In vivo endoscopic autofluorescence microspectro-imaging of bronchi and alveoli

Autofluorescence bronchoscopy has been extensively avaluated during the past decade 1 : fluorescences bronchoscopy is based on the observation that premalignant and malignant bronchial mucosae fluoresce less than normal tissue. This allows detection of lesions that may have a normal appearance during conventional whitelight bronchoscopy 2 . However, the technique is hampered by the low specificity of the fluorescence defect, which ranges from 25 to 50% 1 . Coupled with autofluorescence bronchoscopy, the use of a method that would allow real-time non invasive histologic imaging-a principle that is also referred to as "optical biopsy"-may help to ensure higher yield biopsy samples, increase the specificity of the endoscopic technique, and potentially avoid unnecessary biopsy sampling or repeated procedures. Until now, the external diameter of the thinnest commercially available bronchoscope does not make it possible to image the respiratory tract beyond bronchioles smaller than 3mm in diameter in-vivo 4 . Therefore, the pathology of the terminal respiratory unit, from the small terminal and respiratory bronchioles down to the alveolar acini, was until recently assessed only in vitro, using techniques such as bronchoalveolar lavage and histology from transbronchial or open lung biopsies. No real time imaging was available. Fibered confocal microscopy is a new technique that can be used to image the microscopic structure of a living tissue 3 . Fibered confocal microscopy is based on the principle of confocal microscopy, which provides a clear, in-focus image of a thin section within a biological sample, where the microscope objective is replaced by a flexible fiberoptic miniprobe. In its fluorescence mode (fibered confocal fluorescence microscopy [FCFM]), the technique makes it possible to obtain high-quality images from endogenous or exogenous tissue fluorophores, through a fiberoptic probe of 1.4 mm diameter or less that can be introduced into the 2 mm working channel of a flexible bronchoscope. Human connective tissue of the peripheral lung is made of about 50% elastin in humans (5, 6). We therefore hypothesize that FCFM in conjunction with bronchoscopy is able to assess the microstructure of the alveolar elastin network in vivo. In this study, we use FCFM to obtain real time in-vivo imaging of the respiratory bronchi and alveoli in humans during bronchoscopy: we describe the in vivo autofluorescence microscopic structure of the normal bronchial mucosae and the alterations of the bronchial microstructure in precancerous lesions and we characterize the confocal microanatomy in alveoli in both smoker and non smoker healthy subjects.

The FCM imaging instrument, the F400S, is based on the fibered confocal fluorescence microscopy technology system developed by Mauna Kea Technology (Paris, France) : it is basically identical to the commercially available FCFM device called “Cellvizio ® -Lung (Mauna Kea Technologies) except that a spectroscopic channel to the device allows simultaneous recording of the spectrum and the microscopic images of the observed field of view.

As can be seen on figure 1, this device consists of four main elements : a Laser Scanning Unit, a miniaturized flexible fibered probe, a spectroscopic channel and a dedicated control and acquisition software. The laser scanning unit is composed mainly of a solid state laser diode (Laser Sapphire, Coherent, Santa Clara, CA, USA) emitting at 488 nm, an XY scanning system injecting the laser beam into the fiber bundle one fiber at a time and a photodetector. Each fiber, of 1.9 µm core diameter, acts as a point source and a point detector, thereby ensuring the confocal properties of the system. The bundle is scanned at high speed resulting in an frame rate of 12 images per second for a 896 X 640 pixels image. The illumination of the tissue is thus assured in turn by only one fiber which, in return, collects the fluorescence light. On return, the optical path is divided into 2 parts by a beamsplitter, 80% of the fluorescence signal being used for imaging and 20% for spectral analysis. The processed images are displayed on the monitor in real-time. The fluorescence signal, for image and spectral analysis as well, is recorded between 500 nm and 750 nm.

For the studies reported here, the microprobe is a Alveoflex Confocal Miniprobe ® (Mauna Kea Technologies) with an overall diameter of 1.4 mm compatible with the operating channel of standard bronchoscopes for in vivo proximal and distal lung tissues exploration. The fiber bundle is made of 30,000 fiber cores. This probe produces images from a layer of 0 to 50 µm in depth below surface with a lateral resolution of 3.5 µm. Because the probe is devoid of distal optics, the field of view used in the study strictly aquals the scanned surface (600 µm).

Raw data images are processed by a dedicated image processing software developed in-house by MKT (Medicell) which removes the fiber bundle pattern and provides s

…(Full text truncated)…

This content is AI-processed based on ArXiv data.