AbstractRespiratory diseases, such as pulmonary infections, are an important cause of morbidity and mortality worldwide. Preclinical studies often require invasive techniques to evaluate the extent of infection. Fibered confocal fluorescence microscopy (FCFM) is an emerging optical imaging technique that allows for real-time detection of fluorescently labeled cells within live animals, thereby bridging the gap between in vivo whole-body imaging methods and traditional histological examinations. Previously, the use of FCFM in preclinical lung research was limited to endpoint observations due to the invasive procedures required to access lungs. Here, we introduce a bronchoscopic FCFM approach that enabled in vivo visualization and morphological characterisation of fungal cells within lungs of mice suffering from pulmonary Aspergillus or Cryptococcus infections. The minimally invasive character of this approach allowed longitudinal monitoring of infection in free-breathing animals, thereby providing both visual and quantitative information on infection progression. Both the sensitivity and specificity of this technique were high during advanced stages of infection, allowing clear distinction between infected and non-infected animals. In conclusion, our study demonstrates the potential of this novel bronchoscopic FCFM approach to study pulmonary diseases, which can lead to novel insights in disease pathogenesis by allowing longitudinal in vivo microscopic examinations of the lungs.