Intravital Imaging of Attenuated Auxotrophic Mycobacterium tuberculosis in Murine Lung during Early Intravascular Infection.

Intravital microscopy enables direct visualization of dynamic cellular processes within intact tissues. However, its application to Mycobacterium tuberculosis (Mtb) has been limited by Biosafety Level 3 (BSL-3) containment requirements and the technical challenges of stabilizing the lung for high-resolution imaging. Here, we present a protocol that combines the thoracic Window for High-Resolution Imaging of the murine Lung (WHRIL) with mc27902, a genetically defined triple-auxotrophic Mtb strain approved for use under BSL-2 conditions. We also describe the generation of mc28471, a tdTomato-expressing derivative of mc27902, its preparation for intravenous infection, and application for intravital imaging in reporter mice. This system enables real-time visualization of early infection dynamics, including rapid bacillary entry into the pulmonary vasculature, aggregation, dissemination into the lung parenchyma, and macrophage uptake in the same lung microenvironment over the first 3 days post-infection. By enabling longitudinal imaging of bacilli and host-pathogen interactions at single-cell resolution, this approach overcomes key limitations associated with conventional imaging and high-containment models. This protocol provides a practical BSL-2-compatible platform for real-time intravital imaging of attenuated mycobacteria in the lung and establishes a foundation for mechanistic studies of bacterial physiology, host recognition, and immune-mediated clearance.