Luminescent markers play a key role in imaging techniques for life sciences since they provide a contrast mechanism between signal and background. We describe a new type of marker using second harmonic generation (SHG) from noncentrosymmetric BaTiO3 nanocrystals. These nanoparticles are attractive due to their stable, non-saturating and coherent signal with a femtosecond-scale response time and a broad flexibility in the choice of excitation wavelength. We use the coherent SHG signal from BaTiO3 nanoparticles for three-dimensional (3D) imaging without scanning. We built a harmonic holographic (H2) microscope which records digital holograms at the second harmonic frequency. Highresolution 3D distributions of these SHG markers in mammalian cells are successfully captured and interpreted by the H2 microscope. © 2009 SPIE.