As part of a strategy to achieve large two-photon absorptivity in fluorene-based probes, a series of donor-acceptor-donor (D-A-D) type derivatives were synthesized and their two-photon absorption (2PA) properties investigated. The synthesis of D-A-D fluorophores was achieved by efficient preparation of key intermediates for the introduction of central electron acceptor groups. To accomplish the synthesis of two of the new derivatives, a high-yield method for a one-step direct dibromomethylation of phenyl sulfide was developed. The linear and nonlinear optical properties, including UV-vis absorption, fluorescence emission, fluorescence anisotropy, and two-photon absorption (2PA), of the new D-A-D compounds were measured and compared to their D-A or D-D counterparts. Fully conjugated acceptor moieties in the center of the D-A-D fluorophore led to the greatest increase in the 2PA cross section, while weakly conjugated central acceptors exhibited only a modest increase in the 2PA cross section relative to D-A diploar analogs. Encapsulation of the new probes in Pluronic F 108NF micelles, and subsequent incubation in HCT 116 cells, resulted in very high lysosomal colocalization (>0.98 colocalization coefficient) relative to commercial Lysotracker Red, making the micelle-encapsulated dyes particularly attractive as fluorescent probes for two-photon fluorescence microscopy lysosomal imaging.