Blue-violet excited autofluorescence spectroscopy and imaging of normal and cancerous human bronchial tissue after formalin fixation1

Autofluorescence (AF) imaging is a powerful tool for the detection of (pre-)neoplastic lesions in the bronchi. Several endoscopic imaging systems exploit the spectral and intensity contrast of AF between healthy and (pre-)neoplastic bronchial tissues, yet, the mechanisms underlying these contrasts are poorly understood. In this report, the effect of formalin fixation on the human bronchi AF, hence on the contrast, was studied by spectrofluorometric point measurements and DAFE (Diagnostic AutoFluorescence Endoscopy) broad field imaging. Generally, formalin-fixed samples have higher AF intensity than in vivo, whereas the emission spectral shape is similar. Additionally, the spectrofluorometric data showed a moderate decrease of the AF intensity on (pre-)neoplastic lesions relative to the healthy bronchial samples. However, this decrease was lower than that reported from in vivo measurements. Neither spectral measurements nor imaging revealed spectral contrast between healthy bronchial tissue and (pre-)neoplastic lesions in formalin. These results indicate that epithelial thickening and blood supply in the adjacent lamina propria are likely to play a key role in the generation of the AF contrast in bronchial tissues. Our results show that the AF contrast in bronchial tissues was significantly affected by standard, 10% buffered, formalin fixation. Therefore, these samples are not suited to AF contrast studie

Design of an endoscopic optical reference to be used for autofluorescence bronchoscopy with a commercially available diagnostic autofluorescence endoscopy (DAFE) system

We present the design of a sterilizable optical reference to characterize and quantify the inter-patient variations in tissue autofluorescence during autofluorescence bronchoscopy with Richard Wolf's diagnostic autofluorescence endoscopy (DAFE) system. The reference was designed to have optical and spectral properties similar to those of the human bronchial wall in spectral conditions corresponding to autofluorescence bronchoscopy conducted with the DAFE system (fluorescence excitation at 390-470 nm and red backscattering light at 590-680 nm). The reference's effective attenuation coefficient and reflectance were measured at 675 nm. In addition, its fluorescence emission spectrum was determined under 430 nm wavelength excitation. The reference is photostable, reproducible, biocompatible and small enough to be easily inserted through the working channel of a conventional bronchofibrescope. This cylindrical (length: 2 mm; diameter: 2 mm) optical reference was validated in a clinical environment

In vivo time-resolved spectroscopy of the human bronchial early cancer autofluorescence.

Time-resolved measurements of tissue autofluorescence (AF) excited at 405 nm were carried out with an optical-fiber-based spectrometer in the bronchi of 11 patients. The objectives consisted of assessing the lifetime as a new tumor/normal (T/N) tissue contrast parameter and trying to explain the origin of the contrasts observed when using AF-based cancer detection imaging systems. No significant change in the AF lifetimes was found. AF bronchoscopy performed in parallel with an imaging device revealed both intensity and spectral contrasts. Our results suggest that the spectral contrast might be due to an enhanced blood concentration just below the epithelial layers of the lesion. The intensity contrast probably results from the thickening of the epithelium in the lesions. The absence of T/N lifetime contrast indicates that the quenching is not at the origin of the fluorescence intensity and spectral contrasts. These lifetimes (6.9 ns, 2.0 ns, and 0.2 ns) were consistent for all the examined sites. The fact that these lifetimes are the same for different emission domains ranging between 430 and 680 nm indicates that there is probably only one dominant fluorophore involved. The measured lifetimes suggest that this fluorophore is elastin

Design of a light delivery system for the photodynamic treatment of the Crohn's disease

Crohn's disease is an inflammatory bowel disease originating from an overwhelming response of the mucosal immune system. Low dose photodynamic therapy (PDT) may modify the mucosal immune response and thus serve as a therapy for Crohn's disease. Most patients with Crohn's disease show inflammatory reactions in the terminal ileum or colon where PDT treatment is feasible by low-invasive endoscopic techniques. However, the tube like geometry of the colon, it's folding, and the presences of multiple foci of Crohn's lesions along the colon require the development of adequate light delivery techniques. We present a prototype light delivery system for endoscopic clinical PDT in patients with Crohn's disease. The system is based on a cylindrical light diffuser inserted into a diffusing balloon catheter. Homogenous irradiation is performed with a 4 W diode laser at 635 nm. Light dosimetry is performed using a calibrated integrating sphere, The system can be used with conventional colonoscopes and colonovideoscopes having a 3.8 mm diameter working channel. The feasibility of PDT in colon with our prototype was demonstrated in first clinical trials. © 2007 SPIE-OSA

Optimization of the spectral design used to detect early carcinoma in the human tracheo-bronchial tree by autofluorescence imaging

The early detection and localization of bronchial cancer remains a challenging task. Autofluorescence bronchoscopy is emerging as a useful diagnostic tool with improved sensitivity and specificity. Evidence exists that the native fluorescence or autofluorescence of bronchial tissues changes when they turn dysplastic or to carcinoma in situ (CIS). Early lesions in the bronchi tend to show a decrease in autofluorescence in the green region of the spectrum when excited with violet light and a relative increase in the red region of the spectrum. Several endoscopic imaging devices relying on these optical properties of bronchial mucosa have been developed. An industrial endoscopic autofluorescence imaging system for the detection of early cancerous lesions in the bronchi has been developed in collaboration with the firm Richard Wolf Endoskope GmbH, Knittlingen (Germany) and its performance has been evaluated in a previous clinical study. A second study, presented in this article, aims to optimize the spectral design of the device. Twenty-four lung cancer or high risk patients were enrolled in this study to assess the influence of additional backscattered red light on the tumor-to-healthy tissue contrast and to compare the effect of a narrow band violet excitation to a large band violet excitation. In our study we observed a three times higher contrast between cancer and healthy tissue, when backscattered red light was added to the violet excitation. The comparison between a narrow and a large band violet excitation indicated an increase of the tumor-to-healthy tissue contrast by the narrow band excitation

Detection of early bronchial carcinoma by imaging of the tissue autofluorescence

Early detection and localisation of bronchial cancer remains a challenging task. One approach is to exploit the changes in the autofluorescence characteristics of the bronchial tissue as a diagnostic tool with improved sensitivity. Evidence exists that this native fluorescence or autofluorescence of bronchial tissues changes when they turn dysplastic and to carcinoma in situ. There is agreement in the literature that the lesions display a decrease in autofluorescence in the green region of the spectrum under illumination with violet light and a relative increase in the red region of the spectrum is often reported. Imaging devices rely on this principle to detect early cancerous lesions in the bronchi. Based on a previous spectroscopic study, an industrial imaging prototype has been developed to detect early cancerous lesions in collaboration with the firm 'Richard Wolf Endoskope GmbH'. A preliminary clinical trial, involving 20 patients, with this spectrally optimised system proved that autofluorescence can detect lesions that would otherwise have remained invisible even to an experienced endoscopist under white light illumination. A systematic analysis of the autofluorescence images indicated that real-time decisional functions can be defined in order to reduce the number of false positive results. Using this method, a Positive Predictive Value (PPV) of 75% was achieved using autofluorescence only. A PPV of even 100% was obtained when white light mode and autofluorescence mode were combined under the applied conditions. Furthermore, the sensitivity was estimated to be twice as high in AF mode than in WL mode

Autofluorescence detection of tumors in the human lung: comparison between in-vivo and in vitro measurements

To detect bronchial carcinoma by autofluorescence, we measured in-vivo, in an in-vivo model, and in-vitro the spectra of tumor and normal tissue by a fiber-optic-spectrometer. The main difference between tumor and bronchial tissue is the intensity of the 505 nm main peak. © 2005 SPIE-OSA

Autofluorescence detection of tumors in the human lung - spectroscopical measurements in situ, in an in vivo model and in vitro

To detect bronchial carcinoma by autofluorescence, we measured the spectra of tumor and normal tissue in situ, in an in vivo model and in vitro by fiber optic spectrometer and two-dimensional resolved microspectroscopy. The in situ measurements were performed in bronchi of nine patients with squamous cell carcinoma during regular bronchoscopy with autofluorescence assistance. The fluorescence was monitored with a fiber optical spectrometer under blue light excitation (lambda=405nm). In an in vivo model, the resected lobe of a lung was perfused under physiological conditions. Tumorous and normal tissues were examined spectroscopically during perfusion and after blood removal and substitution with formol. In another setup the wavelength dependency of autofluorescence was examined on resected parts of physiological bronchi and central bronchial carcinomas. Under the variation of the excitation from 385 to 465nm the autofluorescence response was monitored with a fiber optic spectrometer. For investigation of the origin of autofluorescence, two-dimensional resolved spectroscopy was performed with the SpectraCube system on several sections of tumor and normal tissues All measurements, performed in vivo, in the in vivo model and in vitro agreed, that the main difference of the autofluorescence between tumor and normal bronchus tissue is the intensity of the fluorescences' main peak at 505nm. The signal on tumor tissue is in all cases significantly lower than that of normal tissue. The shape of the autofluorescence peaks is in healthy and carcinoma tissue approximately the same with two characteristic minima at 540 and 580nm. After the preparation with formaldehyde those minima disappeared from the spectra. A comparison with the absorption spectra of hemoglobin showed, that the variation of the spectra may be due to the blood content in the tissue. Two-dimensional spatially resolved spectroscopy showed, that the lower intensity of fluorescence in tumor tissue is due to the irregular and low-concentrated formation of fluorescent structures, which seen to be the elastic structures of bronchial tissue