Tomographic phase microscopy can provide detailed images of the activity inside a living cell, without the use of fluorescent markers or other contrast agents that could interfere with the results, according to Michael Feld, director of the George R. Harrison Spectroscopy Laboratory at the Massachusetts Institute of Technology (MIT; Cambridge, MA) and a professor of physics. Feld’s research group built imaging devices and interferometers and combined them with a standard CCD camera to develop an imaging technique similar to x-ray CT (computed-tomography) scans that enables scientists to create the first 3-D images of a living cell.
“Light scattering provides important information about the size distribution of substances inside the cell when considering subcellular surgery,” Feld said. “Once we have a tomographic image, we can see how cellular functions change. For the first time, we can study the functional activity of living cells in their native state.”
To create a 3-D image, the researchers combined 100 2-D images taken from different angles and obtained 3-D maps of the refractive index of the cell’s organelles in less than a second. An interferometer provided quantitative phase images from time-dependent interference patterns induced by the frequency shifting of a reference beam relative to the sample beam. A HeNe laser beam (633 nm) was divided into sample- and reference-arm paths by a beamsplitter. A galvanometer-mounted tilting mirror varied the sample’s angle of illumination. In the reference arm, the laser beam passed through two acousto-optic modulators that shifted the frequency by 1250 Hz. A second beamsplitter recombined the sample and reference laser beams, forming an interference pattern at the image plane. For each angle of illumination, a CMOS camera recorded four images at 500 frames. Phase images were calculated by applying phase-shifted interferometry with special software algorithms.
No cell preparation needed
Feld’s team has used the quantitative, high-resolution technique to create 3-D images of cervical-cancer cells (see Fig. 1), showing internal cell structures, as well as the
C. elegans worm and other organism types (see Fig. 2). Because the source of contrast is the refractive index of subcellular components, tomographic phase microscopy can be used to study live cells without any preparation. With other 3-D imaging techniques, the samples must be fixed with chemicals, frozen, stained with dyes, metallized, or otherwise processed to provide detailed structural information.