Abstract:
A method and apparatus for characterizing tissue of epithelial lined viscus in vivo including, for example, the endocervical canal. The method comprises illuminating an interior surface of the viscus with electromagnetic radiation wavelengths to produce a plurality of fluorescence intensity spectra, detecting a plurality of emission wavelengths from the fluorescence intensity spectra, and characterizing the epithelial viscus tissue as a function of the emission wavelengths. The apparatus includes a light source of emitting a plurality of electromagnetic radiation wavelengths, an optical probe connected to the light source, the probe being adapted to apply the plurality of electromagnetic radiation wavelengths to an interior surface of epithelial viscus tissue under test and to gather fluorescence emitted from the tissue, a detector connected to the probe for detecting at least one fluorescence spectrum emitted from the tissue under test and a programmed computer connected to the detector for processing the at least one fluorescence spectrum according to a predetermined algorithm to characterize the tissue under test.

Description:
This is a divisional of application Ser. No. 08/693,471, filed Jun. 22, 1999 (now abandoned). 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates to apparatus and methods for investigating epithelial lined viscus, and more particularly to apparatus and methods for characterizing normal and dysplastic tissue of the endocervical canal. 
     The most prevalent of preinvasive conditions of the female lower genital tract is cervical intraepithelial neoplasia (CIN). The traditional definition calls it a spectrum of intraepithelial changes that begins as a generally well differentiated intraepithelial neoplasm, which has traditionally been classified as a very mild dysplasia, and ends with invasive carcinoma. Neoplastic changes are confined to the squamous epithelium and include nuclear pleomorphism, loss of polarity, and presence of abnormal mitoses. CIN is graded 1 to 3, based on the amount of undifferentiated cells present from the basement membrane to the surface epithelium. When one third of that distance is involved, the grade is 1; when more than one third and up to two thirds is involved, the grade is 2; when more than two thirds is involved, the grade is 3. Full-thickness involvement from the surface epithelium to the basement membrane is referred to as carcinoma in situ (CIS). The median transit time from CIN to CIS depends on the grade of CIN: for grade 1 CIN, the time is approximately 6 years; for grade 2 CIN, approximately 2 years; and for grade 3, approximately 1 year. Despite some debate in the past about CIN and CIS representing two distinct entities, it is currently believed that CIN and CIS are part of a spectrum of disease that leads to invasive cancer of the cervix. The diagnosis and treatment of CIN are thus part of the prevention of invasive cervical cancer. An accepted method to classify cervical tissues is the new Bethesda system as presented in Wright et al. , “Pathology of the Female Genital Tract,” 156-177, Springer-Verlag, (1994). In accordance with that system, lesions with HPV and CIN are classified as squamous intraepithelial lesions (SILs) where they may be further separated as high grade SIL (CIN II, CIN III, CIS) and low grade SIL (CIN I, HPV). Normal, metaplastic and non-specific inflammation tissues are classified as non-SILs. 
     Cervical intraepithelial neoplasia is usually detected by screening Pap smears from asymptomatic women. Patients with abnormal Pap smears are referred for colposcopy and possibly biopsy. Acetic acid is applied to the cervix, and areas with abnormal DNA content, such as those with CIN, turn white. The colposcope, a mounted magnifying lens, is used to direct biopsies of the abnormal white areas. Abnormal configurations of blood vessels, called vascular atypia, signal disordered growth and help the clinician know which other areas require biopsy. An appropriate evaluation of the abnormal Pap smear involves review of the referral and repeat Pap smears, endocervical curettage, and multiple biopsies of the aceto white areas; the results of such analysis will indicate whether the patient has CIN. 
     While the predictive accuracy of colposcopy is a matter of debate in the field with some researchers finding excellent overall accuracy with others finding accuracy to be poor for CIN but good for condyloma. 
     Recently, there has been intensive research to explore the use of optical spectroscopy for the diagnosis of disease in human tissue. Several studies have successfully demonstrated the use of fluorescence, infrared absorption and Raman spectroscopies for disease diagnosis in various organ systems. Auto and dye induced fluorescence have shown promise in recognizing atherosclerosis and various types of cancers. Many groups have utilized autofluorescence for differentiation of normal and abnormal tissues from the human breast and lung, urinary bladder and gastrointestinal tract. 
     Copending application Ser. No. 08/666,021, filed Jul. 19, 1996, assigned to the same assignee as the present application, discloses a system that uses fluorescence spectroscopy to discriminate diseased (pre-cancerous and cancerous) from non diseased (normal tissues and inflammation) tissue as well as differentiate cancer and high grade pre-cancers from low grade precancerous lesions of the human cervix in vivo. This system provides more effective patient management, as 1) fluorescence measurements, and hence diagnostic information, can be obtained in real time and 2) the technique is non-invasive. In vitro studies in which fluorescence was measured from cervical biopsies over the UV and visible regions of the spectrum have shown that the fluorescence intensity of histologically abnormal cervix is significantly lower than that of the normal cervix from the same patient. In accordance with the above-referenced copending application, the system includes a fiber optic probe, illumination source and optical multi channel analyzer. The probe is inserted through the vaginal canal until its tip is flush with the surface of the cervix. The probe delivers light at specific excitation wavelengths and collects fluorescence from the entire emission wavelength range from a predetermined area of the cervix. During colposcopy, spectra are collected from each colposcopically abnormal area of the cervix prior to biopsy and from 1 to 4 colposcopically normal areas. Using this system, laser induced fluorescence acquired from human cervical tissues in vivo at 337, 380 and 460 nm excitation is analyzed to identify cervical intraepithelial neoplasia (CIN). 
     A limitation of previous colposcopic and fluorescence spectroscopic systems is that they are not capable of sampling the endocervix. It is known that atypical colposcopic tissue patterns occur with some frequency at the transformation zone between the squamous and columnar epithelium in the endocervical canal. See, Burke L, Antonioli D A and Ducatman B S.  Colposcopy, Text and Atlas,  pp. 47, 48, 61 and 62, Appleton and Large, Norwalk Conn. (1991) This transformation zone (also known as the squamocolumnar junction) is often located well within the endocervical canal and is not easily subjected to colposcopy or fluorescence spectroscopy using existing systems which are intended primarily to assess the ectocervix. In addition, cervical lesions that exist on the ectocervix often extend into the endocervical canal, and characterization of the lesion within the endocervical canal is often an important matter. 
     It would therefore be desirable to provide a means to subject the endocervical canal, including the transformation zone, to fluorescence spectroscopy. 
     SUMMARY OF THE INVENTION 
     The present invention avoids the above noted drawbacks of the prior art by providing a method and apparatus for characterizing tissue of epithelial lined viscus including, for example, the endocervical canal. In particular, in accordance with a method embodying the present invention, endocervical canal tissue is characterized in vivo, by illuminating endocervical canal tissue in vivo with electromagnetic radiation wavelengths to produce a plurality of fluorescence intensity spectra, detecting a plurality of emission wavelengths from the fluorescence intensity spectra, and characterizing the endocervical canal tissue as a function of the emission wavelengths. The characterizing step may distinguish squamous epithelium and columnar epithelium tissue, normal squamous and abnormal tissue, normal columnar epithelium and abnormal tissue, inflamed and abnormal tissue, low grade SIL and high grade SIL tissue, or normal and high grade SIL tissue. 
     In addition, the illuminating and detecting steps may comprise, illuminating a substantially cylindrical area of the endocervical canal tissue, and detecting the plurality of emission wavelengths from selected portions of the cylindrical area. The illuminating and detecting steps may further comprise illuminating an area of the endocervical canal in a vicinity of a single pixel, and detecting the plurality of emission wavelengths from the single pixel, and repeating the illuminating and detecting steps to substantially cover the cylindrical surface. In another embodiment, the illuminating and detecting steps may further comprise illuminating a substantially ring-shaped area of the endocervical canal, detecting the plurality of emission wavelengths from the substantially ring-shaped area, and repeating the illuminating and detecting steps to substantially cover the cylindrical surface. In yet another embodiment, the illuminating and detecting steps may further comprise, illuminating a substantially line-shaped area of the endocervical canal, detecting the plurality of emission wavelengths from the substantially line-shaped area, and repeating the illuminating and detecting steps to substantially cover the cylindrical surface. 
     In addition, the electromagnetic radiation wavelengths used to practice the method of the present invention may be in the ranges of 317-357 nm, 360-400 nm and 440-480 nm. 
     In addition, an apparatus embodying the present invention for characterizing endocervical tissue, comprises, a light source for emitting a plurality of electromagnetic radiation wavelengths; a probe connected to the light source, the probe adapted to apply the plurality of electromagnetic radiation wavelengths to an interior surface of endocervical canal tissue under test and to gather fluorescence emitted from the tissue under test; a detector, connected to the probe, for detecting at least one fluorescence spectrum emitted from the tissue under test, and a programmed computer connected to the detector means, for processing the at least one fluorescence spectrum according to a predetermined algorithm to characterize the tissue under test. 
     The light source may be a laser light source or a filtered white light source and the plurality of electromagnetic radiation wavelengths may be about 337 nm, about 380 nm and about 460 nm. The probe may include excitation optical fibers for applying the plurality of electromagnetic wavelengths to an interior surface of the endocervical tissue under test, and collection optical fibers for gathering the fluorescence emitted from the endocervical tissue under test. 
     These and other features and advantages of the present invention will become apparent to those of ordinary skill in this art with reference to the following detailed description. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is an exemplary apparatus in accordance with the present invention usable to perform the method of the present invention. 
     FIG. 2 is another exemplary apparatus in accordance with the present invention usable to perform the method of the present invention. 
     FIGS. 3A-3E illustrate various states of the endocervical canal. 
     FIGS. 4A and 4B are an exemplary single pixel probe usable in the present invention. 
     FIG. 5 is another exemplary embodiment of a single pixel probe usable with the present invention. 
     FIGS. 6-11 are various exemplary embodiments of a ring probe useable in the present invention. 
     FIGS. 12A and 12B are an exemplary embodiment of a line probe useable in the present invention. 
     FIG. 13 is a graphical representation of a study of endocervical canal size. 
     FIGS. 14 and 15 are graphs showing the optical transmission and excitation emission of cervical mucus. 
     FIGS. 16 and 17 are graphs showing the optical transmission and excitation emission of fluorinated ethylene-propylene (FEP). 
     FIGS. 18,  19  and  20  are exmplary fluoresence spectra obtained from endocervical canal tissue. 
    
    
     DETAILED DESCRIPTION 
     Measurement Apparatus 
     FIGS. 1 and 2 present exemplary embodiments of the apparatus of the present invention which are useable to practice the method of the present invention. 
     Referring first to FIG. 1, an apparatus is disclosed using a single pixel optical probe. Exemplary embodiments of the single pixel probe are presented in more detail below with reference to FIGS. 4 and 5. The apparatus includes endocervical probe  11  which, as described below in more detail, incorporates a number of optical fibers including excitation fibers  12 ,  13  and  14  and collection fiber  16 . The excitation fibers are connected to an illumination source which may be, for example, two nitrogen lasers  17 , 18  (LN300C, Laser Photonics) with a dye module. Other illumination sources, for example a Xenon lamp and filter wheel (disclosed in more detail with reference to FIG.  2 ), may also be used. Other illumination sources may also be acceptable, including, for example, various types of lasers (for example, HeCd or Ag lasers) used with or without dye modules, and various types of so-called-white light sources (for example, Xe, Hg, or XeHg lamps) used with filter wheels. This illumination source produces light at frequencies that have been selected for their ability to produce fluorescence in tissue that permits characterization of the tissue, For example light at approximately 337, 380 and 460 nanometers has proven useful. This light is coupled into excitation fibers  12 ,  13 ,  14 . For coupling, standard Microbench components (Spindler Hoyer) and planoconvex lenses  19  were used. The light coming out of the two dye modules is bandpass filtered by bandpass filters  21  to minimize fluorescence from the dye being coupled into the excitation fibers  12 ,  13  and  14 . Collection fiber  16  collects the fluorescence which is projected through a coupling optics  22  (for example, Microbench, magnification 50/30) into a detector  24 , for example an F/3.8 spectrograph (Monospec 18, Thermo Jarrel Ash, Scientific Measurement Systems, Inc.). In the coupling optics  22 , longpass filter  23  (for example, color glass filters, Schott) block the reflected excitation light from entering the detector. The spectrograph disperses the light onto an intensified diode array  26 . Exemplary diode array  26 , electronics and controller  27  are manufactured by Princeton Instruments. The system also includes gate pulser  28  which is used to control the operation of lasers  17  and  18 . Lasers  17  and  18  may be controlled, for example at a 30 Hz repetition rate with a 5 nanosecond pulse duration, but other repetition rates and pulse durations may also be acceptable. 
     The apparatus also includes programmed computer  29  which operates to energize lasers  17  and  18  and to analyze the fluorescence spectra collected by collection fiber  16  in order to characterize the tissue sample under study. Details of this control and analysis may be found in copending application Ser. No. 08/666,021, filed Jul. 19, 1996, the disclosure of which is incorporated herein by reference. 
     Referring now to FIG. 2, an apparatus embodying the present invention is disclosed using a multiple pixel optical probe. Exemplary embodiments of multiple pixel optical probes are presented in more detail below with reference to FIGS. 6-12. The apparatus includes a multiple pixel optical probe  21  which incorporates excitation optical fibers  22  and collection optical fibers  23 . Excitation optical fibers  22  are connected to receive light from illumination source  24  which may be, for example, a Xenon lamp  26  in combination with a filter wheel  27 . Once again, other illumination sources, including for example, the laser source disclosed with reference to FIG. 1, would also be acceptable. As with the apparatus of FIG. 1, illumination source  24  produces light at frequencies that have been selected for their ability to produce fluorescence in tissue that permits characterization of the tissue. 
     Collection fibers  23  from probe  21  are connected to detector  28  which includes, for example, an imaging spectrograph  29  (for example, a Chromex 250 IS), and a CCD array  31  (for example, a thermoelectric cooled CCD Princeton Instruments EV 578x384). The output of detector  28  is applied to computer  32  which is programmed to control illumination source  24  and to analyze the fluorescence spectra collected by collection fibers  23  and detected by detector  28  using, for example, the analysis methods disclosed in the aforementioned copending application. 
     Endocervical Canal Morphology 
     Referring now to FIGS. 3A-F, shown are simplified representations of the cross section of the os of the endocervical canal and surrounding tissue illustrating the locations of the squamous epithelium (SE), columnar epithelium (CE) and transformation zone (TZ) of the uterus at various stages of maturity and for various medical conditions. Specifically, FIG. 3A shows the neonate uterus, FIG. 3B shows the premenarchal uterus, FIG. 3C shows the menarchal uterus, FIG. 3D shows the menstruating uterus, FIG. 3E shows the menopausal uterus and FIG. 3F shows the postmenopausal uterus. As can be seen, the transformation zone TZ can appear on the ectocervix (for example, menstruating, FIG.  3 D), or well within the edocervical canal (for example, postmenopausal, FIG.  3 F), or anywhere in between. Since the most common location for CIN and metaplasia is at or near the transformation zone, it is critical that the transformation zone be imaged when conducting fluorescence spectroscopy. This is of particular importance in menopause and postmenopause because most cervical carcinomas occur at this age, and this is when the transformation zone is most deeply within the endocervical canal. 
     Other general observations of the morphology of the endocervical canal are worthy of note. After the external os, which follows a funnel type opening, the endocervical canal enlarges and gets smaller again at the inner os. The uterus opens to its full size after the internal os by a small angle. The canal can be filled inside with non-neoplastic additional tissue like polyps and synechia. Polyps may fill the canal. Atrophy may be present, which results in an abnormal form of the wall (missing folds). In addition, It is known that stenosis may occur after LEEP treatments. 
     The folds of the columnar epithelium may typically be several centimeters deep with varying shapes. For example, in one uterus that was studied after removal by hysterectomy, the folds were a maximum of 7.83 mm with a mean depth of 3.38 mm. The folds were observed to have two main directions: axial and with an angle of approximately 30 degrees to the axis of the canal. The top of this pine tree-like form points outwards the canal. The folds are filled with mucus that sticks strongly to the tissue. Flushing with saline solution will not remove the mucus. A study of the fluorescence characteristics of cervical mucus are presented below with reference to FIGS. 14 and 15. 
     Optical Probes 
     FIGS. 4A and 4B are a single pixel probe  11  that may be used in the apparatus of FIG. 1 in accordance with the present invention. Referring to FIG. 4A, optical probe  11  includes a bundle of optical fibers  41  which are packed in a fluorinated ethylene-propylene (FEP) tubing  42  that is substantially transparent to visible light and that also transmits in the ultraviolet. The FEP tubing  42  containing the fibers  41  is flexibly mounted within a second tubing  43  which may also be made of FEP. The outer diameter of tubing  43  is preferably less than 2 mm, however other dimensions may be used. The outer diameter of tube  43  is determined primarily by anatomical constraints of the endocervical canal, and is discussed in more detail below with reference to FIG.  13 . This dimension allows the passage of the probe through an endocervical canal with a stenosis at the outer os. The fiber  41  within tubing  42  may be rotated and axially displaced within tubing  43  in order to permit the testing of several tissue sites without moving tubing  43 . 
     Although FEP has proven useful for use as the material for the tubings used in the optical probes of the present invention, other materials may also be acceptable, including, for example, other plastics such as polytetrafluorethylen (PTFE), glass and quartz. 
     In the embodiment of FIGS. 4A and B, a short piece of a large diameter fiber  45  is used. as a reflector and the end surface  47  of fiber  45  is polished with an oblique angle (for example, 40°) relative to the axis of probe  11 . Reflection of light emitted by fibers  41  toward the tissue sample under study (downward in FIG. 4A) and reflection of light emitted by the tissue sample back toward fibers  41  occurs because of total internal reflection. An alternative reflector may be made using an angled mirrored surface of polished metal, glass, sapphire, or the like. 
     FIG. 4B is a cross section through section  4 B— 4 B of FIG. 4A, and shows the configuration of fibers  41 . In the exemplary embodiment there are seven fibers  41 , six illumination fibers  48 - 53 , and one collection fiber  54 , however any number of fibers may be used. In the exemplary embodiment, illumination fibers  48  and  51  are used for 337 nm, fibers  49  and  52  are used for 380 nm and fibers  50  and  53  are used for 460 nm. and collection fiber  54  provides a single pixel collection for fluorescence spectroscopy. It should be noted that any combination of illumination and collection fibers may be used without departing from the scope of the invention. For example, three illumination fibers and one collection fiber may be used, three illumination fibers and three collection fibers may be used., one illumination fiber and one collection fiber may be used, one fiber used for the combined purpose of illumination and collection may be used, and so forth. Fibers  48 - 54  may be, for example, type SFS320/385T optical fibers, and fiber  46  may be a type SFS1500/1650N optical fiber, both available from Fiberguide Industries, however other types may also be used. The single pixel embodiment results in a single substantially elliptical measurement and illumination spot, or pixel. 
     The part of probe  11  that extends outside the vagina preferably has a rigid tube with markings which may be used as an aid in positioning the probe both axially and rotationally. Saline solution may be flushed though the openings  61  in tip  62  of probe  11  before or during a testing procedure. 
     FIG. 5 is an alternative embodiment of the single-pixel probe of the present invention. Referring to FIG. 5, probe  11  includes fiber bundle  41  like that of the embodiment of FIG.  4 A. light emitted from the end of fibers  41  is focused by lens  66  and reflected by reflecting surface  67  toward a tissue sample  68  under study. Similarly, light emitted by a tissue sample  68  is focused by lens  66 , and reflected by reflective surface  67  back toward fibers  41 . Other structural details remain substantially as in FIG.  4 A. 
     Referring now to FIGS. 6 and 7, a ring optical probe  21  is disclosed that may be used in the apparatus of FIG. 2 Probe  21  includes a number of optical fibers  72  coaxially arranged in a ring shape. In one embodiment every other one of fibers  72  are used for illumination, with the remaining fibers being used for collection. Alternatively, each fiber  72  may be used for both illumination and collection. In the embodiment of FIGS. 10 and 11, reflection of both illuminating light and collected light is done by a metal plug  73  with a polished reflecting surface  74  Alternately, a sapphire tip  81  may be used as shown in FIG.  8 . In yet another embodiment, the ends of fibers  72  may be cleaved and polished as shown in FIG.  9 . In the embodiment of FIG. 9, every other fiber may be used as an illumination fiber with all remaining fibers being collection fibers. This would result in adjacent fibers (for example, fibers  72 ′ and  72 ″) acting together to illuminate and detect from a single tissue area  77 . Alternately, each of fibers  72  may have the combined function of illumination and collection. It should be noted that for the sake of clarity, the surrounding tube is not shown in the embodiment of FIG.  9 . 
     In all embodiments of the ring probe  21 , light is reflected from fibers  72  toward a tissue sample located adjacent the exterior wall of probe  21  and light emitted by the tissue sample is reflected back toward fibers  72 . This results in a plurality of substantially elliptical measurement and illumination spots, or pixels distributed in a ring shape. 
     In the ring probe  21  embodiment of FIGS. 6,  7  and  8 , channel  76  may be included to permit the flushing of the tissue under test with saline either before or during a test . . . The diameter of probe  21  may be, for example, approximately 2.8 mm, however other diameters may also work. 
     Referring now to FIGS. 12A and 12B disclosed is yet another embodiment of optical probe  21  usable in the apparatus of FIG.  2 . The optical probe  21  of FIGS. 12A and 12B is a line probe. The probe  21  includes of an illuminator that serves to illuminate a tissue sample under study, and a collector that serves to collect light emitted by the tissue sample under study. The collector in the exemplary embodiment is made of 19 100 micrometer optical fibers  122  (type SFS100/110T available from Fiberguide Industries), however, any number of optical fibers may be used. In the disclosed embodiment, The collection of the fluorescence occurs every 1.5 mm with one of the collection fibers  122 . The collection fibers  122  are polished at an oblique angle relative to the longitudinal axis of the fiber  122  (for example, 40°). The ends  123  of the collection fibers  122  are positioned at different axial locations along the probe as shown in FIG.  12 A. In the exemplary embodiment this results in a simultaneous collection every 1.5 mm along a line approximately 2.5 cm. long. The diameter of the line probe  21  of FIG. 12A is approximately 3 mm, however other diameters would also be acceptable 
     The illuminator of probe  21  in FIG. 12A includes a diffuser  124 . Diffuser  124  is mounted on the top of a bundle of fibers  127 . Fibers  127  may be for example type SFS200 200 micrometer optical fibers available from Fiberguide Industries, however other types of fibers may be acceptable. A reflective coating  128  over 270 degrees of diffuser  124  allows a directed illumination over approximately 90 degrees of the circumference. of probe  21 . The diffuser  124  is available, for example, from Rare Earth Medical. 
     The diffuser  124  is packed in a FEP tubing  131  that is substantially transparent in the visible and also in the ultraviolet. Included within tubing  131  is the collection bundle  122 , the diffuser  124  and flushing channels  132  used to carry saline to ports  133  in probe  21  thus permitting flushing of the tissue either before or during testing. The outer diameter of tubing  131  is preferably less than 3 mm, however, other diameters may also be acceptable. This allows the passage of the probe through most endocervical canals. 
     The probe is manually placed into the endocervical canal. Because of its stiffness the whole probe can be pressed against the walls of the endocervical canal while still keeping a minimal bendability. 
     Because 100 micron fibers  122  are used for collection the size of each measured spot or pixel in the exemplary embodiment of FIG. 12A will be smaller than approximately 0.5 mm. The diameter depends on the distance of the collection fiber  122  to the tissue. This distance may not be constant for all fibers and typically varies from approximately 0.3 to 1 mm. 
     The illumination light passes perpendicular through the collection fibers  122 . Therefore the jacket of these fibers  122  should be removed. Collection fibers  122  will then act as cylindrical lenses. 
     Referring now to FIG. 13, presented in graphical form are the results of a study of cervical size. Because the design of the probe used in the present invention depends on the canal properties the geometrical aspects of the endocervical canal were studied. A database of 362 patients at the MD Anderson Cancer Center contained measurements of the diameter of the external os. The obtained diameter is based on the size of a dilator used at MD Anderson to measure the endocervical canal prior a LEEP or LEEP cone treatment. In a following checkup visit the canal is checked again to assure no stenosis occurred. This parameter was measured in another series of 22 patients with similar results. 
     From FIG. 13 it can be seen that the endocervical canal has a mean diameter at the outer os of 5 mm. In most patients the outer os is larger than 3 mm and smaller than 7 mm. The length of the canal was estimated from a uterus removed by hysterectomy. The endocervical canal was measured to be approximately 4 cm long. These mechanical dimensions may then be considered in determining a size of the optical probes used in the present invention. For example, the study reflected in the graph of FIG. 13 indicates that the optical probe should preferably be less than 3 mm in diameter if a single sized probe is to be used for all patients. Of course, probes of different sizes may also be used. 
     In addition, in order to determine the possible effects of mucus in the endocervical canal, the transmission and fluorescence of several samples of mucus was measured, and the results are presented in graphical form in FIGS. 14 and 15. To produce these graphs, small amounts of mucus were diluted in 10 ml of normal buffered saline solution and placed in a 1 cm pathlength. 
     As can be seen with reference to FIGS. 14 and 15, the strongest emission of mucus is at 340 nm emission with an excitation at 280 nm. This will not interfere with the measurements performed by the disclosed exemplary embodiments of the present invention. 
     In addition, the transmission and fluorescence of FEP tubing (the presently preferred material for use as the housing for the probes of the present invention) was measured and the results are presented in FIGS. 16 and 17. As can be seen with reference to FIGS. 16 and 17, the fluorescence of the FEP tubing is low. However the autofluorescence of the FEP tubing is about {fraction (1/10)} of the tissue fluorescence at 337 nm excitation. There is a main emission peak at 400 nm with 320 nm excitation. It was determined that this contribution could be accommodated during a probe calibration procedure, discussed in more detail below. 
     Clinical Procedure 
     In a clinical application, the present invention has as its purpose the characterization of epithelial viscus tissue, such as, for example, tissue of the endocervical canal. In general, when applied to the characterization of endocervical tissue, the present invention has as its purposes to: a) identify lesions extending from the ectocervix into the endocervical canal; b) detect the position of the transformation zone if present inside the endocervical canal; and c) identify squamous lesions with columnar involvement inside the endocervical canal. In general, these purposes are accomplished by measuring fluorescence spectra at spatially resolved locations inside the endocervical canal over a substantially cylindrical area of the interior surface of the tissue of the canal, and using mathematical models to characterize that tissue as a function of the measured spectra. 
     Before beginning a clinical procedure, the measuring apparatus should be calibrated. To calibrate the present invention (as shown, for example in FIGS.  1  and  2 ), the background signals are obtained without any excitation which reflects the dark current of the device. This background is stored and is automatically subtracted from any fluorescence measurement. Next, the autofluorescence of the probe is determined, for example, by placing the probe in a brown bottle containing sterile H2O and measuring fluorescence spectra with the excitation light on. This signal is not subtracted from the tissue fluorescence, however it may be subtracted if desired. In order to confirm calibration, a standard rhodamine solution (OD 0.446725, (=550 nm, 1 cm pathlength) may be measured. Based on previous clinical work, Rhodamine has been shown to have approximately twice the intensity of squamous cervical tissue fluorescence. 
     During spectral measurement of tissue, if improvement in the signal to noise ratio is desired, the spectra may be accumulated 100 and 200 times, respectively at 380 and 460 nm At 337 nm 50 accumulations have proven sufficient. However, other methods to improve the signal to noise ratio may also be used. For all three wavelengths a different background subtraction file may be used with the corresponding accumulations. 
     During a clinical procedure, it is desired to obtain fluorescence spectra at 3 excitation wavelengths along the substantially cylindrical surface of the entire endocervical canal with a spatial resolution of approximately 1.5 mm. This may be accomplished by use of either of the apparatus of FIG. 1 or  2 , using any of the optical probes of FIGS. 4-12. During a procedure, the outer housing of the probe is placed and advanced to the internal os of the endocervical canal. Fluorescence measurement are then started. In the case of the single pixel probe (FIGS.  4  and  5 ), the single measuring pixel is advance both axially and angularly within the housing in order to image a sufficient number of pixels over the substantially cylindrical tissue surface. When using the ring probe (FIGS.  6 - 11 ), the measuring ring of pixels is advance axially in order to image a sufficient number of pixels over the substantially cylindrical tissue surface. Finally, when using the line probe (FIG.  12 ), the measuring line of pixels is incremented angularly in order to image a sufficient number of pixels over the substantially cylindrical tissue surface For example, when using the line probe, four individual measurement may be taken, one each at 12, 3, 6, and 9 o&#39;clock (i.e., every 90°). This procedure takes approximately 3 minutes to complete. 
     Either before or during a procedure, saline solution may be flushed over the tissue in order possibly to improve measurement accuracy by removing mucus or blood or loose tissue form the measurement site. 
     In general, if the margin of the first specimen at the endocervical side is free of dysplasia or cancer and the second specimen shows no changes it may be assumed that the canal is in a normal condition. If this margin is involved with changes it may be assumed that the first 5 mm of the canal are in an abnormal state. If the margin of the endocervical specimen contains no changes it may be assumed that the margins extend no deeper than 2 cm. If this specimen shows abnormal cells it may be assumed that the measurements in the canal were abnormal even after 5 nm. If the second specimen is marked as metaplasia it may be assumed that the transformation zone is inside the endocervical canal. If the first specimen shows metaplasia the transformation zone is located around the os or on the ectocervix. 
     FIGS. 18,  19  and  20  present groups of normalized fluorescence intensity spectra obtained in vivo from endocervical canals of several different patients using the method and apparatus of the present invention In particular, FIG. 18 is a group of normalized fluorescence intensity spectra obtained with 337 nm excitation, FIG. 19 is a group of fluorescence intensity spectra obtained using 380 nm excitation, and FIG. 20 is a group of normalized fluorescence intensity spectra obtained using 460 nm excitation. 
     Based upon the foregoing disclosure, these and other features and advantages of the present invention will become apparent to those of ordinary skill in this art and it will be appreciated that additions, deletions and changes nay be made to the disclosed embodiments without departing from the scope of the invention. 
     The following references, to the extent that they provide exemplary experimental details or other information supplementary to that set forth herein, are incorporated by reference: 
     1. Wright T C, Kurman R J, and Ferenczy A in  Pathology of the Female Genital Tract  (eds. A. Blaustein), 156-177, Springer-Verlag, New York (1994). 
     2. Barron B A, Richart R M, “Statistical model of the natural history of cervical carcinoma: II. Estimates of the transition time from dysplasia to carcinoma in situ,” JNCI 45: 1025-1030 (1970). 
     3. Burke L, Antonioli D A and Ducatman B S.,  Colposcopy, Text and Atlas,  Appleton and Large, Norwalk Conn. (1991). 
     4. Mitchell M F, “Diagnosis and Treatment of Preinvasive Disease of the Female Lower Genital Tract” The Cancer Bulletin. 42: 71-76 (1990). 
     5. Reid R, Stanhope C R, Herschman B R, Crum C P, Agronow S J, “Genital warts and Cervical cancer,” Am J Obstet Gynecol, IV: 815-823 (1984). 
     6. Reid R, Scalzi P, “Genital Warts and Cervical Cancer,” Am J Obstet Gynecol, 153(6): 611-618 (1985). 
     7. Barrasso R, Coupez F, Ionesco M, DeBrux J, “Human Papilloma Viruses and Cervical Intraepithelial Neoplasia: The Role of Colposcopy,” Gynecologic Oncology, 27: 197-207 (1987). 
     8. Alfano R R, Pradhan A and Tang C G, “Optical spectroscopic diagnosis of cancer in normal and breast tissues,” J Optic Soc Am B, 6: 1015-1023 (1989). 
     9. Andersson E S, Johansson J, Svanberg K and Svanberg S, “Fluorescence imaging and point measurements of tissue: applications to the demarcation of malignant tumors and atherosclerotic lesions from normal tissue,” Photochem Photobiol, 53: 807-14 (1991). 
     10. Richards-Kortum R R, Rava R P, Petras R E, Fitzmaurice M, Sivak M V and Feld M S, “Spectroscopic diagnosis of colonic dysplasia,” Photochem Photobiol, 53: 777-786 (1991). 
     11. Rava R P, Richards-Kortum R R, Fitzmaurice M, Cothren R M, Petras R E, Sivak M and Feld M S, “Early detection of dysplasia in colon and urinary bladder tissue using laser-induced fluorescence”, Optical methods for tumor treatment and early diagnosis: mechanisms and technique, SPIE 1426: 68-78 (1991). 
     12. Wong P T T, Wong R K, Caputo T A, Godwin T A and Rigas B, “Infrared spectroscopy of human cervical cells: Evidence of extensive structural changes during carcinogenesis,” Proc Natl Acad Sci USA, 88: 10988-10992 (1991). 
     13. Alfano R R, Lui C H, Sha W L, Zhu H R, Akins D L, Cleary J, Prudente R and Cellmer E, “Human breast tissues studied by IR fourier transform Raman spectroscopy,” Lasers in Life Sc, 4: 23-28 (1991). 
     14. Baraga J J, Feld M S and Rava R P, “Rapid near-infrared Raman spectroscopy of human tissue with a spectrograph and CCD detector.” Appl. Spectr, 46: 187-190 (1992). 
     15. Schomacker K T, Frisoli J K, Compton C C, Flotte T J, Richter J M, Nishioka N S and Deutsch T F, “Ultraviolet laser-induced fluorescence of colonic tissue: Basic biology and diagnostic potential,” Lasers in Surg Med, 12: 63-78 (1992). 
     16. Mahadevan A, Mitchell M F, Thomsen S, Silva E and Richards-Kortum R R, “A study of the fluorescence properties of normal and neoplastic human cervical tissue,” Lasers Surg Med 13:647-655, (1993). 
     17. Ramanujam N, Mitchell M F, Mahadevan A, Thomsen S, Malpica A, Wright T C, Atkinson, N and Richards-Kortum; In Vivo Diagnosis of Cervical Intraepithelial Neoplasia Using 337 Excitation, PNAS 91:10193, 1994. 
     18. Ramanujam N, Mitchell M F, Mahadevan A, Thomsen S, Richards-Kortum R R, “Spectroscopic Diagnosis of Cervical Intraepithelial Neoplasia (CIN) in vivo Using Laser Induced Fluorescence Spectra at Multiple Excitation Wavelengths,” Lasers Surg Med, (in press) (1996). 
     19. Brookner C K, Agrawal A, Trujillo E V, Mitchell M F and Richards-Kortum R R, “Relative Risk of UV-Fluorescence Spectroscopy and Endoscopy are comparable,” 24th. Annual Meeting of the American Society for Photobiology, Photochem Photobiol Supp. (in press) (1996).