In vivo intravital endoscopic confocal fluorescence microscopy of normal and acutely injured rat lungs

Abstract

We present a new lung imaging technique based on endoscopic confocal fluorescence microscopy (ECFM), which is a new method that is able to provide cellular and structural assessment of living tissue using a small confocal probe in direct contact with the visceral pleura. To observe distal airspace structure and cellular condition in normal and injured lungs (hyperoxic and bleomycin challenged), we used fluorescent-specific marker contrast and ECFM. Alveolar space ECFM with spectral analyses were performed at 488-nm excitation using FITC-labeled markers or naturally fluorescent dyes. The normal lung was compared with the sick lung, where our in vivo imaging experiments correlated well with results obtained with corresponding ex vivo conventional assays. Four main elements pertaining to the acute lung injury/acute respiratory distress syndrome (ALI/ARDS) pathophysiology and established early key events were specifically studied: alveolar epithelial membrane phenotype, lung cell apoptosis, neutrophil recruitment, and edema. ECFM allowed visualization of (i) fine-tuned ultrastructural lectin (RCA-1) and sialoglycoprotein (RTI40) epithelial cell membrane expression, (ii) YO-PRO-1-related DNA linking of lung cell apoptosis, (iii) PKH2 green fluorescent cell linker-labeled neutrophil tracking in lung microcirculatory network and airspaces, (iv) FITC-dextran plasma contrast and extravasation with edema formation. ECFM provides reliable results to corresponding ex vivo fluorescent methods. ECFM, using the minimally invasive Five-1® optical instrument and specific fluorescent markers, is able to provide real-time potentially useful imaging of live unfixed normal and injured lung tissue with promising developments for improving bedside diagnostic and decision-making therapeutic strategy in patients with ALI.