Three-dimensional harmonic holographic microcopy using nanoparticles as probes for cell imaging

Luminescent markers play a key role in imaging techniques for life science since they provide a contrast mechanism between signal and background. We describe a new type of marker using second harmonic generation (SHG) from noncentrosymmetric BaTiO_3 nanocrystals. These nanoparticles are attractive due to their stable, non-saturating and coherent signal with a femtosecond-scale response time and broad flexibility in the choice of excitation wavelength. We obtained monodispersed BaTiO_3 nanoparticles in colloidal suspensions by coating the particle surface with amine groups. We characterized the SHG efficiency of 90-nm BaTiO_3 particles experimentally and theoretically. Moreover, we use the coherent SHG signal from BaTiO_3 nanoparticles for three-dimensional (3D) imaging without scanning. We built a harmonic holographic (H^2) microscope which records digital holograms at the second harmonic frequency. For the first time, high-resolution 3D distributions of these SHG markers in mammalian cells are successfully captured and interpreted by the H^2 microscope