Bacteria rely on protein systems for regulation in response to external environmental signals. Single-molecule fluorescence imaging and tracking has elucidated the complex mechanism of these protein systems in a variety of bacteria. We recently investigated Vibrio cholerae, the Gram-negative bacterium responsible for the human cholera disease, and its regulation of the production of toxins and virulence factors through the membrane-localized transcription factors TcpP and ToxR. These experiments determined that TcpP and ToxR work cooperatively under steady-state conditions, but measurements of how these dynamical interactions change over the course of environmental perturbations were precluded by the traditional preparation of bacterial cells confined on agarose pads. Here, we address this gap in technology and access single-molecule dynamics during real-time changes by implementing two alternative sample preparations: microfluidic devices and chitosan-coated coverslips. We report the first demonstration of single-molecule tracking within live bacterial cells in a microfluidic device. Additionally, using the chitosan-coated coverslips, we show that real-time environmental changes impact TcpP-PAmCherry dynamics, activating a virulence condition in the bacteria about 45 min after dropping to pH 6 and about 20 min after inducing ToxR expression. These new technology advances open our ability for new experiments studying a variety of bacteria with single-molecule imaging and tracking during real-time environmental perturbations.