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-1s 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.

Identification of ex‐vivo confocal scanning microscopic features and their histological correlates in human skin

“…These include studies exploring digital staining, confocal‐histological correlations and methods to assimilate images of ex‐vivo CLSM and histology . Furthermore studies from medical fields other than dermatology have been concentrating on pathologies of different organ tissues and their evaluation with ex‐vivo confocal microscopy . To date there have been only few published dermatological articles which evaluated ex‐vivo CLSM images of basal cell carcinoma, its diagnosis and monitoring of therapy , as well as other types of non‐melanoma skin cancer, including squamous cell carcinoma .…”

Evaluation of YO-PRO-1 as an early marker of apoptosis following radiofrequency ablation of colon cancer liver metastases

“…Boffa et al (2005) used YP1 in the evaluation of apoptosis in pancreatic islets that were harvested and isolated from human cadaveric organs. Chagnon et al (2010) added YP1 directly to lung tissues within anesthetized mice to study cell survival following acute lung injury. YP1 staining performed in gastric glands, pieces of dissected skin, and isolated retina samples from mice and rats has resulted in the identification of a clear distinction between live and apoptotic cells (Ito et al 2010;Kohler et al 2010;Liao and Puro 2006).…”

“…Currently, YP1 is used primarily to stain cells in culture and rarely in ex vivo or in vivo tissue samples (Chagnon et al 2010;Kohler et al 2010;Santos et al 2006). However, YP1 can penetrate unsectioned, whole-mount, live tissue specimens and target dying cells from the very onset of the apoptotic process (Boffa et al 2005).…”

Using silkworms to establish alternative animal models for evaluation of drug-induced tissue injury

“…Thus, establishment of a new silkworm model has focused on a model that allows external observation of a silkworm while collecting continuous data from the same animal. Recently, in vivo imaging techniques based on fluorescence have progressed markedly and various animal models have been developed (21)(22)(23). A transgenic silkworm that displays GFP fluorescence has been developed using the GAL4-UAS system (24).…”