires 338.
  • the fibers 20 are held within tube 16 by plug material 11 such that a light spot pattern 27b is formed on the distal surface of an optical shield 12 positioned on the end of the catheter when a laser beam is connected to the proximal ends of the fibers 20 to project light onto the shield along paths 29a-c.
  • Low energy diagnostic laser radiation can be used to perform the diagnostic procedure described herein or high power laser radiation can be used to remove "nibbles" of material 335 a and b.
  • Figure 29A shows the average fluorescence emission spectrum of another set of samples of normal colon obtained with the spectral catheter system
  • excitation wavelength was 369.9nm.
  • Two intense emission peaks are present near 460 and 480nm. Valleys can be observed near 420, 540 and 580nm.
  • the small peak at 520nm is an artifact due to a large decrease in the spectral response of the detection system at this
  • Figure 29B also shows the average fluorescence emission spectrum of samples of adenomatous colon. Here, absolute fluorescence intensities have been preserved. This spectrum shows fluorescence peaks at 460 and 500nm with valleys near 420, 540 and 580nm. This spectrum is similar to that obtained with the fluorimeter except the valleys are less pronounced. Although, there is
  • the shoulder present at 450nm in the spectra some of the adenomas obtained with the fluorimeter is not prominent in the adenoma spectra obtained with the catheter.
  • This ratio spectrum is characterized by four regions: a downward sloping region from 400-480nm, a flat region from 400-480nm, an upward sloping region from 480-650nm where the ratio is ⁇ 1 and an upward sloping region from 650-700nm where the ratio is >1.
  • the downward sloping region from 400-420nm reflects the blue region of the spectrum in which the relative fluorescence intensity of the adenomas is relatively greater than that of normal tissues.
  • the flat region from 430-480nm represents peak at 460nm, where the fluorescence intensity of normal tissue is greater than that of adenomatous tissue. The relatively flat ratio in this region indicates that the fluorescence lineshape of this peak is the same in normal and adenomatous tissue.
  • the upward sloping region from 480-650nm represents a region where the absolute difference in the fluorescence intensity of normal and adenomatous tissue is decreasing, and is due to the red shift in the position of the second most intense maximum in the spectra of adenomatous tissues. Above 680nm, the absolute fluorescence
  • Figure 30B shows the difference of the average adenoma and normal tissue spectra. This spectrum
  • Empirical methods for the presence of adenoma were defined with the fluorescence intensities at emission wavelength listed in Table 5 using the method outlined above.
  • Figures 31 a, b, c shows three diagnostic methods which are defined with this procedure. The emission intensity at 480nm proved to be an effective diagnostic method for adenoma.
  • Equally effective binary diagnostic methods could also be defined using the emission
  • the patients in which in vivo measurements were taken were prepared for colonoscopy by ingestion of an oral lavage solution (colyte).
  • the Colyte was tested in vitro and does not interfere with LIF spectra collection.
  • Colonoscopy was performed using a standard procedure and colonoscope.
  • the probe 10 was passed through the accessory channel of the colonoscope and its outer shield 15 was placed in direct contact with the surface of mucosal polyps and/or control nonpolypoid normal-appearing mucosa. This contact displaced residual colonic contents and/or mucous. Direct contact was also necessary to fix the distance between the mucosa and the distal end 35 of the probe's optical fibers, so that reliable calibrated fluorescence intensity information could be obtained.
  • Fluorescence emission spectra were collected from 350 - 700nm with a resolution of 0.6nm. After three spectra were obtained, the probe was removed and then repositioned two to four additional times with three spectra obtained at each placement. This process yielded nine to 15 individual spectra per site. No appreciable fluorescence photo-bleaching was observed. A biopsy for histologic examination was then performed of the mucosal site analyzed by the probe. Polyps were treated by standard electrosurgical snare polypectomy or
  • Spectra were corrected for non-uniform spectral response of the detection system by using a calibrated lamp.
  • fluorescence paper was measured prior to study in each patient and was used to calibrate the fluorescence intensity of the tissue spectra.
  • the spectral baseline was corrected to zero by substracting a constant
  • LIF spectra were obtained in vivo and analyzed from adenomas, hyperplastic polyps, and his tologically normal areas.
  • the adenomas ranged in size from 2 - 11mm
  • the adenomas were classified as tubular, villous, or tubulovillous. The laser caused no tissue damage that could be detected at the light
  • Figures 34, 35 and 36 illustrates typical average LIF spectra ⁇ standard deviation for spectra obtained from a representative normal colon, adenoma, and
  • fluorescence intensity information can be measured most easily at 460 nm, as this corresponds to the peak fluorescence intensity for both adenoma and normal mucosa and there is less noise in the measurement of fluorescence in regions of high fluorescence
  • Figure 39 demonstrates the average fluorescence spectrum of all hyperplastic polyps superimposed on normal and adenoma spectra.
  • the hyperplastic polyp fluorescence intensity at 460nm lies intermediate between those of adenoma and normal mucosa and closely
  • Table 6 lists the mean fluorescence intensities at 460 nm and at 680nm for all samples in each histologic.
  • FIG. 40 A single 2-dimensional scatter plot of the average fluorescence intensity at 460nm versus 680nm for each specimen is shown in Figure 40. The scatter plot was divided into two regions corresponding to adenoma and nonadenomatous tissues (normal colon and
  • hyperplastic polyp using a straight line decision surface chosen to minimize the number of misclassified samples.
  • This decision surface correctly classified 97% of the 67 samples as adenoma or nonadenoma. No adenomas were misclassified.
  • This procedure retrospectively diagnosed adenoma with a sensitivity of 100%, and a specificity of 97%. The predictive value of a positive test for adenoma was 94%.
  • adenomas cannot be reliably distinguished from nonadenomatous mucosa using macroscopic evaluation through the endoscope, especially when dealing with small lesions.
  • the accuracy rate for diagnosis based on gross observation alone generally approximates 75%.
  • LIF spectra can be used in the recognition and differential diagnosis of mucosal abnormalities during endoscopy. Both in vivo and in vitro spectral results demonstrated that the diagnosis of adenoma can be made using LIF spectroscopy with a high degree of accuracy.
  • the in vivo LIF spectroscopy lineshapes were in general substantially similar to the in vitro observations except for a 440nm peak that was observed in some of the
  • a fluorescence spectroscopy diagnostic system capable of detecting adenoma (dysplas tic/neoplastic) transformation is of great practical importance.
  • the fiberoptic device described herein can identify dysplasia
  • spectroscopy is a useful technique for diagnosing the presence of colonic adenoma.
  • diagnostic procedures presented thus far have been achieved with an empirical analysis of tissue fluorescence spectra.
  • the following demonstrates that the fluorescence spectra of tissue are directly related to the his tochemical
  • tissue fluorophores at 370nm excitation are identified at the morphologic level, using fluorescence and light microscopy of stained and unstained sections of tissue. The optical properties of these morphologic constituents of tissue are measured with 370nm excited fluorescence microspec tros copy. This provides the basis for applying models of tissue
  • tissue fluorescence spectra contain contributions from both intrinsic fluorescence and attenuation.
  • tissue fluorescence spectra contain contributions from both intrinsic fluorescence and attenuation.
  • Total reflectance spectra provide a measure of the attentuation contributions to tissue EEMs. In a total reflectance spectrum, valleys indicate peaks in
  • Attenuation peaks can be related to the tissue EEMs in the following way. Attenuation peaks act to produce valleys in the fluorescence spectra of optically thick tissue samples. As attenuation effects are important both for exciting and emitted radiation, valleys will be produced in both excitation and emission spectra. Thus, at the location of these attenuation peaks, one expects to see valleys in the tissue EEMs parallel to both the excitation and emission axes.
  • Attenuation effects which include both scattering and absorption, were recorded independently by measuring total reflectance spectra of eight (four normal, four adenoma) full thickness colonic specimens from four patients using a standard absorption spectrophotometer equipped with an integrating sphere. Three of the normal samples were matched controls from patients with familial adenomatous polyposis. Percent total reflectance was recorded from 250 - 700nm with a resolution of 5nm FWHM .
  • Figure 43 shows average total reflectance spectra of four samples of normal colon and four colonic adenomas.
  • reflectance valleys are located at 270, 355, 420, 540, 575, and 635nm. These valleys are superimposed on a gently upward sloping background, which is slightly steeper for normal tissue.
  • the valleys in the total reflectance spectra can be correlated with the valleys in the average EEMs shown in Figs. 42 and 42.
  • Figure 41 illustrates an average EEM of four normal human colon samples. Excitation wavelength is plotted on the ordinate, emission wavelength on the abscissa.
  • Contour lines connect points of equal fluorescence intensities. Three sets of linearly spaced contours are shown: twenty from .5 to 10 unites; eighteen from 15 to 100 units; and two from 150 to 200 units. Although fluorescence intensities are given in arbitrary units, the same scale of units is maintained throughout the paper. Figure 42 illustrates an average EEM of 11
  • Contour lines connect points of equal fluorescence intensities. Three sets of linearly spaced contours are shown: twenty from .5 to 10 units; eighteen from 15 to 100 units; and two from 150 to 200 units. Although fluorescence intensities are given in arbitrary units, the same scale of units is maintained throughout the paper.
  • the strongest attenuation peak at 420nm gives rise to valleys in the tissue EEMs at 420nm along both the excitation and emission axes. Although not as prominent, valleys are also present along the emission axis at 540 and 575nm. A small valley along the excitation axis near 355nm can also be appreciated.
  • oxy-hemoglobin The presence of oxy-hemoglob in could be attributed to the vascularity of the bowel wall. The gently upward sloping background in the tissue
  • tissue fluorophores have been identified by comparing excitation/emission peaks in tissue EEMs to those in EEMs of individual tissue constituents as well as peaks cited in the literature. EEMs of these
  • Table 7 contains a compendium of excitation/emission maxima from the EEMs of each of the biochemical compounds considered which might contribute to normal and
  • Attenuation acts to alter both the observed location of the excitation/emission maxima and observed lineshape of individual tissue fluorophores in the multi - component tissue EEMs when the excitation and emission of individual chromophores closely overlap.
  • Fluorophores for the tissue peak at (345, 465nm) include NADH and NADPH. These molecules function as co-enzymes in oxidation-reduction reactions, and both have an excitation/emission maximum at (350, 460nm) in aqueous solution. It should be noted that the (345, 465nm) peak is bounded by attenuation valleys at 420nm along the exciation and emission axes. Thus, the precise location of its excitation/emission maximum can be significantly shifted.
  • peaks which appear in tissue EEMs are near peaks associated with chromophores related to vitamin B 6 .
  • the peak unique to normal tissue at (315, 430nm) is near that of 4-pyridoxic acid at (300, 430nm).
  • the shoulder in adenomatous tissue at (370, 420nm) is near the
  • excitation/emission maximum is at (410, 520nm); the shift in the tissue excitation maximum could be attributed to the Soret band attenuation of oxy-hemoglobin.
  • the effects of oxy-hemoglobin attenuation are reduced in the ratio map, and the peak assigned to pyridoxal
  • 5'-phosphate is observed at (400, 480nm).
  • pyridoxal 5'-phosphate exhibits a second peak at (305, 385nm) which is near the peak found at (330, 385nm) in the normal tissue EEM.
  • Collagen I and collagen III fluorescence show peaks at (340, 395nm) and (330, 390nm), respectively.
  • Microspectrofluor ime try studies described later, can separate contributions of extra-and intra-cellular fluorescence, and provide the definitive answer.
  • Hematoporphyrin derivative for example, which is a mixture of several biologically relevant porphyrins, exhibits fluorescence excitation emission maxima near (400, 610nm) and (400, 675nm).
  • the peak assigned to NADH or NADPH is twice as intense in normal tissue as in adenomatous tissue EEMs. It is known that the absoluate concentrations of NAD+ and NADH decreased 2-3 fold following murine sarcoma virus transformation in rat kidney fibroblasts.
  • the peaks assigned to pyridoxal 5'-phosphate are also approximately twice as intense in the normal tissue as in adenomatous tissue EEMs. Decreased levels of serum pyridoxal
  • chromophores identified from the EEMs include the structural proteins, elastin, and type I and III collagen, NADH, NADPH, pyridoxal 5'-phosphate and
  • Figure 39 shows emission spectra of each of these chromophores (except pyridoxic acid lactone) excited at 370nm. These spectra represent the 370nm excited spectrum from the corresponding chromophore fluorescence EEM.
  • a combination of fluorescence microscopy and light microscopy were used to morphologically identify the fluorescent structures contributing to the 370nm excited fluorescence emission spectra of colon.
  • Autofluorescent structures within normal and adenomatous colon were identified from unstained frozen sections of tissue using a fluorescence microscope. Serial sections were then stained with various his tochemical stains and viewed under the light microscope in order to identify these fluorescent structures at the morphologic level.
  • Tissue autofluorescence was viewed using an inverted fluorescence microscope adapted for laser illumination.
  • Multi-line excitation light from 351 -364nm was provided from a CW argon ion laser via a quartz optical fiber.
  • the distal tip of the fiber was positioned at an angle of approximatley 30° to the stage, achieving approximately trans-illumination.
  • a mm diameter field of view was illuminated with this system, the excitation intensity was mW/mm 2 .
  • a barrier filters (long-pass filter) with a
  • Collagen fibers in the connective tissue of the lamina basement membrane contributed a blue fibrillar autofluorescence, similar in color, but of weaker intensity relative to that of the submucosa.
  • epithelial cells differed remarkable from that of
  • non- dysplas tic cells A relatively homogeneous blue green fluoresence was observed within the cytoplasm of these cells. The intensity of this fluorescence appeared to correlate with the grade of dysplasia as determined from serial sections stained with H&E.
  • Fluorescence spectra were also recorded from four adenomatous polyps obtained from resection specimens of four patients with familial adenomatous polyposis. For each sample, several fluorescence emission spectra were recorded for each of the morpholophores described in the previous section.
  • the 40X objective was used in all cases except for recording fluorescence spectra from absorptive cells, In which case the 100X objective was used.
  • adenomatous mucosa was unique, exhibiting a broader fluorescence peak, with a maxima near 460nm.
  • Mucosal eosinophils in normal and adenomatous colon showed similar fluorescence lineshapes, consisting of a broad peak, centered at 480nm.
  • Absorptive cells in adenomatous mucosa displayed a broad fluorescence peak, with a max imum near 520nm.
  • the fluorescence emission spectra of absorptive cells in normal mucosa was exceedingly weak. The recorded signal was indistinguishable from that of lamina limba connective tissue, indicating possibly that the signal was too weak to be accurately recorded with this system.
  • morpholophores have an emission which peaks around 430nm, the Soret absorption band of oxyhemoglob in acts to shift the observed location of the maximum.
  • the subsidiary maxima at 480nm is this average spectrum is likely due to the fluorescence of eosinophils within the mucosa.
  • the peak at 460nm can also be attributed to collagen fibers within the submucosa and lamina basement. Relative to normal colon, the intensity of this peak is decreased in adenomatous tissues. This is likely due to the increase in mucosal thickness of adenomatous polyps. The brightly
  • fluorescent collagen fibers in the submucosa are further from the lumen, and thus, contribute less to the overall spectrum. Again, the Soret band of oxy-hemoglob in acts to shift the observed potion of this maximum. Eosinophil fluorescence also contributes to the fluorescence
  • the 480nm peak in adenoma spectra is less prominent than that in normal colon spectra for potentially two reasons.
  • the fluorescence emission of collagen in the adenomatous lamina basement membrane is quite broad, and substantially overlaps the eosinophil emission.
  • the distribution of eosinophils Is more uniform in adenomas, thus their overall contribution to the fluorescence spectrum is decreased.
  • the peak at 520nm observed in the adenoma spectrum can be attributed to the fluorescence of dysplastic absorptive cells.
  • microspectroscopy can be used to identify and characterize tissue morpholophores, this technique provides limited insight about the chemical basis of tissue fluorescence.
  • EEMs we were able to provide potential chemical identification of tissue fluorophores. A comparison of these identifications should provide valuable insight into definitively
  • Identifications can be tested. Chemically identified fluorophores, this identification can be supported.
  • fluorophores identified chem i c al ly matches the emission lineshape observed from eosinophils at this excitation wavelength. Eosinophil autofluorescence is associated with eosinophil granules. Finally, the emission maxima of dysplastic absorptive cells matches that of pyridoxal 5' phosphate. However, there is more fluorescence observed in the blue region of this morpholophore than is observed in the chromophore. The weak level of

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Abstract

L'invention ici présentée concerne une méthode de diagnostic de l'état du tissu gastrointestinal, et plus particulièrement, d'une méthode utilisant la fluorescence induite du laser au niveau des tissus pour dépister la présence de tissu malade dans le corps humain.
EP19900906721 1989-04-14 1990-04-09 Diagnostic spectral des tissus malades Ceased EP0466828A1 (fr)

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CA2042075C (fr) * 1991-05-08 2001-01-23 Branko Palcic Systeme d'imagerie endoscopique
US5769792A (en) * 1991-07-03 1998-06-23 Xillix Technologies Corp. Endoscopic imaging system for diseased tissue
US5421339A (en) * 1993-05-12 1995-06-06 Board Of Regents, The University Of Texas System Diagnosis of dysplasia using laser induced fluoroescence
US5590660A (en) * 1994-03-28 1997-01-07 Xillix Technologies Corp. Apparatus and method for imaging diseased tissue using integrated autofluorescence
US5579773A (en) * 1994-09-30 1996-12-03 Martin Marietta Energy Systems, Inc. Laser-induced differential normalized fluorescence method for cancer diagnosis
US6258576B1 (en) 1996-06-19 2001-07-10 Board Of Regents, The University Of Texas System Diagnostic method and apparatus for cervical squamous intraepithelial lesions in vitro and in vivo using fluorescence spectroscopy
US5991653A (en) * 1995-03-14 1999-11-23 Board Of Regents, The University Of Texas System Near-infrared raman spectroscopy for in vitro and in vivo detection of cervical precancers
US5697373A (en) * 1995-03-14 1997-12-16 Board Of Regents, The University Of Texas System Optical method and apparatus for the diagnosis of cervical precancers using raman and fluorescence spectroscopies
US5615673A (en) * 1995-03-27 1997-04-01 Massachusetts Institute Of Technology Apparatus and methods of raman spectroscopy for analysis of blood gases and analytes
US5612540A (en) * 1995-03-31 1997-03-18 Board Of Regents, The University Of Texas Systems Optical method for the detection of cervical neoplasias using fluorescence spectroscopy
FR2737845B1 (fr) * 1995-08-16 1998-01-02 Centre Nat Rech Scient Dispositif d'imagerie endoscopique pour la detection precoce de lesions superficielles cancereuses ou precancereuses
US5647368A (en) * 1996-02-28 1997-07-15 Xillix Technologies Corp. Imaging system for detecting diseased tissue using native fluorsecence in the gastrointestinal and respiratory tract
US6185443B1 (en) 1997-09-29 2001-02-06 Boston Scientific Corporation Visible display for an interventional device
DE69838813T2 (de) * 1997-10-10 2008-11-27 Boston Scientific Limited Miniatur-spektrometer-anordnung
US6289229B1 (en) 1998-01-20 2001-09-11 Scimed Life Systems, Inc. Readable probe array for in vivo use
US6377841B1 (en) 2000-03-31 2002-04-23 Vanderbilt University Tumor demarcation using optical spectroscopy
US20070122344A1 (en) 2005-09-02 2007-05-31 University Of Rochester Medical Center Office Of Technology Transfer Intraoperative determination of nerve location
US20080161744A1 (en) 2006-09-07 2008-07-03 University Of Rochester Medical Center Pre-And Intra-Operative Localization of Penile Sentinel Nodes
US8406860B2 (en) 2008-01-25 2013-03-26 Novadaq Technologies Inc. Method for evaluating blush in myocardial tissue
US10219742B2 (en) 2008-04-14 2019-03-05 Novadaq Technologies ULC Locating and analyzing perforator flaps for plastic and reconstructive surgery
WO2009135178A2 (fr) 2008-05-02 2009-11-05 Flower Robert W Procédés de production et d'utilisation d'érythrocytes chargés d'une substance (s-les) pour l'observation et le traitement de l'hémodynamique vasculaire
US10492671B2 (en) 2009-05-08 2019-12-03 Novadaq Technologies ULC Near infra red fluorescence imaging for visualization of blood vessels during endoscopic harvest
JP6028096B2 (ja) 2012-06-21 2016-11-16 ノバダック テクノロジーズ インコーポレイテッド 血管造影及びかん流の定量化並びに解析手法
WO2016049756A1 (fr) 2014-09-29 2016-04-07 Novadaq Technologies Inc. Imagerie d'un fluorophore cible dans une matière biologique en présence d'auto-fluorescence
AU2014408488B2 (en) 2014-10-09 2019-07-18 Stryker European Operations Limited Quantification of absolute blood flow in tissue using fluorescence-mediated photoplethysmography
JP6931705B2 (ja) 2017-02-10 2021-09-08 ノバダック テクノロジーズ ユーエルシー オープンフィールドハンドヘルド蛍光イメージングシステムおよび方法
US11446055B1 (en) 2018-10-18 2022-09-20 Lumoptik, Inc. Light assisted needle placement system and method

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AT387860B (de) * 1985-09-16 1989-03-28 Optical Sensing Technology Verfahren und vorrichtung zur tumordiagnose mittels sera
GB2203831B (en) * 1986-07-07 1991-02-06 Academy Of Applied Sciences Apparatus and method for the diagnosis of malignant tumours
JPS63255072A (ja) * 1987-03-23 1988-10-21 イーストマン・コダック・カンパニー 二つのレーザーを用いる治療および診断装置
DE3718202C1 (de) * 1987-05-29 1988-11-17 Wolfgang Prof Dr Lohmann Anordnung zur Messung eines Zustandswertes fuer organische Gewebeflaechen
ATE187315T1 (de) * 1987-09-24 1999-12-15 Massachusetts Inst Technology Kathetersystem zur abbildung

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9012536A1 *

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