Prostate cancer is the and, for men in the United States, it’s the second leading cause of death.

Some prostate cancers might be slow-growing and can be monitored over time whereas others need to be treated right away. To determine how aggressive someone’s cancer is, doctors look for abnormalities in slices of biopsied tissue on a slide. But this 2D method makes it hard to properly diagnose borderline cases.

Now a team led by the ����Ӱ�Ӵ�ý has developed a new, non-destructive method that images entire 3D biopsies instead of just a slice. In a proof-of-principle experiment, the researchers imaged 300 3D biopsies taken from 50 patients — six biopsies per patient — and had a computer use 3D and 2D results to predict the likelihood that a patient had aggressive cancer. The 3D features made it easier for the computer to identify the cases that were more likely to recur within five years.

The team Dec. 1 in Cancer Research.

“We show for the first time that compared to traditional pathology — where a small fraction of each biopsy is examined in 2D on microscope slides — the ability to examine 100% of a biopsy in 3D is more informative and accurate,” said senior author , a UW professor of mechanical engineering and of bioengineering. “This is exciting because it is the first of hopefully many clinical studies that will demonstrate the value of non-destructive 3D pathology for clinical decision-making, such as determining which patients require aggressive treatments or which subsets of patients would respond best to certain drugs.”

The researchers used prostate specimens from patients who underwent surgery more than 10 years ago, so the team knew each patient’s outcome and could use that information to train a computer to predict those outcomes. In this study, half of the samples contained a more aggressive cancer.

To create 3D samples, the researchers extracted “biopsy cores” — cylindrically shaped plugs of tissue — from surgically removed prostates and then stained the biopsy cores to mimic the typical staining used in the 2D method. Then the team imaged each entire biopsy core using an open-top light-sheet microscope, which uses a sheet of light to optically “slice” through and image a tissue sample without destroying it.

The 3D images provided more information than a 2D image — specifically, details about the complex tree-like structure of the glands throughout the tissue. These additional features increased the likelihood that the computer would correctly predict a cancer’s aggressiveness.

Shown here is a video of a volume rendering of glands in two 3D biopsy samples from prostates (yellow: the outer walls of the gland; red: the fluid-filled space inside the gland; purple: what researchers called the “gland skeleton,” a stick-like model of the fluid-filled spaces inside the glands). The cancer sample (top) shows smaller and more densely packed glands compared to the benign tissue sample (bottom). Credit: Xie et al./Cancer Research

The researchers used new AI methods, including deep-learning image transformation techniques, to help manage and interpret the large datasets this project generated.

“Over the past decade or so, our lab has focused primarily on building optical imaging devices, including microscopes, for various clinical applications. However, we started to encounter the next big challenge toward clinical adoption: how to manage and interpret the massive datasets that we were acquiring from patient specimens,” Liu said. “This paper represents the first study in our lab to develop a novel computational pipeline to analyze our feature-rich datasets. As we continue to refine our imaging technologies and computational analysis methods, and as we perform larger clinical studies, we hope we can help transform the field of pathology to benefit many types of patients.”

The lead author on this paper is , a UW mechanical engineering doctoral student. Other co-authors on this paper are , , and , all UW mechanical engineering doctoral students; , a UW bioengineering doctoral student; , a clinical instructor in the laboratory medicine and pathology department in the UW School of Medicine; Hongyi Huang, UW research staff in mechanical engineering; , a UW doctoral student in the chemistry department; , a research scientist in the laboratory medicine and pathology department in the UW School of Medicine; , a UW assistant teaching professor in the mechanical engineering department; Qinghua Han, a UW undergraduate student studying bioengineering; Jonathan Wright, a professor in the urology department in the UW School of Medicine; and , both professors in the laboratory medicine and pathology department in the UW School of Medicine; , a UW associate professor of chemistry; , a senior scientist at the Allen Institute who completed this research as a UW mechanical engineering postdoctoral researcher; , , and , all at Case Western Reserve University; at Genentech, who completed this research as a doctoral student at Case Western Reserve University; and Sarah Hawley at the Canary Foundation.

This research was funded by the Department of Defense Prostate Cancer Research Program; the National Cancer Institute; the National Heart, Lung and Blood Institute; the National Institute of Biomedical Imaging and Bioengineering; the National Institute of Mental Health; the VA Merit Review Award; the National Science Foundation; the Nancy and Buster Alvord Endowment; and the Prostate Cancer Foundation Young Investigator Award.

Nicholas Reder, Adam Glaser, Lawrence True and Jonathan Liu are co-founders and shareholders of the UW spinout This company has licensed the technology used in this paper.

For more information, contact Liu at jonliu@uw.edu.

Grant numbers: W81XWH-18-10358, W81XWH-19-1-0589, W81XWH-15-1-0558, W81XWH-20-1-0851, K99 CA24068, R01CA244170, U24CA199374, R01CA249992, R01CA202752, R01CA208236, R01CA216579, R01CA220581, R01CA257612, U01CA239055, U01CA248226, U54CA254566, R01HL151277, R01EB031002, R43EB028736, R01MH115767, IBX004121A, 1934292 HDR: I-DIRSE-FW, DGE-1762114, DGE-1762114

When women undergo lumpectomies to remove breast cancer, doctors try to remove all the cancerous tissue while conserving as much of the healthy breast tissue as possible.

But currently there’s no reliable way to determine during surgery whether the excised tissue is completely cancer-free at its margins — the proof that doctors need to be confident that they removed all of the tumor. It can take several days for pathologists using conventional methods to process and analyze the tissue.

That’s why between 20 and 40 percent of women have to undergo second, third or even fourth breast-conserving surgeries to remove cancerous cells that were missed during the initial procedure, according to

A new microscope invented by a team of ����Ӱ�Ӵ�ý mechanical engineers and pathologists could help solve this, and other, problems. It can rapidly and non-destructively image the margins of large fresh tissue specimens with the same level of detail as traditional pathology — in no more than 30 minutes.

“Surgeons are sort of flying blind during these breast-conserving surgeries,” said mechanical engineering professor . “Oftentimes they’ve left some tumor behind which they don’t know about until a few days later when the pathologist finds it.”

“If we can rapidly image the entire surface or margin of the excised tissue during the procedure, we can tell them if they still have tumor left in the body or not. And that would be a huge benefit to cancer patients,” Liu said.

The new light-sheet microscope — which is described in a published June 26 in — offers other advantages over existing processes and microscope technologies. It conserves valuable tissue for genetic testing and diagnosis, quickly and accurately images the irregular surfaces of large clinical specimens, and allows pathologists to zoom in and “see” biopsy samples in three dimensions.

“The tools we use in pathology have changed little over the past century,” said co-author , chief resident and clinical research fellow in UW Medicine’s Department of Pathology. “This light-sheet microscope represents a major advance for pathology and cancer patients, allowing us to examine tissue in minutes rather than days and to view it in three dimensions instead of two — which will ultimately lead to improved clinical care.”

Current pathology techniques involve processing and staining tissue samples, embedding them in wax blocks, slicing them thinly, mounting them on slides, staining them, and then viewing these two-dimensional tissue sections with traditional microscopes — a process that can take days to yield results.

Another technique to provide real-time information during surgeries involves freezing and slicing the tissue for quick viewing. But the quality of those images is inconsistent, and certain fatty tissues, such as those from the breast, do not freeze well enough to reliably use the technique.

By contrast, the UW open-top light-sheet microscope uses a sheet of light to optically “slice” through and image a tissue sample without destroying any of it. All of the tissue is conserved for potential downstream molecular testing, which can yield additional valuable information about the nature of the cancer and lead to more effective treatment decisions.

“Slide-based pathology is still an analog technique, much like radiology was several decades ago when X-rays were obtained on film. By imaging tissues in 3-D without having to mount thin tissue sections on glass slides, we are trying to transform pathology much like 3-D X-ray CT has transformed radiology,” Liu said.  “While it is possible to scan microscope slides for digital pathology, we digitally image the intact tissues and bypass the need to prepare slides, which is simpler, faster and potentially less expensive.”

“If we can do this without consuming any tissue, so much the better,” said co-author , professor of pathology at UW Medicine. “We want to use that valuable tissue for purposes which are becoming ever more important for treating patients — such as sequencing the tumor cells and finding genetic abnormalities that we can target with specific drugs and other precision medicine techniques.”

The light-sheet microscope also offers advantages over other non-destructive optical- sectioning microscopes on the market today, which process images slowly and have difficulty maintaining the optimal focus when dealing with clinical specimens, which always have microscopic surface irregularities.

The UW microscope can both image large tissue surfaces at high resolution and stitch together thousands of two-dimensional images per second to quickly create a 3-D image of a surgical or biopsy specimen. That additional data could one day allow pathologists to more accurately and consistently diagnose and grade tumors.

“Pathologists are currently very limited in how much they can look at on a glass slide,” said co-author , a postdoctoral fellow in the UW . “If we can give them three-dimensional data, we can give them more information to help improve the accuracy of a patient’s diagnosis.”

The UW team achieved these improvements by configuring various optical technologies in new ways and optimizing them for clinical use. Their open-top arrangement, which places all of the optics underneath a glass plate, allows them to image larger tissues than other microscopes.

The team is currently working on speeding up the optical-clearing process that allows light to penetrate biopsy samples more easily. Future areas of research include optimizing their 3-D immunostaining processes, as well as as continuing a collaboration formed during the UW eScience Institute’s Winter Incubator program with Dr. Ariel Rokem to develop algorithms that can process the vast amounts of 3-D pathology data that their system generates, with the ultimate goal of helping pathologists zero in on suspicious areas of tissue.

The research was funded by the National Institutes of Health and the ����Ӱ�Ӵ�ý.

Additional co-authors include Ye Chen, Chengbo Yin, Linpeng Wei and Yu Wang of the UW Department of Mechanical Engineering and Erin F. McCarty of UW Medicine’s Department of Pathology.

For more information on the light-sheet microscope, contact Jonathan Liu at jonliu@uw.edu. To reach Larry True or Nicholas Reder at UW Medicine, contact Leila Gray at 206-685-0381 or leilag@uw.edu.

Surgeons removing a malignant brain tumor don’t want to leave cancerous material behind. But they’re also trying to protect healthy brain matter and minimize neurological harm.

Once they open up a patient’s skull, there’s no time to send tissue samples to a pathology lab — where they are typically frozen, sliced, stained, mounted on slides and investigated under a bulky microscope — to definitively distinguish between cancerous and normal brain cells.

But a handheld, miniature microscope being developed by ����Ӱ�Ӵ�ý mechanical engineers could allow surgeons to “see” at a cellular level in the operating room and determine where to stop cutting.

The new technology, developed in collaboration with Memorial Sloan Kettering Cancer Center, Stanford University and the Barrow Neurological Institute, is outlined in a published in January in the journal Biomedical Optics Express.

“Surgeons don’t have a very good way of knowing when they’re done cutting out a tumor,” said senior author , UW assistant professor of mechanical engineering. “They’re using their sense of sight, their sense of touch, pre-operative images of the brain — and oftentimes it’s pretty subjective.

“Being able to zoom and see at the cellular level during the surgery would really help them to accurately differentiate between tumor and normal tissues and improve patient outcomes,” said Liu.

The handheld microscope, roughly the size of a pen, combines technologies in a novel way to deliver high-quality images at faster speeds than existing devices. Researchers expect to begin testing it as a cancer-screening tool in clinical settings next year.

For instance, dentists who find a suspicious-looking lesion in a patient’s mouth often wind up cutting it out and sending it to a lab to be biopsied for oral cancer. Most come back benign.

That process subjects patients to an invasive procedure and overburdens pathology labs. A miniature microscope with high enough resolution to detect changes at a cellular level could be used in dental or dermatological clinics to better assess which lesions or moles are normal and which ones need to be biopsied.

“The microscope technologies that have been developed over the last couple of decades are expensive and still pretty large, about the size of a hair dryer or a small dental x-ray machine,” said co-author , associate faculty member in the dermatology service at the Memorial Sloan Kettering Cancer Center in New York City. “So there’s a need for creating much more miniaturized microscopes.”

Making microscopes smaller, however, usually requires sacrificing some aspect of image quality or performance such as resolution, field of view, depth, imaging contrast or processing speed.

“We feel like this device does one of the best jobs ever — compared to existing commercial devices and previous research devices — of balancing all those tradeoffs,” said Liu.

The miniature microscope uses an innovative approach called “dual-axis confocal microscopy” to illuminate and more clearly see through opaque tissue. It can capture details up to a half millimeter beneath the tissue surface, where some types of cancerous cells originate.

In the video below, for instance, researchers produced images of fluorescent blood vessels in a mouse ear at various depths ranging from 0.075 to 0.125 millimeters deep.

https://youtu.be/EzMoZMfXsDo

“Trying to see beneath the surface of tissue is like trying to drive in a thick fog with your high beams on – you really can’t see much in front of you,” Liu said. “But there are tricks we can play to see more deeply into the fog, like a fog light that illuminates from a different angle and reduces the glare.”

The microscope also employs a technique called line scanning to speed up the image-collection process. It uses micro-electrical-mechanical — also known as MEMS — mirrors to direct an optical beam which scans the tissue, line by line, and quickly builds an image.

Imaging speed is particularly important for a handheld device, which has to contend with motion jitter from the human using it. If the imaging rate is too slow, the images will be blurry.

In the paper, the researchers demonstrate that the miniature microscope has sufficient resolution to see subcellular details. Images taken of mouse tissues compare well with those produced from a multi-day process at a clinical pathology lab — the gold standard for identifying cancerous cells in tissues.

The researchers hope that after testing the microscope’s performance as a cancer- screening tool, it can be introduced into surgeries or other clinical procedures within the next 2 to 4 years.

“For brain tumor surgery, there are often cells left behind that are invisible to the neurosurgeon. This device will really be the first to let you identify these cells during the operation and determine exactly how much further you can reduce this residual,” said project collaborator , professor of neurosurgery at the Barrow Neurological Institute in Phoenix. “That’s not possible to do today.”

The research was funded by the National Institutes of Health through its National Institute of Dental and Craniofacial Research and National Cancer Institute.

Co-authors include UW mechanical engineering Chengbo Yin, Ye Chen, Linpeng “Peter” Wei, Steven Leigh and Michael Rosenberg; postdoctoral researchers Adam K. Glaser and Prasanth Pillai; Memorial Sloan Kettering Cancer Center’s Sanjeewa Abeytunge, Gary Peterson and Christopher Glazowski; and Stanford University research scientist Michael Mandella.

For more information, contact Liu at jonliu@uw.edu.

Grant Numbers: NIH / NIDCR – R01DE023497 and NIH / NCI – R01CA175391.