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One of the most exciting features of fluorescent probes is their rapid evolution from simple tracers to functional indicators in diverse research fields [1]. In mammalian cells, fluorescence has been coupled to synaptic transmission [2,3], neuronal differentiation [4], apoptotic cell death [5], fate mapping in different immune cells [6,7], and functional changes in cell physiology, for example, the induction of cytokines [8]. In microbial cells, fluorescence has long been utilized as a tracer for pathogenic microbes, revealing microbial localization, residence, and spread in host tissues [9]. However, measuring functional outcomes during individual encounters with host immune cells remains challenging. In this Pearl, we describe fluorescence-based approaches, which we term “functional microbial reporters,” designed to relay changes in microbial physiology that occur in host cell and tissue environments. This class of microbial reporters typically harnesses fluorescence emission at two wavelengths. One of these signals functions as an invariant tracer of microbial cells and is generally unaffected by hostile conditions that are encountered during interactions with host cells, such as reactive oxygen species or acidified compartments. The other signal varies according to a change in microbial physiology or residence in the host. This second signal may act as a ratiometric indicator (i.e., a shift in the excitation or emission spectrum) to reflect a continuous variable such as the microbial growth rate. Alternatively, the second signal may act as an on-off indicator (i.e., extinction in the emission spectrum) to reflect a binary variable such as microbial viability. Three specific types of functional microbial reporters will be discussed, including reporters that distinguish live and killed fungal cells [10], indicate the mode of host cell entry by an intracellular parasite [11], and measure bacterial growth rates in host tissues [12–14].

Deploying FLAREs to Visualize Functional Outcomes of Host—Pathogen Encounters