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.