JULY 23, 2009--New York University scientists have developed a technique to record three-dimensional movies of microscopic systems, such as biological molecules, using holographic video. The label-free flow cytometry approach could improve medical diagnostics and drug discovery. It is described in a paper published by Optics Express, "Flow visualization and flow cytometry with holographic video microscopy."
To generate and record images, researchers in Professor David Grier's lab created a holographic microscope, based on a conventional light microscope. Instead of an incandescent illuminator, though, this system uses a collimated laser beam--a beam made of parallel light rays. A specimen is placed in the beam's path scatters light, creating a complex diffraction pattern. The scattered light overlaps with the original beam to create an interference pattern like overlapping ripples in a pool of water. The microscope magnifies the pattern of light and dark, and records it with a conventional digital video recorder (DVR). Each snapshot in the resulting video stream is a hologram of the specimen. Unlike a conventional photograph, each holographic snapshot stores information about the specimen's three-dimensional structure and composition.
To analyze the images, the researchers based their work on the quantitative Lorenz-Mie theory, which maintains that the way light is scattered can reveal the size and composition of the object that is scattering it. "We use that theory to analyze the hologram of each object in the snapshots of our video recording," explained Grier, who is part of NYU's Center for Soft Matter Research. "Fitting the theory to the hologram of a sphere reveals the three-dimensional position of the sphere's center with remarkable resolution. It allows us to view particles a micrometer in size and with nanometric precision--that is, it captures their traits to within one billionth of a meter."
"That's a tremendous amount of information to obtain about a micrometer-scale object, particularly when you consider that you get all of that information in each snapshot," Grier added. "It exceeds other existing technology in terms of tracking particles and characterizing their make-up-and the holographic microscope can do both simultaneously."
Because the analysis is computationally intensive, the researchers leverage the graphical processing units (GPUs) used in high-end computer video cards.
The team has already employed the technique for a range of applications, from research in fundamental statistical physics to analyzing the composition of fat droplets in milk.
More broadly, the technique creates a more sophisticated method to aid in medical diagnostics and drug discovery. At its most basic level, research in these areas seeks to understand whether or not certain molecular components--the building blocks of pharmaceuticals--stick together.
One approach, called a "bead-based assay," creates micrometer-scale beads whose surfaces have active groups that bind to the target molecule. Because of their small size, the challenge for researchers is to determine if these beads actually stick to the target molecules. The way this is traditionally done is to create yet another molecule--a fluorescent tag--that binds to the target molecule. This tag molecule is typically time-consuming and costly to produce.
The holographic imaging technique, with its magnification and recording capabilities, allows researchers to observe molecular-scale binding without a tag, saving both time and money. Requiring just one microscopic bead to detect one type of molecule, holographic video microscopy promises a previously unattainable level of miniaturization for medical diagnostic tests and creates possibilities for running very large numbers of sensitive medical tests in parallel.
For more information see the paper, Flow visualization and flow cytometry with holographic video microscopy, at Optics Express.
Posted by Barbara G. Goode, [email protected], for BioOptics World.