Donor-acceptor BODIPY dyads, functionalized at the 2 and 6 positions with benzyl ester (BDP-DE) or carboxylic acid (BDP-DA) groups, were synthesized, and their optoelectronic properties were investigated. Carbonyl groups were found to increase the reduction potential of the BODIPY core by 0.15-0.4 eV compared to regular alkyl-substituted BODIPYs. These compounds exhibited efficient intramolecular charge separation and triplet state formation through the spin-orbit charge transfer intersystem crossing (SOCT-ISC) process, achieving singlet oxygen quantum yields of up to 92 %, depending on the solvent polarity. Notably, the fluorescence and singlet oxygen generation of BDP-DAs were found to depend on the ionization state of the carboxylic groups. Time-resolved fluorescence measurements revealed that complexation of BDP-DAs with bovine serum albumine (BSA) significantly extended their excited state lifetimes. Fluorescence lifetime imaging microscopy (FLIM) studies of human colorectal carcinoma (HCT116) cells and pig small intestinal organoids (enteroids) provided insights into subcellular localization. The diacid with 2,4-dimethoxyphenyl group at the meso-position (DA1) displayed longer lifetimes in lipid-droplet-like structures and shorter lifetimes in cytoplasmic regions. The diacid containing a meso-anthracenyl group (DA2) formed 'islands' in cell monolayers, exhibiting a distinct lifetime gradient from the periphery to the center. These results highlight the potential of donor-acceptor BODIPYs as fluorescent probes for biological imaging, particularly in revealing subtle differences in cellular environments.