are responsible for enzymatic conversion between serine and glycine, reversibly catalyzing the transfer of a hydroxymethyl group from serine to tetrahydrofolate (THF). The resulting folate derivative from SHMT activity, 5,10-methylene-THF, is a functional 1C unit in cellular metabolism. 53, Interestingly, we have found that 5,10-methylene-THF also spontaneously decomposes to produce approximately two equivalents of formaldehyde in vitro, providing another source of 1C units that can also feed into 1C metabolic cycles via formate production. We sought to test the hypothesis that SHMT1/2 double knockout (dKO) cells would have depleted mitochondrial FA pools relative to wild-type (WT) cells, using an HCT-116 colon cancer cell line model. We also included an HCT-116 ADH5 KO model, as ADH5 is the main enzyme responsible for cytosolic clearance of FA, to determine whether perturbations to the cytosolic FA pool would have any crosstalk with the mitochondrial FA pool. Finally, as a key set of control experiments to assess whether MitoRFAP-2 could provide unique information on subcellular FA pools owing to its mitochondrial targetability, we also compared the fluorescent responses of a previously reported non-targeted parent FA sensor, RFAP-2, in these models under the same conditions. Indeed, the control probe RFAP-2, which does not possess the ability to selectively localize in mitochondria, is unable to detect changes in FA with subcellular resolution. The ability of the triphenylphosphonium targeting moiety to achieve rapid, selective, and efficient mitochondrial localization 48,55 of MitoRFAP-2 compared to the relatively slower rate of aza-Cope FA reactivity, coupled with a ratiometric response with an internal calibration standard to normalize signal, ensures that this probe is responsive to FA fluxes with superior subcellular resolution compared to the non-targeted control.