Most commonly used methods for three-dimensional (3D) fluorescence microscopy make use of sectioning techniques that require that the object be physically scanned in a series of two-dimensional (2D) sections along the z axis. The main drawback in these approaches is the need for these sequential 2D scans. An alternative approach to fluorescence imaging in three dimensions has been developed that is based on optical scanning holography. This novel approach requires only a 2D scan to record 3D information. Holograms of 15-microm fluorescent latex beads with longitinal separation of ~2 mm have been recorded and reconstructed. To our knowledge, this is the first time holograms of fluorescent specimens have been recorded by an optical holographic technique.

Twin-image elimination in the context of optical scanning holography has recently been proposed. The proposed technique involves simultaneously acquiring sine and cosine Fresnel holograms. A complex hologram is then formed by complex addition of the holograms, and twin-image rejection is predicted by computer simulations. An experimental verification of the technique by optical acquisition of the two holograms and subsequent reconstruction of the complex hologram digitally is reported. Three-dimensional image reconstruction without twin-image noise is demonstrated.

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.