Visualizing early Mycobacterium tuberculosis interactions with murine lung macrophages using intravital imaging

Intravital microscopy enables direct visualization of dynamic cellular processes within intact tissues, but 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 a genetically defined, triple-auxotrophic Mtb strain (mc27902) approved for use under BSL-2 conditions. We describe the construction of a tdTomato-expressing derivative (mc28471) preparation of bacteria for intravenous infection and intravital imaging in reporter mice. This system enables visualization of rapid bacterial entry into the pulmonary vasculature, subsequent aggregation, and vascular occlusion, dissemination into the lung parenchyma, and macrophage uptake over three days post-infection. This protocol provides the first practical platform for real-time intravital imaging of mycobacteria in the lung and establishes a foundation for mechanistic studies of bacterial physiology, host recognition, and immune-mediated clearance using safe Mtb surrogates. SummaryThis protocol describes a biosafety level 2 (BSL-2)-compatible intravital imaging platform for visualizing Mycobacterium tuberculosis (Mtb) in the intact murine lung at single cell resolution. By combining the Window for High-Resolution Imaging of the murine Lung (WHRIL) with a fluorescently labeled, genetically defined triple auxotrophic Mtb strain (mc27902), this approach overcomes long-standing biosafety and technical barriers that have prevented real-time imaging of mycobacterial infection in vivo. The method enables direct visualization of early bacterial localization, aggregation, vascular interactions, and macrophage uptake during the initial hours to days following infection, providing a practical foundation for mechanistic studies of host-pathogen interactions under safe experimental conditions.