Geneviève Bourg-Heckly

Confocal Fluorescence Endomicroscopy of the Human Airways

Confocal endomicroscopes aim at providing to the clinician microscopic imaging of a living tissue. The currently available microendoscopic devices use the principle of confocal fluorescent microscopy, in which the objective is replaced by an optical fiber and a miniaturized scanhead at the distal end of the endoscope or by a retractable bundle of optical fibers. Such systems have recently been applied to the explorations of several organs, including the gastrointestinal tract, and more recently to the proximal and distal airways in vivo. Respiratory fluorescence microendoscopes use 488 nm or 660 nm excitation laser light and thin flexible miniprobes that are introduced into the working channel of the bronchoscope. The devices have a lateral resolution of 3 microm, a field of view of 600 microm, and produce real-time imaging at 9 frames per second. For in vivo imaging, the miniprobe is applied onto the bronchial wall surface or advanced into a distal bronchiole down to the acinus. In nonsmokers, the 488-nm excitation device images the autofluorescence of the elastin that is contained in the basement membrane of the proximal airways and that participates to the axial backbone of the peripheral interstitial respiratory system. In smokers, a specific tobacco tar-induced fluorescence allows in vivo macrophage and alveolar wall imaging. Using 660 nm excitation and topical methylene blue, the technique enables cellular imaging of both bronchial epithelial layer and peripheral lung nodules. This article reviews the capabilities and possible limitations of confocal microendoscopy for in vivo proximal and distal lung explorations.

2D PANORAMAS FROM CYSTOSCOPIC IMAGE SEQUENCES AND POTENTIAL APPLICATION TO FLUORESCENCE IMAGING

Abstract This work describes an algorithm for the automatic construction of 2-D panoramas from sequences of images taken from a cystoscopic exam. During a cystoscopy, the existent distance between the cystoscope and the bladder walls allows the clinician to observe only a reduced region of the zone of interest. A panoramic representation of this zone could represent a better visual support to the clinician in the localization of eventual lesions and in the application of further exams. We also explore the application of this algorithm to endoscopic fluorescence imaging, one of the main procedures used in the detection of epithelial cancers.

In vivo assessment of the pulmonary microcirculation in elastase-induced emphysema using probe-based confocal fluorescence microscopy

Introduction: The alveolar capillary bed, which appears essential for the maintenance of alveolar septa, is altered in pulmonary emphysema. Until recently, techniques that allow its analysis in vivo in spontaneously breathing conditions were lacking. Fibered confocal fluorescence microscopy (FCFM) is a new technique that enables distal lung microstructures imaging in vivo. FCFM can be coupled with I.V fluorescein injection to image the pulmonary capillary network. The aim of this study was to assess the lung microcirculation in vivo using FCFM and I.V fluorescein in rats with experimental emphysema. Results: In vivo pulmonary microcirculation imaging was possible in 7/7 elastase animals and in 6/7 controls. Using FCFM, intercapillary distances and alveolar facets diameters were found significantly higher in the elastase group compared with controls (49.5 vs. 41.8 µm p < 0.001, and 118.5 vs. 95.1 µm p < 0.001, respectively). Ex vivo mean interwall distance (MIWD) was correlated with the alveolar facets diameters measured in vivo (rs = 0.65 ; p = 0.016). Methods: 14 Sprague-Dawley rats were assigned to intratracheal instillation of porcine pancreatic elastase (n = 7) or saline (n = 7). The subpleural microcirculation was assessed using FCFM in spontaneously breathing rats, through a 2mm thoracic window using a continuous aspiration system, after I.V. injection of fluorescein-dextran. FCFM sequences were recorded and the image analysis was performed separately by two observers, blindly to the animal group. Fluorescence intensity (FI), maximal intercapillary distances, and alveolar facets diameters measured with FCFM were compared between groups, and to ex vivo lung morphometric measurements (MIWD). Conclusion: FCFM allows the quantitative assessment of the microcirculation alterations due to emphysema in vivo.

Autofluorescence spectro-endomicroscopy of alveoli: comparative spectral analysis of healthy smocker volonteers and amiodarone-induced pneumonitis patients

Fluorescence endomicroscopy with spectroscopic analysis capability was used during bronchoscopy, at 488nm excitation, to record, simultaneously, autofluorescence (AF) images and associated emission spectra of the alveoli of healthy smoking volunteers (HS) and amiodarone treated non-smoker patients (ATNS). Alveolar fluorescent cellular infiltration was observed in both groups. Our objective was to assess the potential of spectroscopy in differentiating these two groups. Each normalized spectrum was modeled as a linear combination of several components: cellular flavins (FAD) and another cellular component, namely lipopigments, modelled by a Voigt profile, extracellular matrix (ECM) elastin and tobacco tar; HbO2 absorption was also taken into account. Besides the FAD and elastin contributions, the addition of a tobacco tar component is necessary to account for HS group spectral shape. The ATNS patients autofluorescence spectra result from the contribution of elastin, FAD and lipopigments, the tobacco tar component being found to zero. In conclusion, spectral analysis is able to differentiate between similar cellular infiltrated images from healthy smokers and ATNS patients.

Characterization of the autofluorescence of normal and tumoral esophageal epithelium cells

The objectives of this study are to characterize the autofluorescence spectra of normal and tumoral esophageal epithelial cells and to link the cellular spectra with a data basis of in vivo tissular spectra. Our preliminary results show that no difference in spectral distribution can be observed between squamous cell carcinoma, adenocarcinoma and normal cells. A statistical significant difference is observed between the average intensity of the raw spectra of the different cell types. Nucleus autofluorescence presents the same spectral shape as cytoplasm, but with lower intensity.