Clinical recording of laser-induced fluorescence spectra for evaluation of tumour demarcation feasibility in selected clinical specialities

Laser-induced autofluorescence spectra from humans were recorded in vivo at three different clinics in a study aimed at investigating the capability of this method to discriminate between malignant tumours and normal surrounding tissues. For the recordings a mobile trolley with the necessary equipment was constructed for use in an examination room or in an operating theatre environment. Laser light was guided through a 600m optical fibre to the target tissue. The fluorescence from the excited tissue was collected with the same fibre and was fed to an optical multichannel analyser. Two excitation wavelengths were used (337 and 405 nm) in order to optimize the fluorescence signals in two interesting wavelength regions (380–500 and 550–700 nm). Oral and oropharyngeal tumours excited with 405 nm light contained detectable endogenous porphyrins and were in this way discriminated from the normal mucosa. Astrocytoma grade III–IV fluorescence different from that of normal brain tissue, while tumours in the bronchial tree were not detectable using the spectral shape of the pure tissue autofluorescence.     

Optical spectroscopy characteristics can differentiate benign and malignant renal tissues: a potentially useful modality

PURPOSE: Promising results of optical signals have been reported in the literature for the diagnosis of Barrett's esophagus, oral cavity lesions, brain tumor margins, cervical intraepithelial neoplasia, skin cancer and bladder cancer. The potential usefulness of these techniques in renal tissues and neoplasms has not been described to date. This initial study examined the feasibility of using fluorescence and diffuse reflectance spectroscopy to differentiate between malignant and benign renal tissues. MATERIALS AND METHODS: An ex vivo study was conducted to identify optical characteristics of various renal tissue types. Pathologically confirmed benign and malignant renal samples were obtained from nephrectomy specimens from patients undergoing radical nephrectomy. Fluorescence and diffuse reflectance spectra were measured from benign and malignant renal tissues. RESULTS: All renal tissues, malignant or benign, contain 2 primary emission peaks-a strong one at approximately 285 nm excitation, approximately 340 nm emission (Peak A), and a weak one at approximately 340 nm excitation, approximately 460 nm emission (Peak B). Peak A of normal renal tissue typically locates at the shorter excitation wavelength region than that of malignant tissue. The intensity of Peak B from benign tissues tends to be greater than that from malignant renal tissues. Diffuse reflectance intensities from malignant renal tissues between 600 and 800 nm are markedly greater than those from normal renal tissue. Empirical discrimination algorithms developed based on selected fluorescence and diffuse reflectance spectral characteristics yields accurate differentiation between benign and malignant renal tissues. CONCLUSIONS: Highly accurate differentiation between normal human renal tissues and renal cell cancers is feasible using combined fluorescence and diffuse reflectance spectroscopy in an ex vivo setting. If successful in future clinical studies, optical spectroscopy could aid in margin detection and tissue discrimination while performing nephron sparing surgery.     

Aspects of tumour demarcation in rats by means of laser-induced fluorescence and haematoporphyrin derivatives

The effects of different parameters of interest for the localization of malignant tumours in situ by means of laser-induced fluorescence and haematoporphyrin derivatives were investigated. Such parameters are drug composition, drug concentration, laser pulse energy and excitation wavelength. In order to assess the relative merits of the two tumor-seeking agents Photofrin (haematoporphyrin derivative) and Photofrin II (dihaematoporphyrin ether) we have performed a comparative study on rat tissues. The results suggest that Photofrin is at least as good as the therapeutically more potent agent Photofrin II. A linear relation between drug dose and recorded porphyrin fluorescence intensity was also found. Using not only the porphyrin fluorescence, but also natural tissue autofluorescence, better tumour demarcation is observed when utilizing an excitation wavelength shorter than the porphyrin excitation peak at 405 nm.     

Fluorescence spectroscopy for in vivo characterization of ovarian tissue

BACKGROUND AND OBJECTIVE: The objective of this study was to explore whether fluorescence spectroscopy signatures differed between normal variations within the ovary, benign neoplasms, and ovarian cancer. STUDY DESIGN/MATERIALS AND METHODS: Ovarian tissue fluorescence emission spectra were collected sequentially at 18 excitation wavelengths ranging from 330 to 500 nm from 11 patients undergoing oophorectomy and assembled into fluorescence excitation emission matrices (EEMs); biopsies corresponded to the area interrogated. Spectral areas that could differentiate normal ovary, benign neoplasms, and cancers were evaluated, using histopathology as the reference standard. RESULTS: The most promising measurements are (1) the integrated fluorescence intensity from 400 to 430 nm excitation at 460 nm emission, and (2) the ratios of fluorescence intensities at 330 nm excitation, 385 and 500 nm emission, and at 375 and 415 nm excitation, 460 nm emission. Simple systems to visualize these optical signatures at laparoscopy could be designed. CONCLUSION: Fluorescence spectroscopy may have the ability to distinguish ovarian cancers from normal ovarian structures and benign neoplasms, as well as differentiate between normal variations and metaplastic structures and should be further explored as a device for the early detection of ovarian cancers.     

Autofluorescence of various rodent tissues and human skin tumour samples

Fluorescence spectra from different organs in rats and mice have been recorded to explore the potential of non-intrusive tissue diagnostics. The fluorescence was induced by a nitrogen laser that emitted at a wavelength of 337 nm. Optical multichannel techniques were used for the detection. Spectra are given from 19 different sites in Wistar/Furth rats, including an inoculated malignant tumour. The spectra seem to be a sum of two wavelength distributions only, each distribution occurring with a different weight in different organs. Spectra obtained from living and dead tissue were compared to verify that the measurements on sacrificed experimental animals were valid. Preliminary results are given for some human tumours, transplanted in nude mice, and for some human skin samples.     

Laser Induced Autofluorescence Diagnosis of Bladder Cancer

Purpose

We assessed the ability of laser induced autofluorescence to differentiate malignant from nonmalignant bladder lesions.

Materials and Methods

We studied 53 patients with bladder cancer undergoing mucosal biopsies or transurethral resection of a bladder tumor. A quartz optical fiber was advanced through the working channel of a cystoscope and placed in gentle contact with the bladder. Tissue fluorescence was excited by 337 nm. light pulses (nitrogen laser). One fiber was used for transmission of the excitation and emission (fluorescence) light. An optical multichannel analyzer system was used to record fluorescence spectra of the sites of interest.

Results

We analyzed the fluorescence spectra of 114 bladder areas (1 carcinoma in situ as well as 28 malignant, 35 inflammatory, 7 dysplastic, 1 squamous metaplastic and 42 normal areas). These lesions included 44 difficult to diagnose suspicious tumors (11 malignant and 33 nonmalignant). We developed an algorithm that used the I385:I455 nm. fluorescence ratio to distinguish malignant from nonmalignant lesions, including inflammatory areas. By analyzing the data on all 114 lesions, we noted the sensitivity, specificity, and positive and negative predictive values of this method for differentiating malignant from nonmalignant bladder lesions to be 97, 98, 93 and 99 percent, respectively.

Conclusions

Under excitation with 337 nm. light a clear differentiation between malignant and nonmalignant bladder tissues can be made using the I385:I455 nm. autofluorescence ratio.     

Integral spectrophotometric analysis of 5-aminolaevulinic acid-induced fluorescence cytology of the urinary bladder

OBJECTIVE: To evaluate whether tumour cells can be detected in bladder lavage fluid samples by an objective spectrofluorometric method, as 5-aminolaevulinic acid (ALA)-induced fluorescence endoscopy (AFE) and cytology are promising valuable tools for detecting transitional cell carcinoma of the urinary bladder (TCCB). MATERIALS AND METHODS: After instilling ALA into the urinary bladder, lavage samples were collected and their sediments analysed spectroscopically under blue excitation at approximately 400 nm wavelength. During AFE, biopsies were taken. From 62 cases, 24 patients had a histologically confirmed TCCB (group A), 28 had a history of TCCB but no evidence of disease (group B) and 10 were negative for TCCB (group C). RESULTS: Lavage sediments of all patients fluoresced in the green spectral range, typical of cellular autofluorescence. Sediments of all patients of group A caused red fluorescence peaking at 635 nm, indicating protoporphyrin IX (PPIX). The PPIX signals derived from bleaching spectra were significantly different between benign and malignant findings (P = 0.001). There was another red fluorescence peak at approximately 620 nm; in 19 cases its intensity exceeded the intensity of the PPIX signal. CONCLUSIONS: Spectrofluorometric analysis of lavage sample sediments can be used to detect tumour-associated red fluorescence of PPIX in TCCB. Immediate significant and objective measurements are possible, which could be further automated for the rapid diagnosis of TCCB.     

In vivo quantification of photosensitizer fluorescence in the skin-fold observation chamber using dual-wavelength excitation and NIR imaging

A major challenge in biomedical optics is the accurate quantification of in vivo fluorescence images. Fluorescence imaging is often used to determine the pharmacokinetics of photosensitizers used for photodynamic therapy. Often, however, this type of imaging does not take into account differences in and changes to tissue volume and optical properties of the tissue under interrogation. To address this problem, a ratiometric quantification method was developed and applied to monitor photosensitizer meso-tetra(hydroxyphenyl) chlorin (mTHPC) pharmacokinetics in the rat skin-fold observation chamber. The method employs a combination of dual-wavelength excitation and dual-wavelength detection. Excitation and detection wavelengths were selected in the NIR region. One excitation wavelength was chosen to be at the Q band of mTHPC, whereas the second excitation wavelength was close to its absorption minimum. Two fluorescence emission bands were used; one at the secondary fluorescence maximum of mTHPC centered on 720 nm, and one in a region of tissue autofluorescence. The first excitation wavelength was used to excite the mTHPC and autofluorescence and the second to excite only autofluorescence, so that this could be subtracted. Subsequently, the autofluorescence-corrected mTHPC image was divided by the autofluorescence signal to correct for variations in tissue optical properties. This correction algorithm in principle results in a linear relation between the corrected fluorescence and photosensitizer concentration. The limitations of the presented method and comparison with previously published and validated techniques are discussed.     

Early differentiation between caries and tooth demineralization using laser-induced autofluorescence spectroscopy

BACKGROUND AND OBJECTIVES: The intrinsic fluorescence of carious human teeth, of different stages of teeth demineralization, and the correspondence of such fluorescence to the mineral and organic distribution within the lesions were investigated. STUDY DESIGN/MATERIALS AND METHODS: Fluorescence spectra of teeth excited with 337 nm nitrogen laser were recorded. Spectra were obtained from healthy enamel, dentine, demineralized areas, and different carious stages of the teeth investigated. RESULTS: Spectra obtained from sound enamel consisted of one intensive peak at 480-500 nm and one secondary peak at 430-450 nm. In dentine, this secondary component had much higher intensity. Fluorescence spectra of normal teeth were similar to those of enamel layer. A significant decrease of the intensity of the fluorescence signal was observed in both cases-in demineralized teeth and in carious lesion. The appearance of a fluorescence peak in the red spectral region was observed in the spectra of the initial carious lesions. In the teeth demineralization process, we observed an increase of the relative fluorescence peak intensity at 430-450 nm related to thinned out of enamel. CONCLUSIONS: A differentiation between initial tooth demineralization and early stages of caries could be made by the laser-induced fluorescence spectroscopy method.     

Autofluorescence and diffuse reflectance properties of malignant and benign breast tissues

Background Fluorescence spectroscopy is an evolving technology that can rapidly differentiate between benign and malignant tissues. These differences are thought to be due to endogenous fluorophores, including nicotinamide adenine dinucleotide, flavin adenine dinucleotide, and tryptophan, and absorbers such as β-carotene and hemoglobin. We hypothesized that a statistically significant difference would be demonstrated between benign and malignant breast tissues on the basis of their unique fluorescence and reflectance properties. Methods Optical measurements were performed on 56 samples of tumor or benign breast tissue. Autofluorescence spectra were measured at excitation wavelengths ranging from 300 to 460 nm, and diffuse reflectance was measured between 300 and 600 nm. Principal component analysis to dimensionally reduce the spectral data and a Wilcoxon ranked sum test were used to determine which wavelengths showed statistically significant differences. A support vector machine algorithm compared classification results with the histological diagnosis (gold standard). Results Several excitation wavelengths and diffuse reflectance spectra showed significant differences between tumor and benign tissues. By using the support vector machine algorithm to incorporate relevant spectral differences, a sensitivity of 70.0% and specifcity of 91.7% were achieved. Conclusions A statistically significant difference was demonstrated in the diffuse reflectance and fluorescence emission spectra of benign and malignant breast tissue. These differences could be exploited in the development of adjuncts to diagnostic and surgical procedures.     

贲门癌与同体正常胃组织的显微自体荧光对比研究

目的探讨贲门癌组织显微自体荧光特征及自体荧光在同体正常胃组织各层的分布和差异。方法采用激光扫描共聚焦显微镜以氩离子(Ar+)激光(Ex=488nm)和氦氖激光(He-Ne)(Ex=543nm)为激发光的双通道法对16例贲门癌手术标本(贲门癌组织与同体胃体组织)进行自体荧光图像分析。结果同体正常胃组织胃壁各层中,以黏膜下层荧光信号最强。固有层呈现较强的荧光信号,主要分布于腺体上。癌组织自体荧光信号与正常组织各层相比均显著减弱(P<0.01)。结论贲门癌组织的显微自体荧光与同体正常胃组织在形态、颜色、分布及荧光强度上都存在明显差异。     

Behaviour of haematoporphyrin derivative in adenomas and adenocarcinomas of the colon: a microfluorometric study

Intravenously injected haematoporphyrin derivative localizes in malignant tissue more than it does in the surrounding normal tissues; this allows cancer to be treated photodynamically. The aim of this study is to determine whether adenomas of the large bowel, which are benign precarcinomatous lesions, accumulate HPD as well as does malignant tissue, and whether such accumulation occurs to a greater extent than it does in the surrounding normal mucosa. For this purpose, microspectrofluorometric analysis was performed on biopsy samples of adenomas, on adenocarcinomas, and on normal mucosa, and the data were compared. The samples were obtained from patients affected by histologically proven adenocarcinomas and synchronous adenomas of the bowel 48h after intravenous injection of HPD (3 mg/kg bodyweight), that is, the time interval usually required for photodynamic therapy. The intensity of fluorescence of the adenomas was a little lower than that observed from adenocarcinomas, but was higher than that from the surrounding mucosa. The fluorescence spectra from adenomas and from adenocarcinomas showed three main emission bands centred at about 630, 670 and 700 nm. The relative amplitudes of the band at 630 and 670 nm in benign and malignant colorectal tumours varied with the excitation wavelength, depending on the different physicochemical state of the accumulated drug. A further analysis, performed 72 h after administration, indicated the existence of an intratissue turnover of the porphyrins.     

Laser Induced Autofluorescence Diagnosis of Bladder Tumors: Dependence on the Excitation Wavelength

Purpose

We assessed the ability of laser induced autofluorescence spectroscopy to distinguish neoplastic urothelial bladder lesions from normal or nonspecific inflammatory mucosa.

Materials and Methods

Three different pulsed laser excitation wavelengths were used successively: 308 nm. (xenium chloride excimer laser), 337 nm. (nitrogen laser) and 480 nm. (coumarin dye laser). The excitation light was delivered by a specially devised multifiber catheter connected to a 1 mm. core diameter silica monofiber introduced through the working channel of a standard cystoscope with saline irrigation. The captured fluorescence light was focused onto an optical multichannel analyzer detection system. Device performance was evaluated in 25 patients after obtaining consent and immediately before transurethral resection of a bladder tumor. Spectroscopic results were compared with histological findings.

Results

At 337 and 480 nm. excitation wavelengths the overall fluorescence intensity of bladder tumors was clearly decreased compared to normal urothelial mucosa regardless of tumor stage and grade. At the 308 nm. excitation wavelength the shape of the tumor spectra, including carcinoma in situ, was markedly different from that of normal or nonspecific inflammatory mucosa. No absolute intensity determinations were required in this situation, since a definite diagnosis could be established based on the fluorescence intensity ratio at 360 and 440 nm.

Conclusions

This spectroscopic study could be particularly useful to design a simplified autofluorescence imaging device for detection of occult urothelial neoplasms.     

Laser induced fluorescence spectroscopy of normal and atherosclerotic human aorta using 306-310 nm excitation

Ultraviolet excited laser induced fluorescence (LIF) was studied in normal and atherosclerotic human arterial wall in vitro. Using excitation wavelengths from 306 to 310 nm, two distinct emission bands were observed in the LIF of both normal and pathologic aorta: a short wavelength band, peaking at 340 nm emission, which was attributed to tryptophan; and a long wavelength band, peaking at 380 nm emission, which was assigned to a combination of collagen and elastin. The intensity of the short wavelength band was quite sensitive to the choice of excitation wavelength, while the long wavelength band was not, so that the relative contributions of the bands could be controlled by the precise choice of excitation wavelength. A valley in the spectra at 418 nm was attributed to fluorescence reabsorption by oxy-hemoglobin. By using 308 nm excitation to observe emission simultaneously from both the short and long wavelength bands, normal and atherosclerotic aorta were spectrally distinct. Two LIF emission intensity ratios were defined to characterize both the relative tryptophan fluorescence content as well as the ratio of elastin to collagen fluorescence in each spectrum. The differences in these two emission ratios among the various histologic tissue types correlated qualitatively with the histologic and biochemical compositions of these tissues. By combining these parameters in a binary classification scheme, normal and atherosclerotic aorta were correctly distinguished in 56 of 60 total cases. Furthermore, atherosclerotic plaques, atheromatous plaques, and exposed calcifications could be classified individually with sensitivities/predictive values of 90%/90%, 100%/75%, and 82%/82%, respectively.     

Fluorescence spectroscopy of endomyocardial tissue post-human heart transplantation: does it correlate with histopathology?

BACKGROUND: A significant correlation between autofluorescence spectroscopy and heart allograft rejection has been described in the rat heterotropic allograft model. However, the use of this technique in human heart transplants has not been validate. METHODS: We obtained fluorescence and reflectance spectra on 37 human endomyocardial biopsy specimens and correlated the spectra with International Society Heart and Lung Transplantation grade for histologic rejection. RESULTS: Using different excitation wavelengths (ultraviolet, lambda = 337 nm; blue, lambda = 440 nm, and green, lambda = 486 nm), we found no significant difference in the fluorescence spectra among the different grades of rejection. CONCLUSIONS: Fluorescence spectroscopy is not a sensitive method for detecting rejection in human heart transplant recipients.     

Laser-induced fluorescence emission: I. The spectroscopic identification of fibrotic endocardium and myocardium.

Laser-induced fluorescence has been developed as a guidance system for laser angioplasty. Laser ablation has been used for resection of arrhythmogenic ventricular scar. We have investigated the use of laser-induced fluorescence for the detection of fibrotic and ischemic changes in endocardium and myocardium. Fluorescence emission spectra from human necropsy specimens were correlated with histologic examination. Normalized fluorescence intensity detected from both the endocardial and the myocardial surfaces of the fibrotic ventricular specimens was significantly higher than that of corresponding normal specimens at 440 to 475 nm. Fibrotic endocardium could be identified by a fluorescence emission intensity ratio less than 1.5 for wavelength ratio 375/450nm. Acutely infarcted endocardium was recognizable by a ratio of 1.5 to 2.0. The specificity and sensitivity of detection of scarred endocardium was 70 and 100%, respectively. Fibrotic myocardium was also consistently identified by fluorescence spectroscopy. Conclusion: Fluorescence emission spectroscopy can differentiate normal and fibrotic endocardium and myocardium, in vitro. This technique may be useful for guidance during laser ablation of arrhythmogenic ventricular scar.     

An affordable, portable fluorescence imaging device for skin lesion detection using a dual wavelength approach for image contrast enhancement and aminolaevulinic acid-induced protoporphyrin IX. Part I. Design, spectral and spatial characteristics

Steady-state fluorescence imaging can be used in conjunction with selective exogenous or endogenous fluorescent compounds for the diagnosis of skin lesions, for example cancerous lesions. Depending on the excitation and emission properties of the fluorescent compound used, various excitation and/or emission wavelengths can be chosen in order to allow fluorescence imaging. Unwanted background signals like autofluorescence and scattering can decrease the image quality and hence the diagnosis potential of this imaging method. A method involving two excitation and/or emission wavelengths was used in order to suppress the unwanted background signal and allow contrast enhanced fluorescence imaging. A fluorescence imaging device prototype was assembled using both the two wavelength excitation method and the two wavelength emission method. Additionally, a white light source was included to allow the collection of images as seen with the naked eye. The prototype was designed to be affordable and portable and was laid out towards the diagnosis of skin lesions using aminolaevulinic acid-induced protoporphyrin IX (PpIX). This paper describes the excitation and detection characteristics of a fluorescence imaging device prototype. This includes spectral and spatial characteristics of the various light sources included in the device as well as specifications of the image detector used. Furthermore, the image analysis procedure used for the dual wavelength excitation/emission is described. Paper received 27 July 2000; accepted after revision 23 October 2000.     

AUTOFLUORESCENCE GUIDED BIOPSY FOR THE EARLY DIAGNOSIS OF BLADDER CARCINOMA

Purpose

We validate the usefulness of laser-induced autofluorescence for the detection of bladder carcinoma.

Materials and Methods

We obtained and analyzed fluorescence spectra from 75 patients in whom bladder cancer was suspected. Tissue fluorescence was excited by a nitrogen laser using a quartz optical fiber placed in gentle contact with the area of interest. The laser-induced autofluorescence spectrum was recorded using an intensified optical multichannel analyzer system. Spectra were corrected for the spectral response of the optical system, and the ratios of laser-induced autofluorescence intensities (I) at 385 and 455 nm. (I₃₈₅/I₄₅₅) were determined. We had previously established this ratio as a diagnostic algorithm. We included only suspicious bladder lesions (erythematous, edematous, raised and so forth) that were difficult to diagnose by cystoscopy as well as areas from which random biopsies were obtained. The fluorescence ratio algorithm was applied to 130 bladder areas.

Results

Of the 130 biopsies obtained during routine cystoscopy 107 (82%) were nonmalignant by histological classification. In contrast, because laser-induced autofluorescence effectively guides biopsies towards malignant lesions, only 30 biopsies (72% fewer) would have been obtained from nonmalignant tissue if the fluorescence ratio that identifies 95% of malignant lesions (95th percentile) had been selected as the decision criterion during standard cystoscopy.

Conclusions

By guiding the surgeon to suspicious lesions that are most likely to be malignant, laser-induced autofluorescence substantially decreases the number of biopsies obtained from nonmalignant tissue during cystoscopy to diagnose bladder carcinoma.     

Using the laser-induced fluorescence spectroscopy in the differentiation between normal and neoplastichuman breast tissue

This article reports results of the in vitro study for potential evaluation of the laser-induced fluorescence spectroscopy in the differentiation between normal and neoplastic human breast tissue. A coumarine dye laser pumped by nitrogen laser generated an excitation light centered at 458 nm. In order to collect the fluorescence signal was used an optical fiber catheter coupled to a spectrometer and CCD detector. Fluorescence spectra were recorded from normal and neoplastic (benign and malignant) human breast tissue, adding up 94 different areas. The discrimination between normal and neoplasm groups reach a sensitivity and specificity of 100%.     

Autofluorescence and diffuse reflectance spectroscopy for oral oncology

BACKGROUND AND OBJECTIVES: Autofluorescence and diffuse reflectance spectroscopy have been used separately and combined for tissue diagnostics. Previously, we assessed the value of autofluorescence spectroscopy for the classification of oral (pre-)malignancies. In the present study, we want to determine the contributions of diffuse reflectance and autofluorescence spectroscopy to diagnostic performance. STUDY DESIGN/MATERIALS AND METHODS: Autofluorescence and diffuse reflectance spectra were recorded from 172 oral lesions and 70 healthy volunteers. Autofluorescence spectra were corrected in first order for blood absorption effects using diffuse reflectance spectra. Principal Components Analysis (PCA) with various classifiers was applied to distinguish (1) cancer and (2) all lesions from healthy oral mucosa, and (3) dysplastic and malignant lesions from benign lesions. Autofluorescence and diffuse reflectance spectra were evaluated separately and combined. RESULTS: The classification of cancer versus healthy mucosa gave excellent results for diffuse reflectance as well as corrected autofluorescence (Receiver Operator Characteristic (ROC) areas up to 0.98). For both autofluorescence and diffuse reflectance spectra, the classification of lesions versus healthy mucosa was successful (ROC areas up to 0.90). However, the classification of benign and (pre-)malignant lesions was not successful for raw or corrected autofluorescence spectra (ROC areas <0.70). For diffuse reflectance spectra, the results were slightly better (ROC areas up to 0.77). CONCLUSIONS: The results for plain and corrected autofluorescence as well as diffuse reflectance spectra were similar. The relevant information for distinguishing lesions from healthy oral mucosa is probably sufficiently contained in blood absorption and scattering information, as well as in corrected autofluorescence. However, neither type of information is capable of distinguishing benign from dysplastic and malignant lesions. Combining autofluorescence and reflectance only slightly improved the results.