Microscopy Technique Images Thick, Multicellular Samples in 3D
Gradient light interference microscopy (GLIM), an add-on module to a commercial differential interference contrast (DIC) microscope, could provide a novel technique for extracting 3D information from thin and thick unlabeled specimens. In contrast to most microscopy techniques, GLIM can probe deep into thick samples by controlling the path over which light travels through the specimen. It can be used to produce images from multiple depths that can then be composited into a single 3D image.
A GLIM image of a rendered cow embryo that was cut through the center to reveal internal structures. Courtesy of Gabriel Popescu.
GLIM uses low-coherence interferometry to extract phase information from the specimen, which in turn can be used to measure cell mass, volume, surface area and the evolution of these measurements in time. Because it combines multiple intensity images that correspond to controlled phase shifts between two interfering waves, GLIM is capable of suppressing the incoherent background due to multiple scattering.
“When looking at thick samples with other methods, your image becomes washed out due to the light bouncing off of all surfaces in the sample,” said researcher Mikhail Kandel. “It is like looking into a cloud.”
At the smallest condenser aperture of the microscope, GLIM gives exact values of the quantitative phase for thin samples. At the largest condenser aperture, it can be used as a tomography method for obtaining time-lapse 3D information of thick samples.
Researchers from the University of Illinois at Urbana-Champaign tested GLIM on various samples, including beads, HeLa cells and live bovine embryos. The researchers believe that the technique could be used to help determine embryo viability before in vitro fertilization in humans.
“One of the holy grails of embryology is finding a way to determine which embryos are most viable," professor Matthew Wheeler said. “Having a noninvasive way to correlate to embryo viability is key. Before GLIM, we were taking more of an educated guess.”
Marcello Rubessa, Gabriel Popescu and Matthew B. Wheeler teamed up to produce 3D images of live cattle embryos that could help determine embryo viability before in vitro fertilization in humans. Courtesy of L. Brian Stauffer.
There is no universal marker for determining embryo health, Wheeler said.
“This method lets us see the whole picture, like a three-dimensional model of the entire embryo at one time,” said researcher Tan Nguyen.
As a label-free method, GLIM can be applied to imaging live cells and thick samples nondestructively over broad temporal and spatial scales. It is not limited by photobleaching and phototoxicity commonly associated with fluorescence microscopy. According to the researchers, it provides excellent optical sectioning. However, similar to other label-free imaging, GLIM lacks specificity. Therefore, the researchers envision that GLIM and fluorescence techniques will co-exist and offer the combined advantages of noninvasiveness and specificity. GLIM operates on the same optical path as the fluorescence channels, allowing a seamless transition between the two modalities.
GLIM could potentially become a valuable tool for in vitro fertilization, where contrast agents and fluorophores may impact the viability of the embryo. It has been used successfully to look at thick samples of brain tissue in marine life for neuroscience studies. The team plans to continue to collaborate with other biomedical researchers.
The research was published in Nature Communications
LATEST FROM PHOTONICS MEDIA