Since holography was invented 60 years ago, an aberration-free, three-dimensional microscope has long been sought. Now such a four-dimensional microscope is within reach that would enable 3-D movies of cellular life at the nano-scale. For the 3-D movies visit: http://ust.caltech.edu/movie_gallery/
The California Institute of Technology (Pasadena, CA) earned U.S. Patent 7,813,016 for its method of nonlinear harmonic holography enabling 4-D microscopy.
Credit: O.-H. Kwon, A. H. Zewail, Science 2010,328, 1668-1673/Caltech
A harmonic holography (H2) technique and system that combines holography and nonlinear optics that enables holographic recording of 3D images with femtosecond framing time are provided. The H2 technique records holograms with second harmonic (SH) signals scattered off specialized nanocrystals that are functionalized to label specific protein or other biomolecules in a living organism.
The capability of generating second harmonic radiations is specific to materials with noncentrosymmetric crystalline structures only, and .chi(2) vanishes for all other types of materials. Therefore, a sharp contrast is formed when particles of noncentrosymmetric structures are dispersed in a medium of other species, pumped at a fundamental frequency, and imaged at the second harmonic frequency. The new scheme provides a sound basis for a new type of contrast microscopy with enormous potential in molecular biomedical imaging., according to inventors Ye Pu (Pasadena, CA) and Demetri Psaltis (St-Sulpice, CH)
Optomechanical and crystallization phenomena visualized with 4D electron microscopy: Interfacial carbon nanotubes on silicon nitride, [Web link] [GIF] [AVI] [MPG] [MP4]
Credit: D. J. Flannigan, A. H. Zewail, Nano Lett. 2010, 10, 1892-1899/Caltech
The harmonic holograph system and method includes the following components/steps: a first optical splitter for separating said an optical pulse excitation an reference pulses; a sample including a target of interest having a nanoparticle incorporated therein for generating one coherent nonlinear optical emission when radiated; an optical filter for blocking all emissions from said sample except nonlinear emissions; a nonlinear homodyne reference generator, such as a frequency doubler, for converting the reference pulse into one coherent nonlinear reference pulse; a second optical splitter for recombining the split pulses into one holographic signal; and a detector for recording the holographic signal. In such an embodiment either multiple or a single excitation pulse may be used.
The current harmonic holography (H2) technique provides a unique means to achieve contrast in the coherent domain, enabling the use of holography in ultrafast four-dimensional contrast imaging with high spatial and temporal resolution. The H2 technique of the current invention records holograms using second harmonic signals scattered from SHG nanocrystals and an independently generated second harmonic reference. Exemplary experiments show that the technique of H.sup.2 has unique advantages over direct imaging, including numerical aberration compensation and device noise canceling.
In short, the Caltech H2technique has proven to be a powerful technique to achieve aberration-free, shot noise-limited performance with low photon count signals, providing a technique that allows for molecular biomedical imaging applications.
When combined with modern high-repetition rate pulsed laser and fast imaging devices, a time sequence of consecutive 3D images can be captured, forming a 4D microscope. Successful 4D holographic imaging has been achieved recently in the context of fluid velocity measurement.
Despite the technical advancements, and prior to Caltech’s work, holographic microscopes have not yet been widely deployed in biomedical research because of the lack of specificity. In a microscopic setting with biological samples, holography alone suffers severe background scatterings from irrelevant cell structures. A holographic microscope would capture all scattering entities in the viewing field faithfully, but indiscriminately. On the other hand, the signals of interest (often from small nanostructures like protein molecules) are usually very weak and buried in the strong ambient scatterings from much larger organelles.
The Caltech discovery may now move 4-D microscopes into mainstream research when they enter into a manufacturing phase.