To this end, we first evaluated the ability of F-7 and catalyst 9 to visualize changes in formate levels in living cells upon exogenous addition of this one-carbon unit (Figure ). We treated two types of representative mammalian cell lines, NIH 3T3 (mouse fibroblast) and HCT 116 (human colorectal carcinoma) cells, with F-7 (10 μM) for 30 min followed by washing to remove excess fluorophore, with subsequent addition of catalyst 9 (10 µM) for 60 min, followed by exposure to various concentrations of formate (50 μM to 500 µM). Visualization by livecell imaging using confocal microscopy showed significant and dose-dependent ratiometic fluorescence changes in response to formate versus control cells (Figure ), demonstrating the ability of catalyst 9 and fluorophore F-7 to detect changes in exogenous formate levels in a cellular context. We next investigated whether catalyst 9 and fluorophore F-7 could be applied to image changes in endogenous formate fluxes based on nutrient-dependent alterations in one-carbon metabolism within the cell. First, we supplemented colon cancer HCT 116 wild-type (WT) cells with the native one-carbon donor serine (500 µM) for 60 min, washed, treated with fluorophore F-7 (10 μM) for 30 min, washed, labeled with catalyst 9 (10 μM) for 60 min, and then imaged the formate-dependent ratiometric responses by confocal microscopy. We observed a significant increase in the reduction of aldehyde F-7 to its alcohol counterpart F-10, indicating elevated levels of cellular formate and NAD(P)H in serine-supplemented cells relative to vehicle-treated controls (Figure ). This result is consistent with our expectations because excess serine may be metabolized to form up to 2 equivalents of NAD(P)H and 1 equivalent of formate (Figure ) in one-carbon metabolic cycles. Specifically, excess cellular serine may be metabolized to glycine and 5,10-methylene-tetrahydrofolate (5,10-CH2-THF) through the activity of SHMT1/2 (cytosolic and mitochondrial isoforms), while 5,10-CH2-THF may be oxidized to 10-formyltetrahydrofolate (10-CHO-THF) via MTHFD1 or MTHFD2/2L and then to formate/formic acid after spontaneous or enzymatic hydrolysis via MTHFD or MTHFD1L. Alternatively, 5,10-CH2-THF can spontaneously release formaldehyde, which can be oxidized directly to formate by ALDH2 and/or scavenged by glutathione (GSH) and oxidized to S-formylglutathione (GS-CHO) by ADH5; GS-CHO releases formate upon hydrolysis (Figure ). With these results in hand, we sought to further decipher formate-generating pathways in the cell by activity-based imaging using colon cancer cell lines with genetic alterations in key onecarbon metabolism enzymes. Specifically, we conducted comparative imaging experiments with HCT116 wild-type (WT) cells and HCT116 lines with either a double SHMT1/2 knockout or triple SHMT1/SHMT2/ADH5 triple knockout (tKO). SHMT1/2 double knockout HCT 116 cells have impaired serine consumption, blocking the predominant source of formate. And since formaldehyde can be an additional source of formate to support one-carbon units in the absence of SHMT1/2, we also tested cells with an additional ADH5 knock out to further deplete basal cellular formate pools. We used our standard imaging protocol (10 µM F-7 for 30 min, wash, 10 µM catalyst 9 for 60 min, wash, then imaging by confocal microscopy) to see the effect of removing enzymes upstream of formate/NAD(P)H production (Figure ). Gratifyingly, we observed a significant decrease in iridium-catalyzed ratiometric responses in the tKO HCT 116 cells (cells lacking SHMT1, SHMT2, and ADH5) relative to the wild-type controls, indicating deficient formate production by blocking multiple one-carbon metabolism pathway inputs in parallel (Figure ).