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In vivo confocal fluorescence endomicroscopy of lung cancer

Abstract : With recent advances in computer and optics engineering, diagnostic endoscopy of the respiratory tract has now entered the era of microscopic imaging.Fibered confocal fluorescence microscopy (FCFM) is based on the principle of confocal microscopy, where the microscope objective has been replaced by a thin fiberoptic probe. FCFM has a lateral resolution of 3µm, a field of view of 600 µm and produces real-time imaging at 9 frames per second in contact with the probe tip. FCFM has the capability to image in real time the epithelial and subepithelial layers of the proximal 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 of FCFM include « optical biopsy » assessment of early bronchial cancers, bronchial wall remodelling evaluation, 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 strategy may help in the future to enable early diagnosis, rapid typing of molecular markers and assessment of therapeutic outcome in many lung diseases. This paper reviews the capabilities and possible limitations of confocal microendoscopy for proximal and distal lung exploration with special focus on lung cancer imaging in-vivo. Introduction : Confocal microscopy allows in-vivo optical sectionning of cells and tissue with enhanced lateral and axial resolutions (1, 2). Translating the principles of confocal microscopy into the clinic for endomicroscopy is currently the subject of significant scientific efforts (1, 2), which recently ended in the availability of commercial systems for both animal (3-5) and human in vivo explorations (6-8).Confocal endomicroscopes aim at providing to the clinician « optical biopsies », i.e. in vivo microscopic imaging, of a living tissue during endoscopy (9, 10). Such systems have recently been applied to the in-vivo microscopic imaging of both the proximal (7) and distal respiratory systems (8). Current commercially available confocal endomicroscopy system for lung exploration (Cellvizio®, Mauna Kea Technologies, Paris, France) uses the principle of proximal scanning in which the illumination light scans the proximal part of a coherent fiber bundle or miniprobe. This bundle conducts the light back and forth from the imaged area at the tip of the miniprobe (11). This fiber bundle or catheter based system, also described as « fibered confocal fluorescent microscopy (FCFM) » uses very thin and flexible miniprobes (300µm to 2 mm in diameter) that can contain up to 30,000 compacted microfibers. Similar to conventional confocal microscopes, FCFM uses two rapidly moving mirrors to scan the microfibers across the coherent fiber bundle in a raster fashion. Each microfiber, which is scanned one at a time by the laser light, acts as a light delivery and collection system and is, in essence, its own pinhole. The main advantages of this design is the very small size and the flexibility of the probe that can reach the more distal part of the lungs (8), as well as the fast image collection speed that helps to avoid artifacts due to tissue movement. Specific miniprobes for bronchial and alveolar imaging have a diameter of 1mm that can enter the working channel of any adult bronchoscope. These miniprobes are devoid of distal optics and have a depth of focus of 0-50 µm, a lateral resolution of 3µm for a field of view of 600 x 600 µm. The system produces endomicroscopic imaging in real time at 9 to 12 frames / second. Two different wave lengths are currently available. The Cellvizio 488 nm is used for autofluorescence imaging of the respiratory tract as well as for fluorescein induced imaging of the GI tract (7, 8, 12). Another device at 660 nm excitation can be used for epithelial cell imaging after topical application of exogenous fluorophores such as methylene blue (13-15). The main limitations are related to the maximal imaging capabilities (30,000 pixels) which restrict the lateral resolution to the fiber intercore distance (3µm), and the fact that the focus point of the system cannot be adjusted. Interpretation of the data also relies on the fluorescence properties of the imaged tissue. Human In-vivo confocal microimaging of the normal lung using FCFM . FCFM imaging of the proximal bronchi. FCFM can easily be performed during a fiberoptic bronchoscopy under local anesthesia (7, 8) . The technique of in-vivo bronchial FCFM imaging is simple : the miniprobe is introduced into the 2mm working channel of the bronchoscope and the probe tip applied onto the bronchial mucosae under sight control. The depth of focus being 50 µm below the contact surface, the system can image the first layers of the bronchial subepithelial connective tissue, presumably the lamina densa and the lamina reticularis (7). At 488 nm excitation, FCFM produces very precise microscopic fluorescent images of the bronchial basement membrane zone (Figure 1). FCFM bronchial microimaging reveals a mat of large fibers mainly oriented along the longitudinal axis of the airways with crosslinked smaller fibers, as well as larger openings – 100µm to 200 µm - corresponding to the bronchial glands origins. In vivo, the technique also makes it possible to record high resolution images of small airways such as terminal bronchioles, which are recognizable by the presence of the helicoidal imprint of the smooth muscle on the inner part of the bronchiole. (7) Fluorescence properties of the bronchial mucosae at 488 nm excitation are determined by the concentration of various cellular and extracellular fluorophores, including the intracellular flavins, that could originate from the epithelial cells, and specific crosslinks of collagens and elastin present in the subepithelial areas (1, 16, 17). Microspectrometer experiments coupled with FCFM imaging, have clearly demonstrated that the main fluorescence signal emitted after 488nm excitation from both bronchial and alveolar human system originates from the elastin component of the tissue (7, 8, 18). Indead, flavin cellular autofluorescence appears too weak to allow imaging of the epithelial layer using 488nm FCFM without exogenous fluorophore (19). Similarly, the collagen fluorescence does not significantly affect the FCFM image produced at 488nm, the fluorescence yield of collagen at this wave length being at least one order of magnitude smaller than that of elastin. As a result, 488nm excitation FCFM specifically images the elastin respiratory network that is contained in the basement membrane of the proximal airways and participates to the axial backbone of the peripheral interstitial respiratory system. In the future, it is possible that a modified FCFM device using several wavelengths (20), or devices based on a multiphoton approach (2) may enable imaging of collagen, elastin and flavins simultaneously. Distal lung FCFM imaging in-vivo : from the distal bronchioles down to the lung acini. In the acinus, elastin is present in the axial backbone of the alveolar ducts and alveolar entrances, as well as in the external sheath of the extra-alveolar microvessels (21, 22). FCFM acinar imaging is easily obtained by pushing forward the probe a few centimeters after the endoscope is distally blocked into a sub-segmental bronchi. When progressing towards the more distal parts of the lungs, the entry into the alveolar space is obtained by penetration through the bronchiolar wall. Alveolar fluorescence imaging in active smokers dramatically differs from imaging in non-smokers. The alveolar areas of smokers are usually filled with highly fluorescent cells corresponding to alveolar fluorescent macrophages, the presence of which appears very specific of active smoking. (8). In situ alveolar microspectrometric measurements have been performed in active smokers, which evidenced that the main fluorophore contributing to the FCFM alveolar signal corresponds to the tobacco tar by itself, explaining this difference (8, 18). Potential clinical applications of bronchoalveolar confocal imaging. Preliminary studies have shown that per endoscopic FCFM could be used to study specific basement membrane remodeling alterations in benign or malignant / premalignant bronchial alterations (7). The FCFM microstructure of the bronchial walls underlying premalignant epithelia is significantly altered. In these precancerous conditions, the elastic fibered pattern of the lamina reticularis is absent or disorganized in almost every preinvasive lesions (Figure 1). This supports the hypothesis of an early degradation of the basement membrane components in preinvasive bronchial lesions. However, while this observation shed some light on the origin of the autofluorescence defect in precancerous bronchial lesions, the absence of epithelial cell visualisation does not allow the technique to differentiate between the different grades of progression of the precancerous bronchial lesions such as metaplasia / dysplasia / carcinoma in situ. In order to be successfully applied to the exploration of precancerous / cancerous bronchial epithelial layer, the FCFM technique would need to be coupled with the use of an exogenous non toxic fluorophore. Ex-vivo studies have shown that the resolution of the system is not a limitation for nuclear or cellular imaging (7, 8) . Exogenous fluorophores that could be activated at 488 nm such as Acriflavin - a putative mutagen agent - or fluorescein solution, which does not stain the nuclei (23) are not approved for intrabronchial use. Recently, Lane et al. have used a confocal microendoscope prototype at 488 nm excitation and topical physiological PH cresyl violet to provide cellular contrast in the bronchial epithelium both in-vitro and in-vivo (24). Methylene blue is a non toxic agent which is commonly used during bronchoscopy for the diagnostic of broncho-pleural fistulae. MB is also used in gastroenterology for chromo-endoscopic detection of precancerous lesions (25-27), as well as for in-vivo microscopic examination of the GI tract and bronchus using a novel endocytoscopic system (28, 29). MB is a potent fluorophore which enters the nuclei and reversibelly binds to the DNA, before being reabsorbed by the lymphatics. In order to give a fluorescent signal, MB needs to be excited around 660 nm, and is therefore accessible to FCFM intravital imaging using this excitation wave length. Preliminary study has demonstrated that Cellvizio 660 / topical methylene blue makes it possible to reproducibly image the epithelial layer of the main bronchi. Future studies using this technique could make it possible to differentiate normal, premalignant and malignant alterations at the microscopic level. If this strategy is successful, FCFM may become a very powerful technique for in vivo diagnostic of early malignant and premalignant conditions of the bronchial tree, allowing the analysis of both the epithelial and subepithelial layers during the same procedure. Coupled to electromagnetic navigation or radial EBUS, FCFM could also image microstructural and cellular patterns of peripheral solide lung nodules in-vivo at both 448nm and 660 nm (14, 15) (figure 2). Until now, confocal microendoscopy of the airways has only used endogenous autofluorescence or simple fluorescent contrast agents to visualize the in-vivo cellular and interstitial organization of the airways and distal lung parenchyma. In the future, using molecular contrast coumponds, it will be possible to extend the range of biomarkers that may be imaged. Pilot studies exploring this strategy have recently been published, that provided specific confocal imaging of molecular probes in precancerous conditions of the oral cavity ex-vivo (30) and of colonic dysplasia in-vivo (31). Coupled to FCFM, molecular imaging may help in the future to enable early diagnosis, rapid typing of molecular markers and assessment of therapeutic outcome in many lung diseases.
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https://www.hal.inserm.fr/inserm-00467854
Contributor : Céline Breton <>
Submitted on : Monday, March 29, 2010 - 1:25:45 PM
Last modification on : Saturday, February 15, 2020 - 1:43:38 AM

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  • HAL Id : inserm-00467854, version 1

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Luc Thiberville, Mathieu Salaün, Samy Lachkar, Stéphane Dominique, Sophie Moreno-Swirc, et al.. In vivo confocal fluorescence endomicroscopy of lung cancer. 13th world conference on lung cancer, Aug 2009, San Francisco, United States. ⟨inserm-00467854⟩

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