"I've probably been labeled creative because the patents on my inventions expire before anyone does anything with them," quips Dr. Greenleaf, a faculty member of the Department of Physiology and Biomedical Engineering, and of the Graduate School 's Biomedical Imaging Program. "Over 30 years ago, we invented computer tomography with ultrasound here at Mayo. There are several companies still working on it but it hasn't really been applied yet."

"We are bringing ultrasound to the forefront of imaging and therapy."
James Greenleaf, Ph.D.

Three-dimensional imaging is now a hot topic in ultrasound—yet, as early as 1986, Dr. Greenleaf was granted a patent for inventing apparatus and a method for recording and displaying 3D images. A wall full of patents and a hefty body of publications including more than 170 original, full-length, and peer-reviewed articles, document a long and successful career. His patent collection includes a set of three for vibro-acoustography.

A New Invention: Vibro-Acoustography

Dr. Greenleaf's ten-year collaboration with colleague Mostafa Fatemi, Ph.D., has also been fruitful. Together they developed vibro-acoustography—a novel technology that mechanically excites an object using known ultrasound methods, then exploits the acoustic response with a fundamentally new imaging method. It works this way:

See how vibro-acoustography works

When ultrasound waves travel through an object, a force is produced. In experiments conducted in a water tank, Drs. Greenleaf and Fatemi focused two continuous ultrasound beams to intersect on an object. By definition, ultrasound is inaudible. However, a small portion of the ultrasound energy converts to a low frequency tapping force that causes the object to vibrate and produce an audible sound. The acoustic response is recorded by a sensitive microphone and used to construct a computer-generated image of the object.

The images produced with vibro-acoustography are very clear—unlike those fuzzy early fetal ultrasound pictures. Scientists refer to that snow-like interference as 'speckle.' The new technology produces images that are speckle-free.

Subsequent investigations also led to an ultrasound-stimulated vibro-acoustic method of spectral analysis. The method measures the mechanical properties of body tissue by analyzing a range of emission frequencies produced by the tissue. 

"It's just like tapping on crystal and listening to its response," explains Dr. Greenleaf. "We scan the beam back and forth over the object to create an oscillatory force that gives us a high resolution picture."

Spectroscopy could be used to help diagnose and follow the treatment of conditions including cancer and infections. Drs. Greenleaf and Fatemi have been awarded three patents in conjunction with this project. They continue to publish results on physical principles and various applications of vibro-acoustography.

From the Lab to the Patient

Dr. Greenleaf is dedicated to Mayo Clinic's research mission to improve patient care and benefit society. He is currently collaborating with industry on five vibro-acoustography projects, and with many Mayo Clinic physicians to put his inventions to use in the clinical arena. One application for vibro-acoustography has already been approved for clinical trials.

Identifying Abnormalities in Breast Tissue

Dr. Fatemi led a collaborative project with Dr. Greenleaf and radiologist Dana Whaley, M.D., to develop breast imaging by vibro-acoustography. The team have successfully imaged microcalcifications and calcified arteries in human breast tissue.

X-ray (left) and vibroacoustic (right) images of breast tissue. The lower arrow indicates a calcified vessel. Even the glue spots used to secure the tissue show up with vibro-acoustography.

In addition, they have developed a new vibro-acoustography machine specifically designed for breast imaging. The new machine is integrated with x-ray mammography to provide simultaneous imaging of the breast via two methods. The machine will be used to test the imaging method's ability to detect breast cancer in a forthcoming Phase I clinical trial.

"We anticipate that vibro-acoustography will give us a new way of looking at tissue quality," says Dr. Fatemi. "Physicians will be able to view both soft and hard tissues and be able to assess their hardness."

Building a New, All-Purpose Vibro-Acoustography System

Being able to identify varying degrees of hardness could be helpful in a variety of ways such as finding malignant tumors, and identifying arterial calcification. To that end, Dr Fatemi has been awarded a grant from National Institutes of Health (NIH) grant to develop a vibro-acoustography system with a contact probe that can image various parts of the human body. This work is conducted in collaboration with industry.

See how vibro-acoustography identifies clogged arteries

Precise implantation of radioactive seeds to treat prostate cancer

Radiation oncologist Brian Davis, M.D., Ph.D., is funded by a National Institute of Aging (NIH/NIA) grant to develop an imaging technique that improves permanent prostate brachytherapy (PPB), a common treatment for early stage prostate cancer. He and the Ultrasound Research Lab have published five abstracts in this area of interest.

Vibro-acoustic image of radioactive 'seeds' that treat prostate cancer.

PPB is an outpatient procedure during which between 40 and 100 rice-sized seeds or pellets are implanted in the prostate. The seeds contain one of several radioactive isotopes that emit radiation to kill the cancer cells. PPB's effectiveness depends upon the correct distribution of the seeds. Current imaging methods are unable to consistently view all seeds during the procedure. Computed tomography (CT) is accurate but not usually possible until after the procedure.

Vibro-acoustography has 3D imaging capability and is particularly sensitive to hard objects — qualities that make vibro-acoustography an ideal imaging method for allowing brachytherapy seeds to be placed, and their position assessed and corrected — all in one procedure. The Ultrasound Research Lab has conducted vibro-acoustography tests on human tissue that produced clear, 3D images of the prostate within a few minutes.

Like Raindrops on a Tin Roof

Everyone knows that ultrasound is a common obstetrical diagnostic tool. What nobody knew until Drs. Fatemi and Greenleaf published their experiments in 2001, is that fetuses can hear, and are disturbed by ultrasound. The finding generated worldwide media attention.

"Raindrops don't make any sound in the air," explains Dr. Fatemi. "But when they hit a tin roof the sound is clearly audible. In the same way, ultrasound waves produce a tapping effect that is sensed by the fetus."

Obstetricians have long observed that fetuses move in response to ultrasound exams. That fact raised the scientists' curiosity.

James Greenleaf, Ph.D. and Mostafa Fatemi, Ph.D. collaborate in the Ultrasound Research Lab to develop novel ultrasound biotechnology.

"We have statistically confirmed that fetuses indeed increase movement during ultrasound," says Dr. Fatemi. "We believe that the ultrasound tapping translates into audible noises that stimulate the fetus to move in the womb."

Drs. Fatemi and Greenleaf have been awarded two patents in conjunction with this project. They continue to publish results on the physical principles involved. To date, there is no evidence to document that fetuses are physically harmed by the ultrasound test.

Watch video from Medical Edge

Broken Bones? How Can Ultrasound Help?

An ultrasound technique that stimulates the healing process in bone fractures exists but is not well understood. Together with Mark Bolander, M.D., a member of the Mayo Clinic Metabolic Bone Disease Core Group, in Orthopedic Surgery's Bone Histomorphometry Lab, Dr. Greenleaf is conducting animal studies to get a better grip on exactly how the process works.

One of their studies was funded by the National Space Biomedical Research Institution to assess the possible use of ultrasound to heal fractures in outer space. The studies uncovered two surprises: microgravity itself appears to accelerate fracture healing; and ultrasound also accelerates the formation of cartilage under microgravity conditions.

Ultrasound and Gene Therapy

In 1995, Drs. Greenleaf and Bolander were among the first to describe transfection using ultrasound—a promising method for transferring genes into cells. ( Journal of Bone Mineral Research, 10:S434, August 1995. Abstract.)

Gene therapy has the potential to treat many diseases. Many molecular scientists are exploiting the characteristic of viruses to efficiently penetrate cells carrying modified genes with them. Although viruses are very effective vectors, they carry with them dangerous risks of infection and immune reaction. Ultrasound holds promise as a simple and safe gene transfection method if its efficiency can be raised to the level of viral vectors—a recent focus of the Ultrasound Research Laboratory.

"By injecting microbubbles mixed with DNA we have enhanced transfection significantly since our original experiments," explains Dr. Greenleaf. "The DNA sits on the surface of the bubble and ultrasound drives the DNA into the cells."

Contrast agents made of tiny microbubbles are routinely used in ultrasound imaging because they scatter ultrasound very effectively. Knowing this, Dr. Greenleaf was intrigued when, while working on an unrelated project, he stumbled on a further enhancement.

From Knocking Bacteria Out of Biofilms to Piggybacking Gene Therapy on Microbubbles

Dr. Greenleaf is working with Robin Patel, M.D., on the use of ultrasound to diagnose infections of artificial joints. The offending bacteria live in communities protected by a slime layer, or biofilm, that covers the surface of the artificial joint. The biofilm prevents bacteria from appearing in fluid drawn from around the prosthesis, so cultures often fail to correctly identify them.

If a prosthetic joint becomes infected and does not respond to antibiotic therapy, it must be removed and the patient treated before it can be replaced. Dr. Greenleaf has developed ultrasound technology that dislodges the bacteria from the biofilm, making them accessible to culture. The removed, infected, artificial joints are placed in ultrasound cleaning baths. These work by cavitation—the agitation of minute bubbles, called cavities, that probably cause tiny tears in the biofilm, forcing the bacteria out of their protective layer.

An exciting project by itself, Dr. Patel made a discovery that enhanced the gene transfection work. Her experiments showed that the entire action of the microbubbles occurred with the first ultrasound application and that repeated exposures failed to produce more bacteria.

"I applied her discovery to our transfection experiments," says Dr. Greenleaf. "Perhaps the key to enhancing transfection is not repeated exposures to ultrasound, but increasing the number of microbubbles in our DNA cocktail. So we tried using contrast agent microbubbles."

The hypothesis, that the bubbles burst and cause tiny tears, could apply both to the biofilm and to the cell membrane, in the latter case briefly opening many tiny windows that provide access for DNA to enter the cell.

Dr. Greenleaf has now brought in another collaborator to investigate how the new development might generate new uses of contrast agent microbubbles.

Preventing Heart Failure Through Ultrasound Transfection

Robert Simari. M.D., is a cardiologist and molecular biologist who is exploring the use of gene therapy to treat heart failure. He is particularly interested in natriuretic peptides (NPs)—hormones that are naturally expressed by the heart and have beneficial effects in early stages of heart failure.

Dr. Simari is collaborating with Dr. Greenleaf on enhancing ultrasound transfection to the point where gene transfer can generate enough NPs to prevent heart failure. Their experiments have shown that ultrasound-enhanced plasmid gene transfer is capable of transducing skeletal muscle.

Computing Blood Flow with Microbubbles

Microbubbles measuring only three microns can be transported into the smallest capillaries coated with a protein, lipid or polymer layer. Using contrast enhanced ultrasound the cardiologist can see which part of the heart muscle has poor blood flow.

Elastography

Richard Ehman, M.D. a radiologist in the Magnetic Resonance Imaging Laboratory and Dr. Greenleaf are collaborating on a magnetic resonance imaging technology called elastography. The goal of elastography technology is to distinguish materials based on their mechanical properties—to ‘palpate' anatomical structures too deep for physicians to access by laying their hands on the skin.

Joel Felmlee, Ph.D., a physicist in the Magnetic Resonance Imaging Laboratory is also collaborating with Dr. Greenleaf. Their project combines two imaging technologies: the use of ultrasound to destroy tumors and magnetic resonance elastography to estimate the optimal time to stop therapy.

Mentoring Young Investigators

One of Dr. Greenleaf's great joys is to mentor young investigators and help set them on the path to a successful career—either at Mayo or at other institutions.

Marek Belohlavek, M.D., Ph.D., is a case-in-point. Dr. Belohlavek began his career at Mayo Clinic in 1990 as a research fellow. Under Dr. Greenleaf's mentorship, he focused on higher-dimensional imaging in cardiac ultrasonography. Five years later he had completed his Ph.D., obtained his first NIH grant, and joined Mayo faculty with appointments in Physiology & Biomedical Engineering and Cardiovascular Diseases.

"Mayo Clinic is extremely fortunate to have a career investigator of Dr. Greenleaf's caliber," says Dr. Belohlavek. "His support of the personal challenges I faced, having just arrived from the Czech Republic, was extraordinary. His mentoring and collaboration is responsible for much of my professional success and has resulted in multiple National Institutes of Health, American Heart Association and other peer-reviewed research grants."

Dr. Belohlavek now directs the Translational Ultrasound Research Unit and the Echocardiography Research Center. He collaborates extensively with James Seward, M.D., former director of Mayo Clinic's renowned Non-invasive Ultrasound Imaging and Hemodynamic Laboratory as well as other investigators in cardiology and bioengineering areas. The basic and clinical ultrasound team of investigators have shepherded ultrasound science into new applications that directly improve patient care.

Ultrasonic images of cardiac tissue after heart attack. The right image has been computer-processed to delineate areas of dead red), reduced blood flow (yellow); and healthy (green) tissue.

Novel cardiovascular research that has evolved from Dr. Greenleaf's mentoring includes:

• Analyses of short-lived functional events occurring during the cardiac cycle and their relationship to the underlying cardiac physiology
• 3D ultrasound imaging of cardiac function, morphology, and perfusion—including images from within the heart produced by an ultrasound-tipped imaging catheter developed in collaboration with industry
• Innovative solutions for the visualization of ultrasound contrast agents

Dr. Greenleaf is grateful for the contributions that his protégés are now making to the field. But he thinks there's still some fruit to be harvested from his own labors.

"People ask me when I'm going to retire and I tell them when it's not fun anymore. I just started three students and it takes me five years to train them," he offers. "I'll retire when I'm no longer excited about getting to the next experiment."