Patent Application: US-51915300-A

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:
fig1 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 fig1 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 fig4 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 fig2 ), 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 fig2 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 fig6 - 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 fig1 would also be acceptable . as with the apparatus of fig1 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 . referring now to fig3 a - 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 , fig3 a shows the neonate uterus , fig3 b shows the premenarchal uterus , fig3 c shows the menarchal uterus , fig3 d shows the menstruating uterus , fig3 e shows the menopausal uterus and fig3 f shows the postmenopausal uterus . as can be seen , the transformation zone tz can appear on the ectocervix ( for example , menstruating , fig3 d ), or well within the edocervical canal ( for example , postmenopausal , fig3 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 fig1 and 15 . fig4 a and 4b are a single pixel probe 11 that may be used in the apparatus of fig1 in accordance with the present invention . referring to fig4 a , 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 fig1 . 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 fig4 a 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 fig4 a ) 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 . fig4 b is a cross section through section 4 b — 4 b of fig4 a , 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 . fig5 is an alternative embodiment of the single - pixel probe of the present invention . referring to fig5 probe 11 includes fiber bundle 41 like that of the embodiment of fig4 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 fig4 a . referring now to fig6 and 7 , a ring optical probe 21 is disclosed that may be used in the apparatus of fig2 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 fig1 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 fig8 . in yet another embodiment , the ends of fibers 72 may be cleaved and polished as shown in fig9 . in the embodiment of fig9 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 fig9 . 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 fig6 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 fig1 a and 12b disclosed is yet another embodiment of optical probe 21 usable in the apparatus of fig2 . the optical probe 21 of fig1 a 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 fig1 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 fig1 a is approximately 3 mm , however other diameters would also be acceptable the illuminator of probe 21 in fig1 a 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 fig1 a 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 fig1 , 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 fig1 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 fig1 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 fig1 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 fig1 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 fig1 and 17 . as can be seen with reference to fig1 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 . 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 fig1 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 fig1 or 2 , using any of the optical probes of fig4 - 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 ( fig4 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 ( fig6 - 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 ( fig1 ), 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 . fig1 , 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 , fig1 is a group of normalized fluorescence intensity spectra obtained with 337 nm excitation , fig1 is a group of fluorescence intensity spectra obtained using 380 nm excitation , and fig2 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 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