DETECTION OF CERVICAL NEOPLASIAS USING FLUORESCENCE SPECTROSCOPY

Abstract: A method for detecting tissue abnormality, particularly precancerous cervical tissue, through fluorescence spectroscopy is disclosed. In vitro fluorescence measurements over a variety of different fluorescence spectra are used to screen tissue samples. Using a principal component analysis (PCA), it is possible to discriminate between normal and dysplastic tissues with relatively low false-positive and false-negative results.

Description: DETECTION OF CERVICAL NEOPLASIAS USING FLUORESCENCE SPECTROSCOPY BACKGROUND OF THE INVENTION The field of the invention relates to optical methods used for the screening and diagnosis of tissue abnormalities. In particular, the invention relates to the use of fluorescent spectroscopy to detect cancerous and precancerous tissues of the cervix. Cervical cancer is the second most common malignancy in women worldwide, exceeded only by breast cancer and in the United States, it is the third most common neoplasm of the female genital tract - 15,000 new cases of invasive cervical cancer and 55,000 cases of carcinoma in situ (CIS) were reported in the U.S. in 1994. In 1994, an estimated 4,600 deaths occurred in the United States alone from cervical cancer. However, in recent years, the incidence of pre-invasive squamous carcinoma of the cervix has risen dramatically, especially among young women. Women under the age of 35 years account for up to 24.5% of patients with invasive cervical cancer, and the incidence is continuing to increase for women in this age group. It has been estimated that the mortality of cervical cancer may rise by 20% in the next decade unless further improvements are made in detection techniques. The mortality associated with cervical cancer can be reduced if this disease is detected at the early stages of development or at the pre-cancerous state (cervical intraepithelial neoplasia (CIN) ) . A Pap smear is used to screen for CIN and cervical cancer in the general female population. This technique has a false-negative error rate of 15-40%. An abnormal Pap smear is followed by colposcopic examination, biopsy and histologic confirmation of the clinical diagnosis. Colposcopy requires extensive training and its accuracy for diagnosis is variable and limited even in expert hands. A diagnostic method that could improve the performance of colposcopy in the hands of less experienced practitioners, eliminate the need for multiple biopsies and allow more effective wide scale diagnosis could potentially reduce the mortality associated with cervical cancer. Recently, fluorescence, infrared absorption and Raman spectroscopies have been proposed for cancer and precancer diagnosis. Many groups have successfully demonstrated their use in various organ systems. Auto- and dye-induced fluorescence have shown promise in recognizing atherosclerosis and various types of cancers and precancers. Many groups have demonstrated that autofluorescence may be used for differentiation of normal and abnormal tissues in the human breast and lung, bronchus and gastrointestinal tract . Fluorescence spectroscopic techniques have also been investigated for improved detection of cervical dysplasia. Despite these advances, there remains a need for diagnostic methods with improved accuracy and ease of application that also provide more rapid results. Such methods will permit earlier diagnosis, more effective patient management and, potentially, reduce mortality. SUMMARY OF THE INVENTION Thus, it is an objective of the present invention to provide improved methods for the early detection of neoplasia. In particular, it is an objective of the present invention to provide improved spectroscopic methods for the identification of abnormal cervical 0746 PC17US96/04305 tissue, thereby providing a rapid, accurate and simple method for detecting cancerous or precancerous cervical tissue. In satisfying these and other objectives, there is provided a method for the optical diagnosis of tissue abnormalities. In one embodiment, the present invention provides for the detection of tissue abnormality in a tissue sample in vi tro by illuminating a tissue sample with a series of electromagnetic radiation wavelengths selected to cause the tissue sample to produce a series of fluorescence intensity spectra indicative of tissue abnormality. The fluorescence intensity spectra emitted from the tissue sample as a result of illumination with the electromagnetic radiation are detected. Then, a probability that the tissue sample is normal or abnormal is calculated from the fluorescence intensity spectra. The invention further contemplates that the calculations include principal component analysis of the spectra, relative to a plurality of preprocessed spectra obtained from tissue samples of known diagnosis. The invention also contemplates normalizing the spectra, relative to a maximum intensity within the spectra, and mean-scaling the spectra as a function of a mean intensity of the spectra. The apparatus of the present invention includes a controllable illumination device for emitting a plurality of electromagnetic radiation wavelengths selected to cause a tissue sample to produce a fluorescence intensity spectrum indicative of tissue abnormality, an optical system for applying the plurality of radiation wavelengths to a tissue sample, a fluorescence intensity spectrum detecting device for detecting an intensity of fluorescence spectra emitted by the sample as a result of illumination by the plurality of electromagnetic radiation wavelengths, a data processor, connected to the detecting device, for analyzing detected fluorescence spectra to calculate a probability that the sample is abnormal. 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 appended drawings and following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph of the λex/λe values for known cell and tissue flurophores. Also indicated are known cell and tissue fluorophores: first and second harmonic Rayleigh Scattering (1) ; H 2 0 Raman Scattering (2) ; PN (NADH) (3) ; Fp (FADH 2 ) (4); tryptophan (5); porphyrins (6) ; collagen (7) ,- elastin (8) ; Hb absorption bands (9) . FIG. 2 is a graph of the average cell pellet EEM. FIG. 3 is a graph of a sample cell pellet EEM with a high (430,520) peak. FIG. 4 is a graph of a sample cell pellet EEM with a high (250,400) peak. FIG. 5 is a scattergram of cell pellet classification based on principal component analysis of EEM's (spectral data only) . (x) indicates samples deemed abnormal by Pap smear reading and (o) indicates samples deemed normal by Pap smear reading; 44 total = 15 normal and 29 abnormal. FIG. 6 is a graph showing classification errors for Fisher's discriminant analysis (• - cross validation error estimates; ■ - Fisher's method errors) . FIG. 7 is a graph of typical ThinPrep versus Pellet Spectra for 280 nm excitation wavelength ( is thin prep slide; — is cell pellet) . FIG. 8 is a graph of typical ThinPrep versus Pellet Spectra for 370 nm excitation wavelength ( is thin prep slide; — is cell pellet) . FIG. 9 is a graph of a hypothetical distribution of test values for hypothetical samples (1 is specificity; 2 is sensitivity) . DETAILED DESCRIPTION I. Introduction Clinical detection of neoplasias can be divided into two different kinds of analysis. First, screening provides a way to identify suspicious samples taken from a rather large pool of subjects. Subjects may be from the population as a whole or they may be part of a group identified as having a higher than average risk for one or more cancers. It is desirable that, because of the sheer number of tests, screening assays be relatively rapid, easy to conduct and inexpensive. It also is desirable that they exhibit a low false-negative rate. Once patients have been screened, it is necessary to proceed with more detailed testing that can be referred to generically as diagnosis. In diagnosis, the neoplasias nature of the sample is confirmed and, in addition, further information on the type and degree of dysplasia is obtained. This provides the clinician with an understanding of the disease state necessary to begin a treatment regimen. For diagnosis, cost, ease of application and rapidity are less important, though always desirable. It is important, however, that diagnostic procedures be accurate with respect to the kind of cancer identified and its clinical stage. The present invention is an example of the first kind of detection, screening. Present screening methods, like Pap smears, are time intensive, require highly trained individuals and are relatively expensive. Even so, the subjective nature of the scoring often results in an unacceptable number of false negatives, the outcome of which can be devastating. It is believed that by using a more objective standard like fluorescent emissions, the accuracy of the screen can be improved. In addition, the possibility for automation has further benefits in terms of time and expense. A. Method for Determining Fluorescent Spectra The present invention is premised on the hypothesis that normal and abnormal cells will emit differing fluorescent spectra in response to stimulating electromagnetic radiation. In its most general form, the methods comprises providing a tissue sample, illuminating that tissue sample with electromagnetic radiation, detecting fluorescence of the sample and comparing the fluorescence of the sample with that of some standard. Each of these steps is described in greater detail, below. Obtaining a tissue sample can be achieved by any one of a variety of different means, largely depending on the nature of the sample to examined. For example, for examination of solid tissues, samples can be taken by biopsy. Alternatively, scrapings of cells can be taken from the tissue of interest. For examination of cells that are not part of solid tissue, liquid samples may be obtained and the cells isolated therefrom. For example, blood samples may be obtained by any normal methodology. Aspiration of fluids also is contemplated, such as thin tissue aspirates. Once obtained, it may be necessary to further process the samples before they are examined. Further processing may include various forms of physical arrangement of the samples. For example, with solid tissues, it may be necessary to prepare thin sections. It also may be desired to dissociate the cells from each other and disperse them as a thin film monolayer. Dissociation may be accomplished by physical or enzymatic means. Similarly, dissociated cells in fluid samples or in scrapings may be concentrated and dispersed in a monolayer. In other instances, it may be desirable to concentrate disperse cells as a pellet. This can be accomplished by centrifugation of the liquid samples. Further pre-illumination processing includes chemical treatments such as fixation steps. In some cases, it will be desirable that the natural autofluorescence of the sample be unaffected. In such a case, the chemical treatment is selected so that the fluorescent species are not altered. In other cases, it may prove useful to use treatments that cause different autofluorescent profiles. Exemplary treatments include alcohol fixation. Suitable alcohols include methanol, ethanol, propanol, isopropanol, n-butanol and t-butanol. Typically, the samples are provided on a surface, though they can be provided in an open or closed container. A typical surface is a glass or quartz microscope slide. With certain surfaces, such as glass slides, there may be variation from item to item, requiring internal recalibration with each sample. There also may be distorting effects, especially for container- enclosed samples, that must be taken into account. Once the samples are prepared, the illumination is effected. In the present invention, a variety of different wavelengths can be used spanning from about 200 nm to over 700 nm. Under most circumstances, a plurality of different wavelengths will be applied, individually, to a single sample. Generally, the greater the number of wavelengths used, the better the ability to discriminate between physiologically distinct tissue samples. Of course, at some point, the intervals between wavelengths will be so small that the information achieved will become redundant. Those of skill in the art, knowing the fluorescent behavior of biological molecules, will be able to select both the appropriate number and range of wavelengths for a given purpose. In one embodiment, wavelengths from 250 nm to 550 nm were applied, with 10 nm intervals. Thus, 31 different wavelengths were applied to a single sample in sequence. Because a series of different illuminations and emissions are required, it is important that the sample not be affected by the illuminating wavelengths. One such effect would be pho obleaching, which has been shown to be significant in arterial tissue above excitation fluences of 80 mJ/mm 2 . The next step in the method is detection. For each illuminating wavelength, an emission spectrum is determined over a range of wavelengths. Again, a series of wavelengths may be used with the greater number examined, the more information with which to detect differences between normal and abnormal tissues. The emission spectra are normalized to an appropriate λex/λem. The excitation-emission matrices (EEM's) may be plotted three dimensionally, with excitation wavelength, emission wavelength and log(1/1 (λex/λem) ) as the three axes. FIG. 2, FIG. 3, and FIG. 4 depict various EEM's. In another embodiment, emissions were monitored from 250 nm to 700 nm at 10 nm intervals. Thus, the spectra comprises 46 different emission readings. These readings were normalized to an λex/λem of (270,330) . Based on the results discussed below, it appears that individual wavelengths do not adequately discriminate between normal and abnormal tissues. Taken as a group, however, there appears to be a correlation between fluorescence values and pathology. In order to maximize this correlation, various statistical manipulations were applied to the data, as discussed in detail below. FIG. 1 is a graph showing the excitation/emission profiles for various known cell and tissue fluorophores. Those of skill in the art are aware of other potential natural fluorophores whose fluorescence may be used to generate emission spectra which may then be used in accordance with the present invention. B. Multi-Variate Statistical Method Development In order to maximize the correlation between fluorescence values and the physiologic state of the sample tissue, multi-variate statistics were applied. The five primary steps involved in the multivariate statistical method are 1) preprocessing of spectral data from each patient to account for interpatient variation, 2) dimension reduction of the preprocessed spectra in the calibration set using principal component analysis, 3) selection of the diagnostically most useful principal components using a two-sided unpaired t-test and other criteria and 4) development of an optimal classification scheme based on Fisher's discriminant analysis using the diagnostically useful principal component scores of the calibration set as inputs with cross-validation. These five individual steps of the multivariate statistical method are presented below in more detail . 1) Preprocessing: The objective of preprocessing is to calibrate tissue spectra for inter-patient variation which might obscure differences in the spectra of different tissue types. A normalization method of preprocessing was invoked on the spectral data. Spectra were normalized by dividing the fluorescence intensity at each emission wavelength by the fluorescence intensity at (280 nm, 330 nm) of that sample. Normalizing a fluorescence spectrum removes absolute intensity information; methods developed from normalized fluorescence spectra rely on differences in spectral line shape information for diagnosis. 2) Principal Component Analysis: Principal component analysis (PCA) is a linear model which transforms the original variables of a fluorescence emission spectrum into a smaller set of linear combinations of the original variables called principal components that account for most of the variance of the original data set. Principal component analysis is described in Dillon W.R., Goldstein M., Multivariate Analysis : Methods and Applications, John Wiley and Sons, 1984, pp. 23-52, the disclosure of which is expressly incorporated herein by reference. While PCA may not provide direct insight to the morphologic and biochemical basis of tissue spectra, it provides a novel approach of condensing all the spectral information into a few manageable components, with minimal information loss. Furthermore, each principal component can be easily related to the original emission spectrum, thus providing insight into diagnostically useful emission variables. Prior to PCA, a data matrix is created where each row of the matrix contains the concatenated preprocessed fluorescence spectra of a sample and each column contains the pre-processed fluorescence intensity at each excitation-emission wavelength pair. The data matrix D (r x c) , consisting of r rows (corresponding to r total samples from all patients in the training set) and c columns (corresponding to intensity at c emission- excitation wavelength pairs) can be written as: The first step in PCA is to calculate the covariance matrix, Z. First, each column of the preprocessed data matrix D is mean-scaled. The mean-scaled preprocessed data matrix, D m is then multiplied by its transpose and each element of the resulting square matrix is divided by (r-1) , where r is the total number of samples. The equation for calculating Z is defined as: z = r-1 ( V D« ) (2) The square covariance matrix, Z (c x c) is decomposed into its respective eigenvalues and eigenvectors. Because of experimental error, the total number of eigenvalues will always equal the total number of columns (c) in the data matrix D assuming that c < r. The goal is to select n < c eigenvalues that can describe most of the variance of the original data matrix to within experimental error. The variance, V, accounted for by the first n eigenvalues can be calculated as follows: The criterion used in this analysis was to retain the first n eigenvalues and corresponding eigenvectors that account for 99.9 % of the variance in the original data set. Next, the principal component score matrix can be calculated according to the following equation: R = D C (4) where, D (r x c) is the preprocessed data matrix and C (c x n) is a matrix whose columns contain the n eigenvectors which correspond to the first n eigenvalues. Each row of the score matrix R (r x c) corresponds to the principal component scores of a sample and each column corresponds to a principal component. The principal components are mutually orthogonal to each other. Finally, the component loading is calculated for each principal component. The component loading represents the correlation between the principal component and the variables of the original fluorescence emission spectrum. The component loading can be calculated as shown below: where, CL 13 represents the correlation between the ith variable (preprocessed intensity at ith emission wavelength) and the jth principal component. C 1D is the ith component of the jth eigenvector, λ 3 is the jth eigenvalue and S X1 is the variance of the ith variable. Principal component analysis was performed on each type of preprocessed data matrix, described above. Eigenvalues accounting for 99.9% of the variance in the original preprocessed data set were retained The corresponding eigenvectors were then multiplied by the original data matrix to obtain the principal component score matrix R. 3) Student's T-Test: Average values of principal component scores were calculated for each histopathologic tissue category for each principal component obtained from the preprocessed data matrix. A two-sided unpaired student's t-test was employed to determine the diagnostic contribution of each principal component. Such a test is disclosed in Devore J.L., ProJaJ ϋty and Statistics for Engineering and the Sciences, Brooks/Cole, 1992, and in Walpole R.E., Myers R.H. , Probabili ty and Sta tistics for Engineers and Scientists, Macmillan Publishing Co., 1978, Chapter 7, the disclosures of which are expressly incorporated herein by reference. The hypothesis that the means of the principal component scores of two tissue categories are different were tested for normal smears and abnormal smears (ASCUS, LGSIL, HGSIL) . Principal components were ranked in order of increasing p value. Fisher's discriminant analysis was performed using the most significant principal components and method performance was evaluated. Principal components were then added one at a time and Fisher's discriminant analysis was again performed. This process was repeated until no further improvement was reached or a decrease in performance was noted. Principal components chosen in this manner were used in the diagnostic method. 4) Logistic Discrimination: Logistic discriminant analysis is a statistical technique that may be used to develop diagnostic methods based on posterior probabilities, overcoming the drawback of the binary decision scheme employed in the two-stage method. This statistical classification method is based on Bayes theorem and may be used to calculate the posterior probability that an unknown sample belongs to each of the possible tissue categories identified. Logistic discrimination is discussed in Albert A., Harris E.K., Mul tivariate Interpretation of Clinical Laboratory Data , Marcel Dekker, 1987, the disclosure of which is expressly incorporated herein by reference. Classifying the unknown sample into the tissue category for which its posterior probability is highest results in a classification scheme that minimizes the rate of misclassification. For two diagnostic categories, G_ and G 2 , the posterior probability of being a member of G l t given measurement x, according to Bayes theorem is: P(x|G 1 )P(G 1 )C(2|l) P(G X) = (6) P(x|G 1 )P(G 1 )C(2|l) +P(x|G 2 )P(G 2 )C(l|2) where P(x|Gi) is the conditional joint probability that a tissue sample of type i will have principal component score x, and PfG is the prior probability of finding tissue type i in the sample population. C(j|i) is the cost of misclassifying a sample into group j when the actual membership is group i. The prior probability P(Gi) is an estimate of the likelihood that a sample of type i belongs to a particular group when no information about it is available. If the sample is considered representative of the population, the observed proportions of cases in each group can serve as estimates of the prior probabilities. In a clinical setting, either historical incidence figures appropriate for the patient population can be used to generate prior probabilities, or the practitioner's colposcopic assessment of the likelihood of precancer can be used to estimate prior probabilities, as is known in the art. The conditional probabilities may be developed from the probability distributions of the n principal component scores for each tissue type, i. The probability distributions may be modeled using the gamma function, which is characterized by two parameters, alpha and beta, which are related to the mean and standard deviation of the data set. The normal function is typically used to model probability distributions and is defined below: f(x) = (l/(V / 2π)Σ)e- ,χ - ,n/tv ' 2,J:, (7) The normal distribution function may be used to calculate the conditional probability that a sample from tissue type i, will exhibit the principal component score, x. If more than one principal component is needed to describe a sample population, then the conditional joint probability is simply the product of the conditional probabilities of each principal component (assuming that each principal component is an independent variable) for that sample population. Fisher's discriminant analysis is a particular statistical technique for classifying individuals or objects in to mutually exclusive and exhaustive groups on the basis of a set of independent variables. In this particular application of Fisher's method, the objects are N patient cytological samples, the groups are the diagnostic classifications (normal versus abnormal) and the P variables are the principal components X derived from the fluorescence spectra of the samples. Fisher's method calculates a score Y for each of the samples, based on a linear combination of the variables, i.e., Y(N) = b X tN) + b 2 * X 2 * (N) + ... + b p * X p (N) The coefficients b x through b p are calculated so that the difference between the scores for the normals and the abnormals is maximized. Assuming that the X is normally distributed for the two groups, and assuming that the covariance s of X is the same for the two groups, then the best choice for b-*. is bl = s "1 (avg. of x x of norm. - avg. of x x for abnorm. and similarly for b 2 through b p . Then, a cutoff value for Y is selected and all samples with scores above the threshold are classified as belonging to the first group, normals, and samples with scores below the threshold are classified as belonging to the second group, abnormals. Since there is overlap in the distributions of Y for the two groups, some samples will be misclassified no matter where the cutoff is chosen. The cutoff is chosen to be the one that results in the lowest misclassification rate. The cutoff value given the above assumptions is Y c = (n 2 Y r + n x Y 2 ) / (n-i + n 2 ) where is the number of samples in group 1, and Y x is the Y score using the average values of the X variables for group 1, likewise for group 2. Y c can be adjusted from this value to reduce the FN rate at the expense of the FP rate, or vice versa , depending on the application. Since both the b and Yc values are calculated from the data, it may be asked how well this method will classify new samples, whose values for X were not used in the above-calculations. This performance can be estimated by using cross-validation techniques. For each sample, b and Yc are calculated using the other sample data, and then the method is used to classify that sample. The misclassification error rate for all samples is measured this way is taken as an unbiased estimate of what one can expect when using Fisher's discriminate analysis to classify new samples. Dillon and Goldstein (1985) . II. Examples In order to evaluate the neoplastic diagnostic potential of cellular autofluorescence, excitation of exfoliated, ethanol-fixed cervical squamous epithelial cells with a plurality of wavelengths was performed. In addition, the effects on the fluorescent spectra of different specimen preparation methods were examined using both cell pellet and monolayer preparations made from the same samples. A. Sample Preparation Exfoliated cervical cells were obtained from patients referred to MD Anderson Cancer Center for routine screening, as well as colposcopy, on the basis of previous abnormal cervical cytology. Conventional Pap smears were obtained and the normally discarded cells remaining on the swab suspended in an ethanol-based fixative (PreservCyt Solution, Cytyc Corp., Marlborough, MA) . From each suspension, two types of samples were prepared. First, a monolayer cell touch prep was prepared onto an ordinary glass microscope slide using the CYTYC Thinprep device (Hutchinson, 1992) . This device extracts an aliquot from the suspension and filters it to remove red blood cells and other debris smaller and larger than epithelial cells. The cells were deposited in an circular area 20mm in diameter. The remaining cells in suspension were centrifuged and resuspended three times in HPLC-grade ethanol to remove the fluorescent preservative solution. The number of cells in suspension was determined using a hemocytometer and found to vary between 1 - 50 x 10 4 cells. The cells were then spun down to a pellet, placed on a spot approximately 3 mm in diameter on a quartz microscope slide and air dried. B. Fluorescent Spectroscopy Apparatus and Conditions All fluorescence measurements were made using a standard scanning fluorimeter (SPEX, Fluorolog II, Edison, NJ) with a spectral resolution of 5 nm FWHM. Beam area was approximately 2 mm 2 . Slides were placed so that the cells were on the side facing the beam, and the beam was focused onto this surface of the slide. Excitation light was incident normally and emission was collected at an angle of 20 degrees from the normal. The signal at each (λex,λem) value was integrated for 2 seconds. Data were corrected for the non-uniform spectral response of the emission monochromator and detector using correction factors supplied with the instrument. Also, spectra were corrected for variability with wavelength in the intensity of the excitation source using a Rhodamine B quantum counter (20) . Fluorescence excitation-emission matrices (EEM's) were recorded for each cell pellet sample. Excitation wavelengths ranged from 250 to 550 nm, in 10 nm increments, and emission was measured from 10 nm above the excitation wavelength to 10 nm below the 2nd harmonic of the excitation wavelength, up to 700 nm, in 10 nm increments. A background EEM was recorded from a quartz slide containing supernatant only from the resuspension of one sample. This background EEM was subtracted from each pellet EEM. For several samples, a second emission spectrum was recorded at 250 nm excitation after the initial EEM to check for photobleaching effects. All EEMs were normalized with respect to the intensity at (270,330) . For the ThinPrep samples, one excitation spectrum from 350 nm to 410 nm at 440 nm emission was recorded, and two emission spectra were recorded, at excitations of 280nm and 370nm, using the same increments and spectral ranges as the EEMs above. Since the UV fluorescence of the glass slides differed between slides, a background spectrum was recorded from each slide from a region with no cells. All spectra were normalized with respect to the intensity at (280,330) . C. Pap Smears Conventional Pap smears were read by staff cytopathologists at M.D. Anderson Cancer Center. Diagnosis was done using the standard diagnostic classifications, where the samples are classified as Normal, HPV, CIN I (mild dysplasia) , CIN II (moderate dysplasia) , or CIN III (severe dysplasia or carcinoma in situ) , and Squamous Carcinoma (Vooijs, 1991) . Samples were obtained from 80 patients, of which 36 were not used due to inconclusive diagnoses, samples too dilute, contamination with blood or other foreign matter such as cotton from swabs used in the sample collection, or errors made in the data collection. Of the remaining 44 samples, 15 had conventional Pap smears which were diagnosed as normal (negative for malignant cells) and 29 were read as abnormal (HPV - 14; CINI - 9; CINII - 10; CINIII - 10) . The ages ranged from 20 to 52, with the average age of 33. There were 21 whites, 7 hispanics, 15 blacks, and 1 oriental. For comparison of the pellet to the ThinPrep preparations, 23 samples were selected from the patients mentioned above, of which 10 were normal and 13 abnormal . D. Fixed Cell Fluorescence FIG. 2 is a graph of the average EEM for all 44 pellet prepared samples used. All plots are normalized to the (270,330) values and plotted on a log scale. Contour plot lines are spaced evenly also on a log scale. The most intense fluorescence peak is at (280,330), characteristic of tryptophan. A peak also is present at (370,450) , and a slight shoulder at (280,450) , both characteristic of PN. A shoulder is also present at (250,400) , due to contaminants of unknown origin. There is a slight shoulder near (430,520) due to flavoprotein as well as a valley in the excitation spectra corresponding to the Soret Hb absorption line at λex = 415 nm. No significant photobleaching of the cells was observed. Although the tryptophan and PN peaks were present in all samples, the intensity of the PN and Fp peaks and other features varied greatly among the pellet sample EEM's. FIG. 3 shows an EEM of a sample with the (430,520) peak more pronounced. The Soret absorption band is also clearly visible, while the (250,400) peak is absent completely. For the sample EEM in FIG. 4, the peak at (250,400) is even more intense than the (280,330) tryptophan peak. Table 1 lists these spectral features, and the average and standard deviation for each. All features showed considerable variation among samples, with the magnitude of the standard deviation exceeding the mean in most cases. The (280,330) peak stability can be attributed to the fact that the spectra were normalized to the nearby (270,330) value. The EEM value with the least percent variance was at (300,360) , between the tryptophan and PN peaks . TABLE 1: SPECTRAL FEATURES OF CELL PELLET EEM's * all samples nor. sample abn. samples nor v abn feature (Λem,Λex) μ σ μ σ μ σ P (280,330) 1.18 0.16 1.16 0.16 1.19 0.17 0.56 (250,400) 0.29 0.61 0.33 0.62 0.28 0.62 0.78 (280,450) 0.08 0.09 0.08 0.09 0.08 0.09 0.93 (370,450) 0.23 0.31 0.21 0.29 0.24 0.33 0.75 (430,520) 0.11 0.13 0.09 0.10 0.12 0.15 0.45 (280,450)/ 0.36 0.21 0.40 0.32 0.34 0.28 0.08 (370,450) (300,360) 0.44 0.13 0.39 0.1 1 0.46 0.13 0.04 * - all values normalized to intensity at (270,330) Table 1 also compares population statistics for the normal and abnormal sample groups. For each feature, the variance within each group exceeded the difference between the two groups, as reflected in the high p- values. The lowest p-scores for all values or ratios of values in the EEMs are at the ratio of (280,450)/(370,450) and at (300,360) . In order to test the hypothesis that a combination of several variables may identify significant differences between normal and abnormal cells, principal component analysis (PCA) was used to generate a reduced set of variables which are linear combinations of the EEM values. From each EEM, 25 principal components (PC's) accounting for 99.99% of the variance between all samples were calculated. None of the PC's had a higher statistical significance than any of the features in Table 1. Table 2 provides this comparison. TABLE 2 pc# %VAR mean-nor mean-ab sidβv-nor sidev-ab p-score * 18 0.0193 0.0207 0.0107 0.0507 0.0324 0.041305 * 5 1.4136 -0.1318 0.0682 0.3042 0.3685 0.063457 * 3 4.3008 -0.2137 0.1105 0.5753 0.6279 0.096108 22 0.0047 0.0050 •0.0026 0.0144 0.0229 0.187096 • 16 0.0364 0.0150 •0.0077 0.0491 0.0605 0.188685 23 0.0036 0.0042 ■ 0.0022 0.0169 0.0184 0.25996 • 10 0.1573 0.0284 -0.0147 0.1183 0.1191 0.263165 19 0.0106 0.0074 •0.0038 0.0338 0.0292 0.28409 13 0.0627 0.0146 -0.0076 0.0651 0.0801 0.329235 25 0.0023 0.0032 -0.0016 0.0181 0.0120 0.363534 14 0.0463 0.0122 ■0.0063 0.0663 0.0641 0.382071 12 0.0801 •0.0139 0.0072 0.0946 0.0806 0.469852 15 0.0442 0.0084 -0.0043 0.0694 0.0605 0.553599 8 0.3687 -0.0211 0.0109 0.1569 0.1962 0.561494 7 0.5198 0.0243 -0.0126 0.2173 0.2192 0.599062 1 66.9477 -0.2033 0.1052 2.1941 2.6173 0.682091 21 0.0062 0.0019 •0.0010 0.0226 0.0246 0.695786 17 0.0224 0.0039 -0.0020 0.0534 0.0408 0.707169 9 0.2596 -0.0104 0.0054 0.1533 0.1555 0.75051 pc# %VAR meaπ nor maan-ab sidev-nor sidev-ab p-score 20 0.0079 -0.0013 0.0007 0.0233 0.0288 0.800828 24 0.0031 -0.0006 0.0003 0.0196 0.0156 0.876723 2 22.881 •0.0348 0.0180 1.4811 1.4414 0.910808 4 1.9629 0.0080 -0.0042 0.4774 0.3980 0.933139 pc# %VAR mβan nor mean-ab sidev-nor sidβv-ab p-score 6 0.7267 0.0039 -0.0020 0.2047 0.2826 0.936508 11 0.112 0.0005 ■ 0.0003 0.1313 0.0832 0.983305 * used in Dx algorithm total %VAR used - 5.927% A discriminant function was found using Fisher's discriminant analysis (FDA) , assuming equal prior probabilities and variable cost of misclassification. This function used 5 of the PCs above which together account for 5.93% of the sample-to-sample variance (3,5,10,16,18) . The discriminant score calculated for each sample is plotted in FIG. 5, where samples above Y c are classified as normal, samples below Y c abnormals. The Pap smear diagnosis is shown as well (o - normals, x - abnormals) . The false positive rate (FP) is 2/15 = 13.4%, while the false negative rate (FN) is 12/29 = 41.4%, where the Y c is 0 (solid line) . Another choice is Y c is 0.6, where the FN is 4/29 = 13.8% and the FP is 5/15 = 33.3% (broken line) . A plot of the FN versus FP, obtained by varying Y c in FIG. 5, is shown in FIG. 6, with the diagonal line representing random classification. The expected performance of the discriminant function on additional samples was estimated using cross validation, with the results shown in FIG. 6. E. ThinPrep VB Pellet Autofluorescence FIG. 7 compares ThinPrep and pellet emission spectra from a typical sample for 280 nm excitation. For all samples, the spectra are similar in shape, except that the ThinPrep spectra are blue shifted by an average of 10 nm. In FIG. 8, the spectra at 370 nm excitation are compared for a typical case. Again, the ThinPrep spectrum is blue-shifted with respect to the pellet spectrum. In addition, the intensity at the ThinPrep PN peak around (370,450) is 5- to 10-times lower than the corresponding pellet spectra. The variance between samples is reduced for the ThinPrep spectra, having a lower standard deviation as a percentage of the mean, 58%, compared to 82% for the pellet data. The excitation spectra of the ThinPrep slides recorded at 440 nm emission showed considerable variance below 350 nm excitation due to the strong UV emission of the glass slides. Above 350 nm excitation, the spectra differed from the pellet spectra in the degree of variance between samples and in the intensity, as described above for the (370,450) peak. APPENDIX I: SPECIFICITY AND SENSITIVITY Summarized from: Albert A., Harris E.K. : Multivariate Interpreta ion of Clinical Labora tory Data , Marcel Dekker Inc., New York, pp. 75-82, (1987) , the disclosure of which is expressly incorporated herein by reference. Assuming a group of T samples which can be categorized as normal (N samples) or diseased (D samples) . A diagnostic test, designed to determine whether the sample is normal or diseased, is applied to each sample. The results of the tests is the continuous variable x, which is then used to determine the sample type. FIG. 9 illustrates a hypothetical distribution of test values for each sample type. A diagnostic method based on this test can easily be defined by choosing a cutoff point, d, such that a sample with an observed value x<d is diagnosed as normal and a sample with an observed value x≥d is diagnosed as abnormal . Several quantitative measures have been defined to 'evaluate' the performance of this type of method. The first type evaluates the test itself (i.e., measures the ability of the test to separate the two populations, N and D) . Sensitivity and specificity are two such measures. The second type is designed to aid in the interpretation of a particular test result (i.e. deciding whether the individual test measurement has come from a normal or diseased sample) . Positive and negative predictive value are two measures of this type. To define these measures, some terminology and notation must be introduced. Referring to Table 3, a sample to be tested can be either normal or diseased; the result of the test for each type of sample can be either negative or positive. True negatives represent those normal with a positive test result. In these cases, the diagnosis based on the rest result is correct . False positives are those normal samples which have a positive test result and false negatives are those diseased samples which have a negative test result. In these cases, the diagnosis based on the test result is incorrect . TABLE 3 Normal Diseased Total Samples Test True False Negatives Negative Negatives Negatives (Neg) (x<d) (TN) (FN) Test False True Positives Positive Positives Positives (Pos) (xad) (FP) (TP) Total N D T Samples With this terminology, Table 4 contains a definition of sensitivity and specificity, the two measures which assess the performance of the diagnostic method. Specificity is the proportion of normal samples with a negative test result (proportion of normal samples diagnosed correctly) . Sensitivity is the proportion of diseased samples with a positive test result (Proportion of diseased samples correctly diagnosed) . FIG. 9 also contains a graphical representation of specificity and sensitivity. Specificity represents the area under the normal sample distribution curve to the left of the cut off point while sensitivity represent the area under the diseased sample distribution curve to the right of the cut off point. TABLE 4 Test Meaning Calculation Measure Specificity Proportion of normal Sp=TN/N samples with negative test result Sensitivity Proportion of Se=TP/D diseased samples with positive test result While sensitivity and specificity characterize the performance of a particular method, another set of statistics is required to interpret the laboratory test result for a given specimen. The positive and negative predictive value quantify the meaning of an individual test result (Table 5) . The positive predictive value is the probability that if the test result is positive, the sample is diseased. The negative predictive value is the probability that if the test result is negative, the sample is normal. Positive and negative predictive value are calculated from Baye's rule as outlined in Albert and Harris. Table 5 contains two equivalent formulas for calculation positive and negative predictive value. TABLE 5 Measure Meaning Calculation Calculation 1 2 Positive The probability PV,=TP/Pos PV,=DSe/ (DSe Predic¬ that, if the +N(l-Sp) ) tive test is Value positive, the sample is diseased Negative The probability PV.=TN/Neg PV. Predic¬ that, if the =NSp/ (NSp+D tive test is (1-Se)) Value negative, the sample is normal APPENDIX II: PRINCIPAL COMPONENTS ex(nm) em(nm) evec3 evec5 eveclO evecl6 eveclδ 250 280 0.155 0.353 -0.031 -0.058 -0.055 250 290 0.058 0.097 -0.020 0.077 0.109 250 300 0.024 0.055 -0.033 0.035 0.096 250 310 0.028 0.106 -0.023 0.064 0.186 250 320 0.037 0.153 0.000 0.119 0.168 250 330 0.016 0.188 -0.001 0.052 0.034 250 340 -0.004 0.197 -0.012 -0.003 -0.064 250 350 -0.014 0.187 -0.046 -0.030 -0.140 250 360 -0.015 0.132 -0.051 -0.029 -0.134 250 370 -0.011 0.060 -0.040 -0.010 -0.110 250 380 -0.004 -0.017 -0.013 0.012 -0.039 250 390 -0.003 -0.069 0.019 0.021 -0.010 250 400 -0.005 -0.088 0.049 -0.001 0.010 250 410 -0.010 -0.082 0.051 -0.009 0.006 250 420 -0.012 -0.061 0.046 -0.008 0.061 250 430 -0.013 -0.047 0.044 -0.006 0.059 250 440 -0.013 -0.031 0.038 0.003 0.063 250 450 -0.013 -0.022 0.041 -0.001 0.060 250 460 -0.010 -0.014 0.034 0.006 0.054 260 290 -0.013 0.017 -0.063 -0.046 -0.101 260 300 -0.030 -0.026 -0.102 -0.134 -0.044 260 310 -0.009 0.007 -0.093 -0.019 0.085 260 320 -0.003 0.035 -0.078 0.054 0.160 ex(nm) em(nm) evec3 evec5 eveclO eveclδ eveclβ 260 330 -0.029 0.060 -0.091 0.036 0.052 260 340 -0.045 0.085 -0.121 -0.009 -0.008 260 350 -0.046 0.087 -0.143 -0.031 -0.088 260 360 -0.037 0.066 -0.136 -0.004 -0.119 260 370 -0.029 0.042 -0.115 -0.005 -0.094 260 380 -0.023 0.015 -0.077 -0.006 -0.088 260 390 -0.021 -0.007 -0.039 -0.011 -0.062 260 400 -0.021 -0.021 -0.010 -0.022 -0.048 260 410 -0.023 -0.025 0.010 -0.030 -0.039 260 420 -0.023 -0.023 0.022 -0.031 -0.009 260 430 -0.022 -0.023 0.031 -0.027 -0.024 260 440 -0.021 -0.020 0.037 -0.029 -0.026 260 450 -0.019 -0.018 0.046 -0.028 -0.026 260 460 -0.016 -0.015 0.044 -0.024 -0.032 260 470 -0.014 -0.014 0.044 -0.023 -0.027 260 480 -0.012 -0.011 0.039 -0.020 -0.020 270 300 -0.032 -0.105 -0.059 0.017 -0.173 270 310 0.008 -0.068 -0.036 -0.005 -0.040 270 320 0.026 -0.035 -0.014 0.006 0.028 270 330 0.000 0.000 0.000 0.000 0.000 270 340 -0.020 0.038 -0.025 0.011 0.002 270 350 -0.026 0.046 -0.058 0.073 -0.024 270 360 -0.021 0.035 -0.062 0.124 -0.039 270 370 -0.017 0.017 -0.058 0.140 -0.048 m) em(nm) evec3 evec5 eveclO evecl6 eveclβ 270 380 -0.016 0.004 -0.036 0.115 -0.042 270 390 -0.020 -0.005 -0.009 0.086 -0.035 270 400 -0.024 -0.012 0.011 0.062 -0.038 270 410 -0.030 -0.016 0.027 0.046 -0.042 270 420 -0.031 -0.014 0.040 0.035 -0.011 270 430 -0.031 -0.016 0.051 0.035 -0.038 270 440 -0.030 -0.015 0.059 0.028 -0.040 270 450 -0.027 -0.015 0.066 0.022 -0.042 270 460 -0.023 -0.015 0.064 0.018 -0.048 270 470 -0.020 -0.013 0.060 0.013 -0.048 270 480 -0.017 -0.012 0.055 0.011 -0.037 270 490 -0.015 -0.012 0.048 0.012 -0.024 270 500 -0.013 -0.011 0.042 0.011 -0.008 280 310 0.168 -0.238 -0.057 0.027 -0.099 280 320 0.225 -0.213 -0.037 0.000 -0.067 280 330 0.223 -0.196 -0.046 0.007 -0.052 280 340 0.200 -0.159 -0.089 0.063 -0.006 280 350 0.161 -0.124 -0.136 0.113 0.008 280 360 0.118 -0.089 -0.137 0.126 0.033 280 370 0.079 -0.065 -0.122 0.104 0.028 280 380 0.045 -0.048 -0.091 0.073 0.024 280 390 0.020 -0.040 -0.049 0.042 0.014 280 400 -0.001 -0.035 -0.022 0.024 0.006 280 410 -0.014 -0.030 0.001 0.009 0.011 m) em(nm) evec3 evec5 eveclO evecl6 eveclβ 280 420 -0.020 -0.023 0.018 -0.004 0.031 280 430 -0.022 -0.022 0.031 0.001 0.000 280 440 -0.023 -0.021 0.041 -0.002 -0.009 280 450 -0.021 -0.020 0.048 0.000 -0.012 280 460 -0.018 -0.019 0.051 0.001 -0.024 280 470 -0.016 -0.018 0.049 0.000 -0.031 280 480 -0.014 -0.017 0.046 0.001 -0.032 280 490 -0.012 -0.016 0.042 0.002 -0.018 280 500 -0.010 -0.014 0.037 0.006 -0.011 280 510 -0.009 -0.013 0.032 0.006 -0.005 280 520 -0.008 -0.012 0.029 0.003 -0.005 290 320 0.348 -0.068 0.019 -0.279 0.024 290 330 0.363 -0.066 0.015 -0.231 -0.020 290 340 0.335 -0.053 -0.048 -0.104 0.010 290 350 0.278 -0.040 -0.113 -0.007 0.012 290 360 0.204 -0.029 -0.137 0.057 0.023 290 370 0.140 -0.027 -0.129 0.065 0.017 290 380 0.083 -0.028 -0.097 0.041 0.002 290 390 0.044 -0.030 -0.064 0.015 0.000 290 400 0.016 -0.032 -0.034 -0.007 0.002 290 410 -0.002 -0.030 -0.015 -0.020 0.007 290 420 -0.010 -0.024 0.002 -0.033 0.030 290 430 -0.012 -0.024 0.014 -0.028 0.006 290 440 -0.013 -0.023 0.023 -0.027 -0.005 ex(nm) em(nm) evec3 evec5 eveclO evecl6 eveclθ 290 450 -0.012 -0.023 0.033 -0.021 -0.012 290 460 -0.010 -0.023 0.036 -0.016 -0.026 290 470 -0.009 -0.022 0.036 -0.013 -0.027 290 480 -0.007 -0.020 0.036 -0.011 -0.025 290 490 -0.006 -0.019 0.034 -0.007 -0.024 290 500 -0.005 -0.019 0.030 -0.003 -0.021 290 510 -0.004 -0.018 0.028 -0.001 -0.018 290 520 -0.004 -0.017 0.026 -0.001 -0.012 290 530 -0.003 -0.016 0.024 -0.004 -0.011 290 540 -0.003 -0.015 0.022 -0.003 -0.007 300 330 0.230 0.211 0.331 -0.028 0.004 300 340 0.221 0.226 0.276 0.058 -0.019 300 350 0.184 0.212 0.168 0.105 -0.024 300 360 0.136 0.171 0.081 0.104 -0.021 300 370 0.093 0.125 0.021 0.075 -0.019 300 380 0.054 0.077 -0.006 0.030 -0.008 300 390 0.027 0.041 -0.015 -0.007 -0.005 300 400 0.007 0.012 -0.017 -0.035 -0.006 300 410 -0.005 -0.002 -0.017 -0.049 -0.008 300 420 -0.010 -0.007 -0.010 -0.057 0.015 300 430 -0.010 -0.012 -0.005 -0.048 -0.010 300 440 -0.009 -0.013 0.001 -0.045 -0.013 300 450 -0.007 -0.015 0.010 -0.037 -0.015 300 460 -0.005 -0.016 0.014 -0.028 -0.026 ex(nm) em(nm) evec3 evec5 eveclO eveclδ eveclβ 300 470 -0.003 -0.016 0.017 -0.024 -0.031 300 480 -0.002 -0.016 0.019 -0.019 -0.028 300 490 0.000 -0.016 0.019 -0.013 -0.026 300 500 0.000 -0.016 0.019 -0.008 -0.021 300 510 0.001 -0015 0.018 -0.006 -0.024 300 520 0.001 -0.015 0.018 -0.006 -0.018 300 530 0.001 -0.014 0.018 -0.007 -0.018 300 540 0.001 -0.013 0.016 -0.007 -0.015 300 550 0.001 -0.012 0.016 -0.007 -0.015 300 560 0.001 -0.010 0.013 -0.007 -0.015 310 340 0.004 0.052 0.029 -0.010 -0.021 310 350 -0.002 0.058 -0.003 -0.035 -0.030 310 360 -0.006 0.051 -0.026 -0.055 -0.010 310 370 -0.009 0.039 -0.042 -0.073 -0.003 310 380 -0.012 0.025 -0.047 -0.084 0.020 310 390 -0.013 0.012 -0.043 -0.088 0.025 310 400 -0.015 0.000 -0.042 -0.080 0.008 310 410 -0.017 -0.007 -0.039 -0.077 -0.002 310 420 -0.017 -0.007 -0.029 -0.073 0.017 310 430 -0.014 -0.012 -0.023 -0.054 -0.019 310 440 -0.012 -0.011 -0.016 -0.047 -0.025 310 450 -0.008 -0.012 -0.006 -0.036 -0.024 310 460 -0.004 -0.013 0.001 -0.024 -0.037 310 470 -0.002 -0.012 0.004 -0.016 -0.038 ex(nm) em(nm) evec3 evec5 eveclO evecl6 eveclβ 310 480 0.000 -0.012 0.007 -0.009 -0.037 310 490 0.001 -0.012 0.009 -0.003 -0.035 310 500 0.002 -0.012 0.009 0.002 -0.032 310 510 0.003 -0.011 0.011 0.004 -0.027 310 520 0.003 -0.011 0.011 0.004 -0.024 310 530 0.003 -0.011 0.013 0.003 -0.023 310 540 0.003 -0.010 0.012 0.002 -0.022 310 550 0.003 -0.009 0.011 0.001 -0.018 310 560 0.003 -0.008 0.009 0.002 -0.016 310 570 0.002 -0.007 0.008 0.001 -0.016 310 580 0.002 -0.006 0.006 0.000 -0.014 320 350 -0.019 0.017 -0.041 -0.101 -0.022 320 360 -0.020 0.015 -0.054 -0.129 0.000 320 370 -0.020 0.013 -0.060 -0.141 0.011 320 380 -0.020 0.007 -0.059 -0.138 0.040 320 390 -0.019 0.000 -0.053 -0.125 0.045 320 400 -0.018 -0.008 -0.047 -0.101 0.023 320 410 -0.019 -0.014 -0.045 -0.084 0.004 320 420 -0.018 -0.013 -0.034 -0.071 0.028 320 430 -0.013 -0.017 -0.029 -0.044 -0.018 320 440 -0.010 -0.016 -0.022 -0.031 -0.025 320 450 -0.006 -0.016 -0.010 -0.018 -0.025 320 460 -0.002 -0.017 -0.005 -0.005 -0.040 320 470 0.000 -0.015 -0.001 0.001 -0.041 m) em(nm) evec3 evec5 eveclO evecl6 eveclθ 320 480 0.002 -0.013 0.003 0.005 -0.036 320 490 0.003 -0.013 0.005 0.013 -0.035 320 500 0.004 -0.012 0.005 0.016 -0.030 320 510 0.004 -0.012 0.007 0.017 -0.026 320 520 0.004 -0.011 0.008 0.016 -0.024 320 530 0.004 -0.010 0.009 0.014 -0.021 320 540 0.004 -0.009 0.009 0.011 -0.023 320 550 0.004 -0.008 0.008 0.010 -0.020 320 560 0.003 -0.008 0.007 0.008 -0.018 320 570 0.003 -0.007 0.006 0.007 -0.016 320 580 0.003 -0.006 0.005 0.005 -0.014 320 590 0.002 -0.005 0.004 0.004 -0.015 320 600 0.002 -0.004 0.003 0.004 -0.012 330 360 -0.015 0.013 -0.046 -0.096 0.010 330 370 -0.016 0.011 -0.053 -0.109 0.031 330 380 -0.016 0.007 -0.053 -0.107 0.071 330 390 -0.016 -0.001 -0.046 -0.099 0.076 330 400 -0.016 -0.010 -0.046 -0.075 0.036 330 410 -0.018 -0.017 -0.047 -0.058 0.000 330 420 -0.017 -0.016 -0.033 -0.049 0.033 330 430 -0.013 -0.022 -0.029 -0.017 -0.028 330 440 -0.009 -0.021 -0.022 -0.006 -0.028 330 450 -0.005 -0.020 -0.008 0.004 -0.022 330 460 -0.001 -0.021 -0.003 0.017 -0.042 m) em(nm) evec3 evec5 eveclO evecl6 eveclθ 330 470 0.002 -0.018 0.001 0.020 -0.038 330 480 0.003 -0.016 0.004 0.023 -0.028 330 490 0.005 -0.015 0.005 0.029 -0.025 330 500 0.006 -0.014 0.006 0.031 -0.024 330 510 0.006 -0.013 0.006 0.030 -0.020 330 520 0.006 -0.012 0.007 0.028 -0.013 330 530 0.005 -0.011 0.009 0.023 -0.014 330 540 0.005 -0.010 0.009 0.020 -0.013 330 550 0.004 -0.009 0.008 0.016 -0.012 330 560 0.004 -0.007 0.007 0.013 -0.012 330 570 0.004 -0.006 0.005 0.011 -0.009 330 580 0.003 -0.006 0.005 0.008 -0.013 330 590 0.003 -0.004 0.003 0.006 -0.011 330 600 0.003 -0.003 0.002 0.006 -0.011 330 610 0.002 -0.003 0.002 0.005 -0.010 330 620 0.002 -0.002 0.001 0.005 -0.009 340 370 -0.005 0.005 -0.032 -0.029 0.062 340 380 -0.007 0.004 -0.035 -0.030 0.114 340 390 -0.009 -0.001 -0.029 -0.025 0.130 340 400 -0.011 -0.011 -0.034 -0.006 0.063 340 410 -0.015 -0.018 -0.042 0.005 0.001 340 420 -0.015 -0.017 -0.023 0.007 0.049 340 430 -0.010 -0.024 -0.020 0.039 -0.041 340 440 -0.006 -0.023 -0.012 0.042 -0.036 ex(nm) em(nm) evec3 evec5 eveclO evecl6 eveclδ 340 450 -0.001 -0.023 0.007 0.048 -0.020 340 460 0.004 -0.023 0.010 0.057 -0.044 340 470 0.006 -0.020 0.012 0.051 -0.039 340 480 0.008 -0.017 0.016 0.050 -0.022 340 490 0.009 -0.016 0.016 0.050 -0.022 340 500 0.009 -0.014 0.014 0.048 -0.013 340 510 0.009 -0.012 0.013 0.043 -0.006 340 520 0.009 -0.011 0.013 0.039 -0.004 340 530 0.008 -0.010 0.013 0.032 -0.002 340 540 0.007 -0.008 0.012 0.026 -0.004 340 550 0.006 -0.007 0.011 0.021 -0.003 340 560 0.005 -0.006 0.010 0.016 -0.006 340 570 0.005 -0.005 0.007 0.013 -0.007 340 580 0.004 -0.004 0.005 0.012 -0.008 340 590 0.004 -0.003 0.004 0.009 -0.007 340 600 0.003 -0.002 0.002 0.007 -0.008 340 610 0.003 -0.002 0.002 0.007 -0.009 340 620 0.003 -0.001 0.002 0.006 -0.010 340 630 0.003 0.000 0.001 0.006 -0.011 340 640 0.002 0.000 0.001 0.004 -0.011 350 3β0 -0.004 0.003 -0.025 0.007 0.119 350 390 -0.005 -0.002 -0.016 0.010 0.154 350 400 -0.007 -0.011 -0.031 0.034 0.082 350 410 -0.013 -0.019 -0.047 0.038 0.016 ex(nm) em (nm) evec3 evec5 eveclO eveclδ eveclβ 350 420 -0.016 -0.017 -0.022 0.029 0.088 350 430 -0.010 -0.027 -0.016 0.071 -0.028 350 440 -0.006 -0.025 -0.007 0.065 -0.005 350 450 0.001 -0.026 0.017 0.070 0.019 350 460 0.007 -0.027 0.021 0.078 -0.008 350 470 0.009 -0.023 0.020 0.068 0.003 350 480 0.011 -0.019 0.024 0.062 0.022 350 490 0.012 -0.017 0.022 0.062 0.023 350 500 0.012 -0.015 0.018 0.059 0.028 350 510 0.011 -0.012 0.015 0.051 0.035 350 520 0.011 -0.011 0.014 0.043 0.035 350 530 0.009 -0.009 0.013 0.036 0.029 350 540 0.008 -0.007 0.012 0.028 0.026 350 550 0.008 -0.006 0.009 0.022 0.023 350 560 0.006 -0.004 0.007 0.017 0.016 350 570 0.006 -0.003 0.006 0.013 0.012 350 580 0.005 -0.003 0.004 0.010 0.007 350 590 0.004 -0.002 0.002 0.008 0.004 350 600 0.004 -0.001 0.001 0.007 0.003 350 610 0.004 -0.001 0.000 0.006 -0.001 350 620 0.003 0.000 0.000 0.005 -0.005 350 630 0.003 0.001 0.000 0.005 -0.004 350 640 0.003 0.001 0.000 0.004 -0.007 350 650 0.003 0.001 -0.001 0.004 -0.008 ex(nm) em(nm) evec3 evec5 eveclO evecl6 eveclβ 350 660 0.002 0.001 -0.001 0.004 -0.009 360 390 -0.005 -0.002 -0.004 0.012 0.131 360 400 -0.008 -0.012 -0.025 0.033 0.051 360 410 -0.016 -0.016 -0.058 0.025 -0.019 360 420 -0.021 -0.012 -0.026 0.007 0.093 360 430 -0.013 -0.025 -0.017 0.059 -0.039 360 440 -0.010 -0.021 -0.009 0.051 -0.010 360 450 -0.001 -0.024 0.024 0.062 0.035 360 460 0.007 -0.028 0.032 0.079 0.002 360 470 0.010 -0.022 0.030 0.067 0.015 360 480 0.012 -0.018 0.034 0.061 0.043 360 490 0.014 -0.017 0.031 0.063 0.050 360 500 0.014 -0.014 0.023 0.060 0.054 360 510 0.013 -0.011 0.018 0.050 0.064 360 520 0.012 -0.009 0.015 0.042 0.062 360 530 0.011 -0.007 0.013 0.034 0.058 360 540 0.010 -0.006 0.010 0.026 0.050 360 550 0.008 -0.004 0.007 0.020 0.045 360 560 0.007 -0.003 0.005 0.015 0.034 360 570 0.006 -0.002 0.004 0.011 0.027 360 580 0.006 -0.001 0.001 0.008 0.022 360 590 0.005 -0.001 0.000 0.005 0.015 360 600 0.004 0.000 -0.001 0.004 0.010 360 610 0.004 0.001 -0.002 0.005 0.06 ex(nm) em(nm) evec3 evec5 eveclO eveclδ eveclβ 360 620 0.003 0.001 -0.001 0.004 0.002 360 630 0.003 0.002 -0.001 0.004 -0.001 360 640 0.003 0.002 -0.001 0.004 -0.003 360 650 0.003 0.002 -0.002 0.005 -0.005 360 660 0.003 0.002 -0.002 0.004 -0.006 360 670 0.002 0.002 -0.002 0.005 -0.008 360 680 0.002 0.002 -0.002 0.004 -0.007 370 400 -0.007 -0.011 -0.031 0.021 -0.018 370 410 -0.017 -0.013 -0.073 -0.002 -0.093 370 420 -0.024 -0.005 -0.039 -0.038 0.052 370 430 -0.016 -0.021 -0.024 0.022 -0.085 370 440 -0.014 -0.016 -0.010 0.010 -0.044 370 450 -0.004 -0.020 0.030 0.026 0.005 370 460 0.007 -0.026 0.042 0.056 -0.023 370 470 0.010 -0.020 0.041 0.047 0.002 370 480 0.012 -0.016 0.044 0.044 0.038 370 490 0.015 -0.015 0.039 0.051 0.045 370 500 0.015 -0.012 0.029 0.049 0.058 370 510 0.014 -0.009 0.023 0.042 0.074 ex(nm) em(nm) evec3 evec5 eveclO eveclδ eveclβ 370 520 0.013 -0.007 0.017 0.033 0.073 370 530 0.011 -0.005 0.013 0.026 0.068 370 540 0.010 -0.003 0.009 0.018 0.061 370 550 0.009 -0.001 0.005 0.014 0.053 370 560 0.008 0.000 0.002 0.008 0.045 370 570 0.007 0.000 0.001 0.006 0.038 370 580 0.006 0.001 -0.002 0.003 0.028 370 590 0.005 0.001 -0.002 0.001 0.023 370 600 0.004 0.002 -0.003 0.002 0.017 370 610 0.004 0.002 -0.003 0.002 0.011 370 620 0.004 0.003 -0.004 0.001 0.007 370 630 0.004 0.002 -0.004 0.002 0.002 370 640 0.003 0.003 -0.003 0.002 -0.003 370 650 0.003 0.003 -0.003 0.002 -0.003 370 660 0.003 0.003 -0.003 0.003 -0.005 370 670 0.002 0.003 -0.003 0.003 -0.006 370 680 0.002 0.003 -0.003 0.003 -0.006 370 690 0.002 0.003 -0.003 0.003 -0.007 380 410 -0.017 -0.003 -0.088 -0.035 -0.123 380 420 -0.026 0.008 -0.053 -0.089 0.040 380 430 -0.017 -0.010 -0.034 -0.030 -0.080 380 440 -0.017 -0.002 -0.021 -0.049 -0.043 380 450 -0.007 -0.008 0.028 -0.028 0.004 380 460 0.005 -0.017 0.044 0.012 -0.023 m) em(nm) evec3 evec5 eveclO evecl6 eveclθ 380 470 0.008 -0.011 0.042 0.008 0.002 380 480 0.011 -0.008 0.047 0.012 0.033 3Θ0 490 0.014 -0.009 0.045 0.025 0.040 380 500 0.014 -0.007 0.031 0.025 0.058 380 510 0.013 -0.004 0.024 0.020 0.071 380 520 0.012 -0.003 0.019 0.017 0.074 380 530 0.011 -0.002 0.014 0.013 0.070 380 540 0.010 0.000 0.009 0.007 0.063 380 550 0.008 0.001 0.005 0.002 0.056 380 560 0.007 0.002 0.002 -0.001 0.048 3Θ0 570 0.006 0.003 0.000 -0.003 0.040 380 580 0.005 0.003 -0.002 -0.004 0.031 380 590 0.005 0.003 -0.004 -0.005 0.026 380 600 0.004 0.003 -0.004 -0.004 0.019 380 610 0.004 0.003 -0.005 -0.002 0.013 380 620 0.003 0.003 -0.005 -0.002 o.ooβ 380 630 0.003 0.003 -0.004 -0.002 0.003 380 640 0.003 0.003 -0.004 -0.001 -0.001 3Θ0 650 0.003 0.003 -0.003 -0.001 -0.005 380 660 0.002 0.003 -0.004 0.000 -0.004 380 670 0.002 0.003 -0.003 0.001 -0.006 380 680 0.002 0.003 -0.002 0.001 -0.007 380 690 0.002 0.003 -0.003 0.001 -0.007 390 420 -0.026 0.019 -0.054 -0.118 0.022 ex(nm) em(nm) evec3 evec5 eveclO evecl6 eveclβ 390 430 -0.016 0.001 -0.031 -0.069 -0.063 390 440 -0.017 0.009 -0.016 -0.094 -0.027 390 450 -0.008 0.004 0.029 -0.074 0.006 390 460 0.004 -0.006 0.047 -0.030 -0.025 390 470 0.006 -0.001 0.047 -0.029 -0.005 390 480 0.009 0.001 0.050 -0.022 0.017 390 490 0.012 -0.001 0.045 -0.005 0.029 390 500 0.012 0.000 0.034 0.000 0.040 390 510 0.012 0.001 0.026 -0.002 0.055 390 520 0.011 0.002 0.020 -0.004 0.059 390 530 0.010 0.003 0.015 -0.005 0.058 390 540 0.008 0.004 0.010 -0.009 0.053 390 550 0.007 0.005 0.006 -0.011 0.048 390 560 0.006 0.005 0.002 -0.012 0.043 390 570 0.005 0.005 0.000 -0.012 0.036 390 580 0.005 0.005 -0.002 -0.013 0.030 390 590 0.004 0.004 -0.003 -0.012 0.025 390 600 0.004 0.004 -0.004 -0.010 0.020 390 610 0.003 0.004 -0.005 -0.009 0.016 390 620 0.003 0.004 -0.004 -0.006 0.011 390 630 0.003 0.004 -0.004 -0.005 0.002 390 640 0.003 0.004 -0.003 -0.003 -0.001 390 650 0.003 0.004 -0.002 -0.003 -0.004 390 660 0.002 0.004 -0.003 -0.002 -0.004 ex(nm) em (nm) evec3 evec5 eveclO evecl6 eveclβ 390 670 0.002 0.003 -0.003 -0.001 -0.005 390 680 0.002 0.003 -0.002 0.000 -0.006 390 690 0.002 0.004 -0.003 -0.001 -0.004 400 430 -0.018 0.017 -0.038 -0.099 -0.028 400 440 -0.020 0.024 -0.024 -0.130 -0.003 400 450 -0.012 0.017 0.020 -0.112 0.018 400 460 0.000 0.008 0.038 -0.068 -0.011 400 470 0.003 0.011 0.038 -0.063 -0.001 400 480 0.006 0.011 0.042 -0.052 0.018 400 490 0.009 0.008 0.040 -0.033 0.026 400 500 0.010 0.008 0.030 -0.024 0.041 400 510 0.010 0.009 0.023 -0.024 0.054 400 520 0.009 0.008 0.017 -0.022 0.061 400 530 0.008 0.008 0.013 -0.022 0.058 400 540 0.007 0.008 0.009 -0.024 0.055 400 550 0.006 0.008 0.005 -0.024 0.054 400 560 0.005 0.008 0.002 -0.023 0.045 400 570 0.004 0.008 0.000 -0.021 0.039 400 580 0.004 0.007 -0.002 -0.021 0.035 400 590 0.003 0.006 -0.003 -0.019 0.029 400 600 0.003 0.006 -0.004 -0.016 0.024 400 610 0.002 0.005 -0.004 -0.013 0.019 400 620 0.003 0.005 -0.004 -0.012 0.012 400 630 0.002 0.005 -0.002 -0.009 0.007 ex(nm) em(nm) evec3 evec5 eveclO evecl6 eveclθ 400 640 0.003 0.004 -0.002 -0.008 0.002 400 650 0.003 0.005 -0.002 -0.005 -0.002 400 660 0.002 0.004 -0.001 -0.004 -0.003 400 670 0.002 0.004 -0.002 -0.002 -0.004 400 680 0.002 0.004 -0.002 -0.002 -0.005 400 690 0.002 0.004 -0.003 -0.001 -0.005 410 440 -0.020 0.035 -0.003 -0.141 0.070 410 450 -0.013 0.029 0.031 -0.129 0.039 410 460 -0.002 0.019 0.050 -0.087 0.006 410 470 0.001 0.022 0.047 -0.082 0.000 410 480 0.005 0.021 0.047 -0.068 -0.004 410 490 0.008 0.017 0.042 -0.046 0.003 410 500 0.009 0.016 0.033 -0.037 0.019 410 510 0.009 0.016 0.025 -0.034 0.033 410 520 0.008 0.015 0.019 -0.031 0.040 410 530 0.007 0.014 0.013 -0.030 0.043 410 540 0.006 0.013 0.009 -0.031 0.043 410 550 0.005 0.012 0.005 -0.029 0.041 410 560 0.005 0.012 0.003 -0.029 0.038 410 570 0.004 0.010 0.001 -0.027 0.030 410 580 0.003 0.009 -0.001 -0.025 0.029 410 590 0.003 0.008 -0.002 -0.022 0.022 410 600 0.002 0.007 -0.003 -0.019 0.020 410 610 0.002 0.006 -0.003 -0.017 0.015 m) em(nm) evec3 evec5 eveclO evecl6 eveclθ 410 620 0.002 0.006 -0.003 -0.014 0.011 410 630 0.002 0.005 -0.003 -0.011 0.004 410 640 0.002 0.005 -0.002 -0.010 0.001 410 650 0.002 0.005 -0.002 -0.007 -0.001 410 660 0.002 0.004 -0.002 -0.006 -0.004 410 670 0.002 0.004 -0.002 -0.004 -0.004 410 6Θ0 0.002 0.004 -0.002 -0.002 -0.005 410 690 0.002 0.004 -0.002 -0.003 -0.005 420 450 0.001 0.013 0.052 -0.056 -0.036 420 460 o.ooβ 0.013 0.060 -0.043 -0.063 420 470 0.011 0.017 0.058 -0.039 -0.074 420 480 0.013 0.019 0.051 -0.035 -0.073 420 490 0.015 0.020 0.041 -0.026 -0.056 420 500 0.015 0.021 0.029 -0.020 -0.031 420 510 0.014 0.021 0.020 -0.019 -0.012 420 520 0.012 0.021 0.012 -0.021 0.007 420 530 0.011 0.020 0.008 -0.025 0.015 420 540 0.009 0.019 0.003 -0.027 0.021 420 550 0.008 0.018 0.000 -0.028 0.023 420 560 0.007 0.017 -0.003 -0.028 0.022 420 570 0.006 0.015 -0.004 -0.028 0.020 420 580 0.005 0.013 -0.005 -0.026 0.018 420 590 0.004 0.012 -0.006 -0.022 0.017 420 600 0.003 0.010 -0.007 -0.021 0.015 ex(nm) em(nm) evec3 evec5 eveclO eveclδ eveclβ 420 610 0.003 0.009 -0.006 -0.018 0.012 420 620 0.003 0.008 -0.006 -0.016 0.009 420 630 0.002 0.007 -0.005 -0.012 0.005 420 640 0.003 0.006 -0.003 -0.010 0.001 420 650 0.002 0.006 -0.003 -0.007 0.000 420 660 0.002 0.005 0.003 -0.006 -0.002 420 670 0.002 0.005 -0.003 -0.005 -0.003 420 680 0.002 0.004 -0.003 -0.004 -0.003 420 690 0.001 0.004 -0.003 -0.002 -0.005 430 460 0.013 0.010 0.050 -0.012 -0.121 430 470 0.017 0.017 0.046 -0.010 -0.143 430 480 0.019 0.022 0.038 -0.006 -0.136 430 490 0.020 0.025 0.025 -0.001 -0.115 430 500 0.020 0.027 0.012 0.003 -0.089 430 510 0.019 0.028 0.004 0.001 -0.064 430 520 0.018 0.028 -0.003 -0.004 -0.040 430 530 0.015 0.027 -0.008 -0.009 -0.024 430 540 0.013 0.027 -0.011 -0.014 -0.016 430 550 0.012 0.025 -0.014 -0.016 -0.004 430 560 0.010 0.023 -0.016 -0.018 -0.003 430 570 0.009 0.021 -0.017 -0.017 -0.003 430 580 0.007 0.018 -0.016 -0.018 0.002 430 590 0.006 0.016 -0.016 -0.016 0.002 430 600 0.005 0.014 -0.015 -0.015 0.003 ex(nm) em(nm) evec3 evecδ eveclO eveclδ eveclβ 430 610 0.005 0.013 -0.015 -0.012 0.003 430 620 0.004 0.011 -0.013 -0.011 0.001 430 630 0.004 0.010 -0.012 -0.007 0.000 430 640 0.003 0.009 -0.009 -0.006 -0.003 430 650 0.003 0.007 -0.009 -0.004 -0.004 430 660 0.003 0.007 -0.008 -0.002 -0.006 430 670 0.003 0.006 -0.007 -0.002 -0.005 430 680 0.002 0.005 -0.006 0.001 -0.006 430 690 0.002 0.005 -0.006 0.000 -0.006 440 470 0.014 0.018 0.025 0.000 -0.127 440 480 0.018 0.023 0.016 0.006 -0.131 440 490 0.020 0.027 0.005 0.013 -0.116 440 500 0.020 0.030 -0.005 0.018 -0.094 404 510 0.020 0.032 -0.012 0.017 -0.079 440 520 0.019 0.032 -0.019 0.013 -0.056 440 530 0.017 0.032 -0.022 0.006 -0.040 440 540 0.015 0.031 -0.023 0.000 -0.029 440 550 0.014 0.029 -0.024 -0.002 -0.022 440 560 0.012 0.027 -0.026 -0.006 -0.017 440 570 0.011 0.024 -0.024 -0.007 -0.016 440 580 0.009 0.022 -0.024 -0.006 -0.012 440 590 0.008 0.019 -0.022 -0.005 -0.010 440 600 0.007 0.017 -0.022 -0.003 -0.004 440 610 0.006 0.015 -0.020 0.003 -0.004 ex(nm) em(nm) evec3 evec5 eveclO evecl6 eveclβ 440 620 0.005 0.013 -0.017 -0.001 -0.004 440 630 0.005 0.012 -0.017 0.000 -0.003 440 640 0.004 0.010 -0.014 0.002 -0.003 440 650 0.004 0.008 -0.013 0.003 -0.003 440 660 0.003 0.008 -0.011 0.004 -0.002 440 670 0.003 0.007 -0.010 0.004 -0.004 440 680 0.003 0.007 -0.010 0.005 -0.002 440 690 0.002 0.006 -0.009 0.005 -0.002 450 480 0.013 0.021 -0.001 0.009 -0.082 450 490 0.016 0.026 -0.010 0.017 -0.079 450 500 0.018 0.030 -0.019 0.024 -0.067 450 510 0.019 0.033 -0.025 0.025 -0.055 450 520 0.018 0.035 -0.028 0.020 -0.045 450 530 0.017 0.035 -0.029 0.013 -0.035 450 540 0.015 0.034 -0.031 0.007 -0.024 450 550 0.014 0.033 -0.032 0.003 -0.019 450 560 0.013 0.031 -0.031 0.002 -0.017 450 570 0.011 0.028 -0.030 0.001 -0.015 450 580 0.010 0.025 -0.029 0.000 -0.012 450 590 0.009 0.022 -0.028 0.001 -0.009 450 600 0.008 0.020 -0.026 0.002 -0.007 450 610 0.007 0.017 -0.024 0.003 -0.006 450 620 0.006 0.015 -0.022 0.004 -0.004 450 630 0.005 0.013 -0.021 0.005 -0.001 ex(nm) em(nm) evec3 evec5 eveclO evecl6 eveclβ 450 640 0.004 0.011 -0.018 0.006 -0.003 450 650 0.004 0.010 0.016 0.005 -0.003 450 660 0.003 0.009 -0.014 0.006 -0.004 450 670 0.003 0.008 -0.012 0.007 -0.003 450 6Θ0 0.003 0.007 -0.011 0.007 -0.004 450 690 0.002 0.006 -0.010 0.006 -0.003 460 490 0.013 0.023 -0.024 0.024 -0.050 460 500 0.015 0.028 -0.033 0.032 -0.049 460 510 0.017 0.033 -0.039 0.035 -0.044 460 520 0.017 0.035 -0.042 0.030 -0.039 460 530 0.016 0.037 -0.044 0.022 -0.034 460 540 0.016 0.037 -0.045 0.017 -0.027 460 550 0.015 0.035 -0.045 0.014 -0.025 460 560 0.014 0.034 -0.044 0.012 -0.022 460 570 0.012 0.031 -0.043 0.011 -0.023 460 580 0.011 0.028 -0.042 0.010 -0.021 460 590 0.010 0.025 -0.039 0.011 -0.018 460 600 0.009 0.022 -0.037 0.012 -0.013 460 610 0.008 0.020 -0.035 0.013 -0.011 460 620 0.007 0.017 -0.031 0.013 -0.006 460 630 0.006 0.015 -0.028 0.014 -0.008 460 640 0.005 0.013 -0.025 0.013 -0.007 460 650 0.005 0.011 -0.022 0.012 -0.006 460 660 0.004 0.010 -0.020 0.012 -0.003 ex(nm) em(nm) evec3 evec5 eveclO evecl6 eveclδ 460 670 0.004 0.009 -0.018 0.011 -0.008 460 680 0.003 0.008 -0.016 0.011 -0.004 460 690 0.002 0.007 -0.012 0.009 0.004 470 500 0.011 0.024 -0.029 0.025 -0.007 470 510 0.012 0.030 -0.034 0.025 -0.005 470 520 0.013 0.035 -0.039 0.021 -0.002 470 530 0.013 0.037 -0.040 0.013 0.003 470 540 0.013 0.038 -0.042 0.008 0.003 470 550 0.012 0.038 -0.042 0.006 0.002 470 560 0.012 0.036 -0.043 0.006 0.001 470 570 0.011 0.034 -0.043 0.006 -0.002 470 580 0.010 0.031 -0.041 0.007 -0.001 470 590 0.009 0.028 -0.040 0.009 -0.002 470 600 0.009 0.025 -0.038 0.011 0.001 470 610 0.008 0.023 -0.035 0.014 0.002 470 620 0.007 0.020 -0.033 0.014 0.002 470 630 0.006 0.017 -0.029 0.015 0.003 470 640 0.005 0.015 -0.027 0.016 0.001 470 650 0.005 0.013 -0.024 0.016 -0.001 470 660 0.004 0.011 -0.021 0.015 0.003 470 670 0.004 0.010 -0.019 0.013 0.000 470 680 0.003 0.009 -0.016 0.012 0.000 470 690 0.003 0.007 -0.014 0.012 -0.002 480 510 0.009 0.024 -0.031 0.024 -0.003 ex(nm) em(nm) evec3 evec5 eveclO evecl6 eveclβ 480 520 0.010 0.031 -0.037 0.017 -0.005 480 530 0.011 0.036 -0.040 0.011 -0.007 480 540 0.011 0.038 -0.043 0.005 -0.009 480 550 0.011 0.038 -0.046 0.006 -0.010 480 560 0.011 0.038 -0.047 0.008 -0.013 480 570 0.011 0.036 -0.047 0.008 -0.014 480 580 0.010 0.033 -0.046 0.010 -0.016 480 590 0.010 0.030 -0.046 0.014 -0.011 480 600 0.009 0.028 -0.043 0.018 -0.011 480 610 0.008 0.025 -0.041 0.020 -0.011 480 620 0.008 0.022 -0.038 0.022 -0.009 480 630 0.007 0.019 -0.034 0.022 -0.007 480 640 0.006 0.017 -0.031 0.022 -0.006 480 650 0.005 0.015 -0.028 0.020 -0.005 480 660 0.005 0.012 -0.025 0.020 -0.005 480 670 0.004 0.011 -0.022 0.018 -0.003 480 680 0.004 0.010 -0.018 0.015 -0.004 480 690 0.003 0.009 -0.017 0.015 -0.003 490 520 0.006 0.026 -0.031 0.008 0.010 490 530 0.007 0.033 -0.036 -0.001 0.010 490 540 0.008 0.037 -0.040 -0.005 0.005 490 550 0.008 0.039 -0.045 -0.003 0.004 490 560 0.009 0.038 -0.04Θ 0.001 0.000 490 570 0.009 0.037 -0.050 0.004 -0.006 ex(nm) em(nm) evec3 evecδ eveclO evecl6 eveclβ 490 580 0.009 0.034 -0.049 0.009 -0.006 490 590 0.009 0.032 -0.048 0.012 -0.006 490 600 0.008 0.029 -0.047 0.017 -0.007 490 610 0.008 0.026 -0.046 0.022 -0.004 490 620 0.007 0.023 -0.042 0.023 -0.004 490 630 0.007 0.021 -0.039 0.024 -0.003 490 640 0.006 0.018 -0.035 0.025 -0.003 490 650 0.006 0.016 -0.032 0.024 -0.003 490 660 0.005 0.014 -0.029 0.023 -0.001 490 670 0.004 0.012 -0.027 0.023 -0.001 490 680 0.004 0.011 -0.021 0.019 -0.002 490 690 0.004 0.010 -0.021 0.019 -0.001 500 530 0.002 0.030 -0.024 -0.017 0.026 500 540 0.004 0.035 -0.029 -0.022 0.026 500 550 0.005 0.038 -0.035 -0.020 0.021 500 560 0.006 0.038 -0.041 -0.014 0.015 500 570 0.007 0.038 -0.044 -0.010 0.012 500 580 0.007 0.035 -0.044 -0.003 0.009 500 590 0.007 0.033 -0.044 0.004 0.008 500 600 0.007 0.030 -0.044 0.010 0.007 500 610 0.007 0.028 -0.043 0.014 0.010 500 620 0.006 0.025 -0.041 0.020 0.007 500 630 0.006 0.022 -0.040 0.021 0.007 500 640 0.006 0.020 -0.036 0.022 0.007 ex(nm) em(nm) evec3 evec5 eveclO evecl6 eveclθ 500 650 0.005 0.017 -0.033 0.025 0.006 500 660 0.005 0.016 -0.030 0.022 0.007 500 670 0.004 0.014 -0.027 0.020 0.003 500 680 0.004 0.011 -0.024 0.021 0.000 500 690 0.004 0.011 -0.022 0.020 0.002 510 540 0.000 0.032 -0.022 -0.036 0.036 510 550 0.002 0.036 -0.029 -0.032 0.034 510 560 0.003 0.037 -0.036 -0.027 0.026 510 570 0.005 0.037 -0.042 -0.018 0.022 510 580 0.005 0.035 -0.044 -0.010 0.017 510 590 0.005 0.033 -0.043 -0.003 0.010 510 600 0.006 0.030 -0.043 0.005 0.013 510 610 0.006 0.028 -0.043 0.012 o.ooβ 510 620 0.006 0.025 -0.041 0.017 0.011 510 630 0.006 0.023 -0.038 0.019 o.ooβ 510 640 0.006 0.020 -0.037 0.023 o.ooβ 510 650 0.005 0.018 -0.034 0.025 0.006 510 660 0.005 0.016 -0.029 0.022 0.001 510 670 0.004 0.014 -0.028 0.022 0.005 510 680 0.004 0.013 -0.024 0.020 0.005 510 690 0.003 0.012 -0.021 0.019 0.006 520 550 -0.001 0.033 -0.024 -0.040 0.042 520 560 0.001 0.035 -0.032 -0.031 0.033 520 570 0.003 0.037 -0.039 -0.024 0.027 m) em(nm) evec3 evec5 eveclO evecl6 eveclβ 520 580 0.004 0.035 -0.041 -0.017 0.019 520 590 0.004 0.033 -0.041 -0.009 0.017 520 600 0.005 0.030 -0.042 -0.004 0.013 520 610 0.005 0.028 -0.041 0.005 0.012 520 620 0.005 0.026 -0.040 0.011 0.011 520 630 0.005 0.023 -0.038 0.016 0.007 520 640 0.005 0.021 -0.037 0.018 0.007 520 650 0.005 0.019 -0.034 0.023 0.006 520 660 0.005 0.017 -0.032 0.021 0.005 520 670 0.004 0.015 -0.029 0.021 0.003 520 680 0.004 0.014 -0.028 0.021 0.004 520 690 0.004 0.013 -0.025 0.017 0.005 530 560 0.000 0.032 -0.030 -0.032 0.040 530 570 0.002 0.033 -0.035 -0.024 0.030 530 580 0.003 0.032 -0.037 -0.018 0.022 530 590 0.003 0.030 -0.037 -0.013 0.015 530 600 0.004 0.029 -0.038 -0.004 0.012 530 610 0.004 0.027 -0.038 0.003 0.010 530 620 0.005 0.025 -0.038 0.011 0.009 530 630 0.005 0.023 -0.037 0.015 0.011 530 640 0.005 0.021 -0.034 0.019 0.005 530 650 0.005 0.019 -0.033 0.022 0.003 530 660 0.004 0.017 -0.031 0.020 0.003 530 670 0.004 0.015 -0.026 0.021 0.000 ex(nm) em(nm) evec3 evec5 eveclO evecl6 eveclβ 530 680 0.004 0.013 -0.024 0.023 -0.001 530 690 0.004 0.015 -0.025 0.021 0.007 540 570 0.003 0.029 -0.038 -0.012 0.031 540 580 0.003 0.028 -0.038 -0.009 0.019 540 590 0.004 0.027 -0.037 -0.007 0.015 540 600 0.004 0.026 -0.036 0.000 0.011 540 610 0.004 0.025 -0.038 0.006 0.010 540 620 0.005 0.024 -0.036 0.012 0.007 540 630 0.005 0.022 -0.035 0.015 0.005 540 640 0.005 0.020 -0.033 0.020 0.006 540 650 0.005 0.018 -0.031 0.021 -0.001 540 660 0.005 0.017 -0.031 0.022 0.004 540 670 0.004 0.015 -0.027 0.024 0.001 540 680 0.004 0.015 -0.025 0.023 0.001 540 690 0.004 0.013 -0.025 0.021 0.004 550 580 0.005 0.025 -0.043 0.005 0.021 550 590 0.004 0.023 -0.038 0.005 0.014 550 600 0.005 0.024 -0.036 0.010 0.013 550 610 0.005 0.023 -0.037 0.011 0.011 550 620 0.005 0.022 -0.037 0.018 0.011 550 630 0.005 0.021 -0.036 0.020 0.008 550 640 0.005 0.019 -0.034 0.024 0.005 550 650 0.005 0.018 -0.032 0.025 0.006 550 660 0.005 0.017 -0.031 0.025 0.005 ex(nm) em(nm) evec3 evec5 eveclO evecl6 eveclθ 550 670 0.005 0.015 -0.027 0.025 0.002 550 680 0.004 0.015 -0.025 0.025 0.004 550 690 0.004 0.015 -0.025 0.024 0.006 APPENDIX III: REFERENCES The disclosures of the following publications, patents and applications are expressly incorporated herein by reference, as are any of the other references mentioned above: A two-stage fluorescence diagnostic method is disclosed in detail in application Serial No. 08/060,432, filed May 12, 1993, and is assigned to the same assignee as the present invention. An application entitled "Optical Method And apparatus for the Diagnosis of Cervical Precancers using Raman and Fluorescence Spectroscopies" (Richards-Kortum et al . ) was filed on March 14, 1995, and is assigned to the same assignee as the present invention. Lui et al . , "Fluorescence and Time-Resolved Light Scattering as Optical Diagnostic Techniques to Separate Diseased and Normal Biomedical Media, " J. Photochem. Photobiol . B : Biol . 16, 187-209, 1992. Wong et al . , "Infrared Spectroscopy of Human Cervical Cells: Evidence of Extensive Structural Changes during Carcinogenesis" , Proc. Nat ' l Acad. Sci . USA, 88, 10988-10992, 1991. Kurman RJ, Henson DE, Herbst AL, Noller KL, Shiffman MH, "Interim guidelines for management of abnormal cervical cytology," JAMA, 271, 1866-1Θ69, 1994. Davison JM, Marty JJ, "Detecting premalignant cervical lesions: Contributions of screening colposcopy to cytology, " J Reproductive Medicine, 39, 38Θ-392, 1994. Banda-Gamboa, Hugo, et al, "Automation in cervical cytology: an overview," Anal Cell Path, 4 (1992) 25-48. Chance B, Thorell L, "Localization and kinetics of reduced PN in living cells by microfluorimetry, " J Biol Chem 234:3044, 1959. Caspersson TC, Cell Structure and Function, ch 4, Norton & Co, New York, 1950. Aubin JE, "Autofluorescence of Viable Cultured Mammalian Cells, " J Histochem Cytochem, 27(1) , 36-43, 1979. Benson RC, et al, "Cellular Autofluorescence : Is It Due to Flavins?" J Histochem Cytochem, 27(1), 44-48, 1979. Tamura M, ... Chance B, "In Vivo Study of Tissue Oxygen ...," Annu J?ev Physiol , 1989, 51:813-814. Hutchinson, ML, Agarwal, P, Denault, T, Berger, B, Cibas, ES, "A New Look at Cervical Cytology," Acta Cytologica , 36, 4:499-504, 1992. Taylor DG, Demas JN, "Light intensity measurements I: Large area bolometers with uW sensitivities and absolute calibration of the rhodamine B quantum counter," Anal Chem, 51, 712-717, 1979. Vooijs GP, "Benign proliferative reactions, intraepithelial neoplasia and invasive cancer of the uterine cervix, " in Bibbo M (ed) , Comprehensive Cytopathology, WB Saunders Company, pp 176-177, 1991. Dillon WR and Goldstein M, "Multivariate Analysis Methods and Applications," Chapter 10. Wiley & Sons, New York (1985)

Claims: CLAIMS : 1. A method of detecting tissue abnormality in a tissue sample in vi tro comprising: (i) providing a tissue sample; (ii) sequentially illuminating said tissue sample in vi tro with a set of at least two electromagnetic radiation wavelengths selected to cause said tissue sample to produce a set of fluorescence intensity spectra indicative of tissue abnormality; (iii) detecting said set of fluorescence intensity spectra emitted from said tissue sample as a result of illumination with each of said wavelengths; and (iv) calculating from said set of fluorescence intensity spectra, a probability that said tissue sample is normal or abnormal. 2. The method of claim 1, wherein said calculating step comprises, conducting principal component analysis of said fluorescent spectra relative to a set of preprocessed spectra obtained from tissue samples of known pathology. 3. The method of claim 2, wherein said principal component analysis does not include the highest and lowest order principal components. 4. The method of claim 3, wherein said principal component analysis comprises a Fisher's determinant analysis. 5. The method of claim 1, wherein said calculating step comprises, normalizing said spectra relative to a maximum intensity within said spectra. 6. The method of claim 5, wherein said calculating step further comprises, mean-scaling said spectra as a function of a mean intensity of spectra. 7. The method of claim 1, wherein said providing step comprises obtaining said tissue sample by biopsy. 8. The method of claim 7, wherein said providing step further comprises ethanol fixation of said tissue sample 9. The method of claim 8, wherein said providing step even further comprises generating a monolayer cell touch preparation or a pellet. 10. The method of claim 1, wherein said set of at least two electromagnetic wavelengths are 250 nm, 550 nm and all wavelengths between 250 nm and 550 nm at 10 nm intervals. 11. The method of claim 1, wherein said detecting step comprises, detecting an intensity of fluorescence at 250 nm, 700 nm and all wavelengths between 250 nm and 700 nm at 10 nm intervals. 12. The method of claim 1, wherein said illuminating comprises, illuminating said sample substantially normal to a surface of said sample, and wherein said detecting step comprises, detecting said spectra at an angle of approximately 20° from normal. 13. The method of claim 1, wherein at least 10 sequential electromagnetic radiation wavelengths are used in said illuminating step to produce a set of at least 10 different fluorescence intensity spectra in said detecting step, each spectra comprising fluorescence intensity at at least 11 wavelengths. 14. The method of claim 13, wherein at least 30 sequential electromagnetic radiation wavelengths are used in said illuminating step to produce a set of at least 30 different fluorescence intensity spectra in said detecting step, each spectra comprising fluorescence intensity at at least 31 wavelengths. 15. The method of claim 14, wherein at least 50 sequential electromagnetic radiation wavelengths are used in said illuminating step to produce a set of at least 50 different fluorescence intensity spectra in said detecting step, each said spectra comprising fluorescence intensity at at least 51 wavelengths.

Inventors: MITCHELL MICHELE FOLLEN, RICHARDS-KORTUM REBECCA, STRAERKEL GREGG, YAZDI YOUSEPH, MITCHELL, MICHELE, FOLLEN, RICHARDS-KORTUM, REBECCA, STRAERKEL, GREGG, YAZDI, YOUSEPH
Applicants: UNIV TEXAS, BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM
Application year: 1996
Main classification: G01N21
IPC codes: A61B5, G01N21, G01N33