25There is an urgent need for point-of-care tuberculosis (TB) diagnostic methods that are fast, 26 inexpensive, and operationally simple. Here, we report on a bright solvatochromic dye trehalose 27 conjugate that specifically detects Mycobacterium tuberculosis (Mtb) in minutes.3-28 hydroxychromone (3HC) dyes, known to yield high fluorescence quantum yields, exhibit shifts in 29 fluorescence intensity in response to changes in environmental polarity. We synthesized two 30 analogs of 3HC-trehalose conjugates (3HC-2-Tre and 3HC-3-Tre) and determined that 3HC-3- 31 Tre has exceptionally favorable properties for Mtb detection. 3HC-3-Tre-labeled mycobacterial 32 cells displayed a 10-fold increase in fluorescence intensity compared to our previously reports on 33 the dye 4,4-N,N-dimethylaminonapthalimide (DMN-Tre). Excitingly, we detected fluorescent Mtb 34 cells within 10 minutes of probe treatment. Thus, 3HC-3-Tre permits rapid visualization of 35 mycobacteria that ultimately could empower improved Mtb detection at the point-of-care in low-36 resource settings. 37 38 With 1.2 million deaths and 10 million new cases in 2018, tuberculosis (TB) is the most 39 lethal infectious disease in the world (1). Early detection of the bacterium Mycobacterium 40 tuberculosis (Mtb), the causative agent of TB, followed by appropriate treatment could prevent 41 most deaths (2). The gold standard for TB diagnosis remains a labor-intensive culture test that 42 requires weeks of incubation time in specialized facilities. Although more rapid tests are available, 43 they present several important limitations. PCR-based tests are expensive and require skilled 44 technicians. Microscopy-based methods are attractive in low-resource settings as they are low-45 cost, have fast turnaround times, and report on people at greatest risk of transmission and death 46 (3). As a result, the sputum smear microscopy test is the most widely used technique for TB 47 diagnosis. The century-old smear test is based on the susceptibility of fluorescent auramine dye 48 or colored Ziehl-Neelson (ZN) stain to accumulate within the highly hydrophobic mycobacterial 49 cell wall (4-7). While effective for identification of Mtb cells, this process requires multiple wash 50 steps to reduce non-specific background fluorescence, or in the case of the ZN test, a rigorous 51counterstaining procedure so that stained Mtb cells can be visualized (8,9). Moreover, the smear 52 test does not distinguish live from dead cells, and this capability is vital in order to assess 53 treatment efficacy early and accurately (2). 54In the last decade, we and others have leveraged the trehalose metabolism of 55 mycobacteria to mark them for detection by various imaging methods (10-20). Exogenous 56 trehalose molecules can be directly mycolylated at the 6 position by antigen 85 (Ag85) enzymes 57 to form trehalose monomycolates (TMM) that are inserted into the mycobacterial cell wall, termed 58 the mycomembrane (10). Researchers have shown that Ag85 enzymes are promiscuous enough 59 to ...