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Investigating small-molecule dynamics within microbes is essential for comprehensive studies of microbial function. Both intra-organism and inter-organism small molecule dynamics play critical roles in microbial physiology, symbiosis, and disease. However, monitoring these dynamics remains highly challenging using most existing techniques. Fluorophore-based genetically encoded biosensors are powerful tools for tracking small-molecule dynamics in vivo and hold high potential for driving new discoveries. These biosensors are most commonly used in fluorescence imaging, often in combination with perfusion devices that allow precise control over environmental conditions. When integrated with advanced imaging techniques, this approach provides high-resolution, spatially and temporally resolved data, enabling insights into single-cell microbial responses. Despite their promise, implementing such biosensors remains technically challenging. Understanding the key steps is crucial for broader adoption. Here, we present a protocol designed to support the effective deployment of newly engineered biosensors into microbes for quantitative ratiometric fluorescence imaging under controlled conditions.

Investigating small-molecule dynamics within microbes is essential for comprehensive studies of microbial function. Both intra-organism and inter-organism small molecule dynamics play critical roles in microbial physiology, symbiosis, and disease. However, monitoring these dynamics remains highly challenging using most existing techniques. Fluorophore-based genetically encoded biosensors are powerful tools for tracking small-molecule dynamics in vivo and hold high potential for driving new discoveries. These biosensors are most commonly used in fluorescence imaging, often in combination with perfusion devices that allow precise control over environmental conditions. When integrated with advanced imaging techniques, this approach provides high-resolution, spatially and temporally resolved data, enabling insights into single-cell microbial responses. Despite their promise, implementing such biosensors remains technically challenging. Understanding the key steps is crucial for broader adoption. Here, we present a protocol designed to support the effective deployment of newly engineered biosensors into microbes for quantitative ratiometric fluorescence imaging under controlled conditions.