Virtual histology method bolsters visualization of mouse embryos

Susannah J. Veith

iologists use gene targeting in mice to further understand the causes of birth defects and childhood cancer. However, the technique they use entails careful and tedious histological sectioning of early embryos, a task that is not only time-consuming, but also quite challenging. Although the introduction of magnetic resonance microscopy has enabled a speedier process and higher-resolution scans, its use remains limited by the requirement for specialized and expensive equipment.

Now researchers at the University of Utah in Salt Lake City and the University of Texas Health Science Center at San Antonio have proved that they can overcome these boundaries by developing a method of obtaining virtual histology using x-ray microscopic CT (microCT).

“I had already developed expertise [in] CT scanning adult mice with intravenous contrast and was compelled to find a high-resolution technique for visualizing 3-D internal structures of intact embryos,” said Dr. Charles Keller, team leader and assistant professor in the department of cellular and structural biology at the University of Texas. “Our results exceeded our expectations.”

Computational sectioning of a 12 1/2-day-old normal mouse embryo was performed using virtual histology. The animal is oriented face forward and to its left. The embryo specimen is intact, but the scan can be “virtually sectioned” in any plane, here shown in sagittal sections. Courtesy of Jerry Chang, Children’s Cancer Research Institute, University of Texas, and Ali Bahadur, Numira Biosciences.

Unlike traditional histology methods, the technique doesn’t involve sectioning. Instead, the mice are stained with specific dyes. The researchers used a 1 percent solution of osmium tetroxide to stain the embryos’ cell membranes and found that it was best suited for gestational ages with limited epidermal layers. The osmium stain enabled detection of features as intricate as developing brain vesicles, the dorsal root ganglia and the anterior cardinal vein.

The investigators employed a General Electric eXplore Locus SP specimen scanner to attain scans of the stained embryos at 27-μm isometric resolution, during a mere 2-h acquisition time. The 27-μm sections are sufficient to perform high-quality segmentation analysis of major organ compartments. However, the team found that the small openings within some organs — for example, the right atrium of the heart — are not as well segmented with the rapid scan as with the higher-resolution scan. If increased definition of smaller structures is necessary, the stained embryo can be scanned at 8-μm resolution. The researchers also achieved a 6-μm resolution with the Scanco uCT40.

To demonstrate the potential value of high-throughput phenotyping for major organ compartments and tissue structures of younger embryos, they used transgenic mice with complex malformations of the developing brain and upper spinal cord.

“The malformations were so severe that, by examining them under the dissecting microscope, it was difficult to determine what structure was what. Even light microscope sections were confusing,” Keller said. “Only after doing the microCT scans did we really understand which part of the brain was overgrown and which part was underdeveloped.”

The embryos were scanned at 27-μm resolution and then rendered with segmentation to visualize the cephalic forebrain, midbrain and hindbrain vesicles, the heart wall and cardiac ventricles as well as the liver. With these renderings, the complex global three-dimensional organization of the mutant brain sections were distinctly visible. These types of findings would not have been possible with real histology derived from paraffin-embedded specimens.

This cutaway of a 12 1/2-day-old mouse embryo shows the animal oriented face forward and to its right with the tail in front and lower limbs at the bottom. Visible in the top right cutaway are the neural tube (rear), heart, tongue and developing nose. Courtesy of John T. Johnson and Chris R. Johnson, Scientific Computing and Imaging Institute, Salt Lake City.

To display the CT scan data, the Scientific Computing and Imaging Institute at the University of Utah designed a graphical user interface called BioImage. The program uses 2-D transfer function, which enables the coloring of boundaries between materials of varying densities. Thus, only the important information can be visualized and the less important ignored.

The researchers believe that using osmium tetroxide staining and microscopic CT-based imaging will be most useful in the high-throughput analysis of potential side effects of chemicals and drugs, evaluation of tissues from adult animals and neocapillary mapping for tumor biopsies of patients undergoing antiangiogenesis therapies. Because multiple samples can be scanned simultaneously, this technology may be especially important given the National Institutes of Health’s initiative to make knockout mice for each of the 25,000 mouse genes.

The technique is being commercialized by Numira Biosciences, founded in San Antonio by Keller and his colleague Michael Beeuwsaert.

PLoS Genetics, published online April 28, 2006.