Reversible Two-Photon Photoswitching and Two-Photon Imaging of Immunofunctionalized Nanoparticles Targeted to Cancer Cells

Both photoswitchable fluorescent nanoparticles and photoactivatable fluorescent proteins have been used for super-resolution far-field imaging on the nanometer scale, but the photoactivating wavelength for such photochemical events generally falls in the near-UV (NUV) region (<420 nm), which is not preferred in cellular imaging. However, using two near-IR (NIR) photons that are lower in energy, we can circumvent such problems and replace NUV single-photon excitations (e.g., 390 nm) with NIR two-photon excitations (e.g., 780 nm). Thus, we have demonstrated that alternating 780 nm NIR two-photon and 488 nm single-photon excitations induces reversible on−off fluorescence switching of immunotargeted nanoparticles in the human breast cancer cell line SK-BR-3. Herein, two-photon absorption not only caused spiropyran−merocyanine photoisomerization within the particles but also imparted red fluorescence. In comparison with single-photon NUV excitations, two-photon NIR laser excitations can potentially reduce absorption-related photodamage to living systems because cellular systems absorb much more weakly in the NIR.