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

Abstract

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.

Spatiotemporal control of light in turbid media

“…The nanocrystals consist of barium titanate (BaTiO 3 ) with a tetragonal crystal structure and an average diameter of 200 nm. Prepared using the method described in [141], about 90% of the nanocrystals are not clustered and are well-separated from other particles on the glass coverslip, which we confirmed by imaging a sample of nanocrystals without the scattering medium. The layer of silica spheres (diameter 400 nm to 500 nm) has been applied on top of the nanocrystals by spray-coating a colloidal dispersion in ethanol, which after drying forms a rough surface with a varying layer thickness between L = 25 µm and L = 50 µm.…”

“…We consider a system with a second-order nonlinearity, which could be realized, e.g., by a nonlinear scatterer, such as microbubbles in acoustics [160,161] or nonlinear nanocrystals [141] in optics, combined with a time-integrated detection of the scattered second-order response. We calculate the detector response by…”

Evanescent-Field-Induced Second Harmonic Generation by Noncentrosymmetric Nanoparticles

“…The strong evanescent field of such waveguide modes allows the design of highly sensitive devices and provides the possibility for two-photon fluorescence excitation on comparatively large areas. [4,5] Inorganic non-centrosymmetric nanocrystals, often referred to as SHRIMPs (Second Harmonic IMaging Probes), have attracted increasing attention and stimulated a wide series of proposals for their applications in bioimaging [6,7,8], micromanipulation [9], and exploitation of their coherent optical response [10,11,12] since the appearance of the first studies on their nonlinear optical properties. Due to their sub-wavelength dimensions and related absence of phase-matching constraints [13], there is no spectral limitation for the nonlinear excitation of such nanoparticles.…”

KTiOPO4 Single Nanocrystal for Second-Harmonic Generation Microscopy

“…Different materials have been considered, e.g. BaTiO3 (Hsieh et al, 2009) and ZnO (Kachynski et al, 2008). In this view, we were among the pioneers in this domain, considering the well-known KTP material.…”