A two-photon excitation difluoroboron dye activated in the near infrared region for biological image analysis was synthesized in this study. Cell affinity, membrane interaction, and the endocytosis pathway of PAMAM dendrons were investigated using only covalent two-photon dyes (TPD) at the periphery of the PAMAM dendrons. Generation 3 TPD-labeled PAMAM dendrons (BG3) exhibited multivalency binding on the HeLa cell membranes from the cell affinity study in the fixation of HeLa cells. Photo-stimulation on the membrane of the living HeLa cell was observed by confocal optical imaging in situ, using the two-photon model, when incubated with BG3. Analyses of cell membrane integrity via lactate dehydrogenase (LDH) assay confirmed membrane damage at two photon excitation model. However, no variation in the cell was observed using the one-photon excitation model. These results indicated a high degree of dendrons uptake by cells through binding to the cell membrane following the endocytotic pathway. Furthermore, the wide excitation fluorescence spectrum of difluoroboron dye provides dual imaging with which to study the endocytosis of TPD-labeled PAMAM dendrons using a single near infrared laser. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.