New Microscopy Technique Could Enable Rapid Tumor Analysis in the Operating Room

Bioengineers at Caltech have developed a new imaging technology that could offer surgeons a fast and cost-effective way to image tissue samples in the operating room to determine whether the entirety of a tumor has been removed or if additional cuts are needed.

The researchers describe the new technique, which they call parallel ultraviolet photoacoustic microscopy (PUV–PAM), in a paper in the December 11 issue of the journal Science Advances.

In the current leading method to quickly sample and image tissue samples obtained during surgery, a biopsy is conducted to obtain a tissue sample that is then frozen, stained to enhance viewability, and sliced into thin sections that are mounted on glass slides. Then, an optical microscope for histology is used for a detailed examination of the tissues. The presence of tumor cells on the surface of a tissue sample indicates that the surgeon has cut through, not around, the tumor—meaning that a portion of the tumor remains inside the patient, who will then need a follow-up surgery to have more tissue removed. Frozen section pathology faces challenges such as tissue artifacts and reduced staining quality, affecting diagnostic accuracy and surgical decision-making.

Caltech's new PUV–PAM technique would simplify and speed up that process, removing the need to freeze, section, or stain tissue samples. Even relatively thick samples with irregular surfaces that are typically too thick to image with microscopy can be directly imaged using the new method. That could give oncologists the ability to analyze biopsy samples right in the operating room, providing them the ability to remove additional tissue as deemed necessary without follow-up surgeries.

"We hope this new imaging system can provide more opportunities for intraoperative pathological examination of slide-free specimens in oncology surgeries. We believe it has the potential to revolutionize intraoperative histology," says Rui Cao, lead author of the new paper, who conducted the work in the lab of Lihong Wang, Caltech's Bren Professor of Medical Engineering and Electrical Engineering. Cao is now an assistant professor of biomedical engineering at Case Western Reserve University.

The new approach is based on a technique pioneered by Wang's lab called photoacoustic microscopy (PAM). Using PAM, a tissue sample is excited with a low-energy laser, which causes the tissue to vibrate. The system measures the ultrasonic waves emitted by the vibrating tissue. Because the nuclei of cells absorbs more light than the surrounding material, PAM reveals the sizes and distribution of those nuclei along with the packing density of cells. Cancerous tissue tends to have larger nuclei and more densely packed cells.

Wang's lab has previously developed several PAM systems for imaging bone and breast tissue samples. But to make the systems viable for use within the operating room, they needed to significantly increase the technique's imaging speed, which has been limited by the speed of the ultraviolet lasers used to excite the tissues.

To bypass this laser speed problem, the researchers divided a single laser beam into eight smaller laser "spots" all operating in parallel. This makes the imaging much faster because the spots can cover the sample area much faster. Furthermore, PUV-PAM uses a combination of two scanning techniques to achieve fast imaging speed for slide-free tissues. Taken together, these enhancements make the new technique about 40 times faster than state-of-the-art methods previously developed in Wang's laboratory.

"With the current system, we can image a 1 cm² sample at 1.3 micron resolution within about five minutes," Cao says. "And we demonstrate in the paper that this technique is effective in a variety of tissue types."

"We believe that leveraging advanced ultraviolet lasers with higher pulse rates and integrating more parallel channels could significantly enhance the speed of imaging further with this technique," says Wang, who is also the Andrew and Peggy Cherng Medical Engineering Leadership Chair and executive officer for medical engineering at Caltech.

Additional authors of the paper, "Optical-resolution parallel ultraviolet photoacoustic microscopy for slide-free histology," are Caltech graduate student Yilin Luo and Caltech senior postdoctoral scholar research associate in medical engineering Yide Zhang; Yushun Zeng and Qifa Zhou of USC; and Jingjing Zhao, now at Huazhong University of Science and Technology, who completed the work while at Stanford University working with co-author Adam de la Zerda, who is also part of the Chan Zuckerberg Biohub in San Francisco.

The work was supported by the National Institutes of Health and the National Science Foundation.