In vivo endoscopic autofluorescence microspectro-imaging of bronchi and alveoli

Fibered confocal fluorescence microscopy (FCFM) is a new technique that can be used during a bronchoscopy to analyze the nature of the human bronchial and alveolar mucosa fluorescence microstructure. An endoscopic fibered confocal fluorescence microscopy system with spectroscopic analysis capability was developed allowing real-time, simultaneous images and emission spectra acquisition at 488 nm excitation using a flexible miniprobe that could be introduced into small airways. This flexible 1.4 mm miniprobe can be introduced into the working channel of a flexible endoscope and gently advanced through the bronchial tree to the alveoli. FCFM in conjunction with bronchoscopy is able to image the in vivo autofluorescence structure of the bronchial mucosae but also the alveolar respiratory network outside of the usual field of view. Microscopic and spectral analysis showed that the signal mainly originates from the elastin component of the bronchial subepithelial layer. In non smokers, the system images the elastin backbone of the aveoli. In active smokers, a strong autofluorescence signal appears from alveolar macrophages. The FCFM technique appears promising for in vivo exploration of the bronchial and alveolar extracellular matrix

IN VIVO CONFOCAL MICROENDOSCOPY: FROM THE PROXIMAL BRONCHUS DOWN TO THE PULMONARY ACINUS

In vivo endoscopic microscopy aims to provide the clinician with a tool to assess architecture and morphology of a living tissue in real time, with an optical resolution similar to standard histopathology. To date, available microendoscopic devices use the principle of fluorescence confocal microscopy, and thereby mainly analyse the spatial distribution of specific endogenous or exogenous fluorophores. Fluorescence microendoscopes devoted to respiratory system exploration use a bundle of optical fibres, introduced into the working channel of the bron- choscope. This miniprobe can be applied in vivo onto the bronchial inner surface or advanced into a distal bron- chiole down to the acinus, to produce in situ, in vivo microscopic imaging of the respiratory tract in real time. Fluorescence confocal microendoscopy has the capability to image the epithelial and subepithelial layers of the pro- ximal bronchial tree, as well as the more distal parts of the lungs, from the terminal bronchioles down to the alveolar ducts and sacs. Potential applications include in vivo microscopic assessment of early bronchial cancers, bronchial wall remodelling evaluation and diffuse peripheral lung disease exploration, as well as in vivo diagnosis of peripheral lung nodules. The technique has also the potential to be coupled with fluorescence molecular imaging. This chapter de- scribes the capabilities and possible limitations of confocal microendoscopy for proximal and distal lung exploration

Autofluorescence endoscopic spectro-imaging and 2D-cartography for in situ localisation and diagnosis of cancerous lesions

Early diagnosis is the most efficient way to struggle against cancer. Among all the existing techniques, optical methods (photodiagnosis from NUV to NIR) show important characteristics required by the physicians : high sensitivity, non-ionising radiations and non-traumatic measurements. They are particularly well suited to the detection of cancers in hollow organs, that are usually superficial and hardly visible with classical endoscopy. This paper describes a methodological approach based on the use of tissue autofluorescence, applicable in clinical endoscopy, and leading to the definition of diagnosis indicators from the spectral parameters. Following a state-of-the-art on autofluorescence spectroscopic (LIFS) and endoscopic imaging methods, we present the efficiency of fibered LIFS in terms of sensitivity and specificity for the diagnosis of esophagus cancerous lesions (clinical study over 25 patients). We then present the technological characteristics of an autofluorescence endoscopic imaging prototype developed in our labs as well as its calibration. A second part is devoted to endoscopic image registration and mosaicing and to optics aberration correction in perspective of the automatic construction of a panoramic image (cartography) of the organ’s explored areas. Finally, exploiting the fluorescence data provided by the imager, the feasibility of the superimposition of spatial and spectral information is validated with a phantom.Le diagnostic précoce est le moyen le plus efficace de lutte contre le cancer. Parmi toutes les techniques possibles, les méthodes optiques (photodiagnostic du proche UV au proche IR) présentent des caractéristiques importantes recherchées par les médecins : grande sensibilité, radiations non ionisantes et mesures atraumatiques. Elles sont particulièrement bien adaptées à la détection des cancers des organes creux, par nature superficiels et difficilement décelables en endoscopie classique. Cet article décrit une approche méthodologique fondée sur l’exploitation de l’autofluorescence tissulaire, applicable en endoscopie clinique, et conduisant à l’élaboration d’indicateurs diagnostiques issus des paramètres spectraux. Après un état de l’art sur les méthodes spectroscopiques (LIFS) et d’imagerie endoscopique d’autofluorescence, nous montrons l’efficacité de la LIFS fibrée en terme de sensibilité et de spécificité pour le diagnostic de lésions cancéreuses de l’oesophage (étude clinique sur 25 patients). Nous présentons ensuite les caractéristiques technologiques et le calibrage du prototype d’imageur endoscopique d’autofluorescence développé. Une seconde partie traite du pré-traitement, du recalage et du mosaïquage des images endoscopiques appliqués à la construction automatique d’une image panoramique (cartographie) à partir de séquences vidéos des zones explorées de l’organe. Finalement, en exploitant les informations de fluorescence fournies par l’imageur, la faisabilité d’une superposition des informations spatiale et spectrale est validée sur fantôme

Perendoscopic alveolar imaging using fluorescent confocal fibered microscopy.

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In vivo endoscopic analysis of the bronchial structure using fluorescence fibered confocal microscopy.

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Fluorescence confocal microimaging of the distal airways during bronchoscopy

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КОНФОКАЛЬНАЯ МИКРОСКОПИЯ IN VIVO: ОТ ПРОКСИМАЛЬНЫХ БРОНХОВ К АЛЬВЕОЛЯРНОМУ ДЕРЕВУ ЛЕГКИХ

In vivo endoscopic microscopy aims to provide the clinician with a tool to assess architecture and morphology of a living tissue in real time, with an optical resolution similar to standard histopathology. To date, available microendoscopic devices use the principle of fluorescence confocal microscopy, and thereby mainly analyse the spatial distribution of specific endogenous or exogenous fluorophores. Fluorescence microendoscopes devoted to respiratory system exploration use a bundle of optical fibres, introduced into the working channel of the bron- choscope. This miniprobe can be applied in vivo onto the bronchial inner surface or advanced into a distal bron- chiole down to the acinus, to produce in situ, in vivo microscopic imaging of the respiratory tract in real time. Fluorescence confocal microendoscopy has the capability to image the epithelial and subepithelial layers of the pro- ximal bronchial tree, as well as the more distal parts of the lungs, from the terminal bronchioles down to the alveolar ducts and sacs. Potential applications include in vivo microscopic assessment of early bronchial cancers, bronchial wall remodelling evaluation and diffuse peripheral lung disease exploration, as well as in vivo diagnosis of peripheral lung nodules. The technique has also the potential to be coupled with fluorescence molecular imaging. This chapter de- scribes the capabilities and possible limitations of confocal microendoscopy for proximal and distal lung exploration. В условиях in vivo эндоскопическая микроскопия направлена на то, чтобы предоставить врачу средство для оценки архитектуры и морфологии живых тканей в режиме реального времени, обеспечив при этом оптическое разрешение, сходное с разрешением при стандартном гистопатологическом исследовании. На сегодняшний день доступные микроэндоскопические устройства используют принцип флуоресцен- тной конфокальной микроскопии и вследствие этого в основном выполняют анализ пространственного распределения специфических эндогенных или экзогенных флуорофоров. Флуоресцентные микроэндос- копы, предназначенные для исследования дыхательной системы, используют пучок оптоволокон, кото- рый вводится в рабочий канал бронхоскопа. Такой мини-зонд может применяться в условиях in vivo для исследования внутренней поверхности бронхов или продвигаться в дистальные бронхиолы, вплоть до ацинуса, чтобы в условиях in situ, in vivo выполнить микроскопическую визуализацию дыхательных пу- тей в режиме реального времени. Флуоресцентная конфокальная микроэндоскопия дает возможность ви- зуализации эпителиальных и субэпителиальных слоев проксимальных отделов бронхиального дерева, а также дистальных отделов легких, от конечных бронхиол до альвеолярных протоков и альвеол. Потенци- альные области применения включают в себя микроскопическую оценку в условиях in vivo ранних стадий рака бронхов, оценку ремоделирования бронхиальной стенки и исследования диффузных заболеваний периферических отделов легких, а также диагностики in vivo узелковых образований в периферических отделах легких. Данный метод также имеет потенциальную возможность совместного использования с флуоресцентной молекулярной визуализацией. В этой статье описаны возможности и вероятные ограни- чения конфокальной микроэндоскопии для исследования проксимальных и дистальных отделов легких.

Bimodal spectroscopic evaluation of ultra violet-irradiated mouse skin inflammatory and precancerous stages: instrumentation, spectral feature extraction/selection and classification (k-NN, LDA and SVM)

This paper deals with the development and application of in vivo spatially-resolved bimodal spectroscopy (AutoFluorescence AF and Diffuse Reflectance DR), to discriminate various stages of skin precancer in a preclinical model (UV-irradiated mouse): Compensatory Hyperplasia CH, Atypical Hyperplasia AH and Dysplasia D. A programmable instrumentation was developed for acquiring AF emission spectra using 7 excitation wavelengths: 360, 368, 390, 400, 410, 420 and 430 nm, and DR spectra in the 390–720 nm wavelength range. After various steps of intensity spectra preprocessing (filtering, spectral correction and intensity normalization), several sets of spectral characteristics were extracted and selected based on their discrimination power statistically tested for every pair-wise comparison of histological classes. Data reduction with Principal Components Analysis (PCA) was performed and 3 classification methods were implemented (k-NN, LDA and SVM), in order to compare diagnostic performance of each method. Diagnostic performance was studied and assessed in terms of sensitivity (Se) and specificity (Sp) as a function of the selected features, of the combinations of 3 different inter-fibers distances and of the numbers of principal components, such that: Se and Sp ≈ 100% when discriminating CH vs. others; Sp ≈ 100% and Se > 95% when discriminating Healthy vs. AH or D; Sp ≈ 74% and Se ≈ 63% for AH vs. D