What appears as a kaleidoscope of shapes and patterns to many people may be tissue from a mouse intestine, bird lung, or even a human brain to the eye trained in microscopy. With modern imaging, scientists are able to observe the cells of living tissue in high resolution.
Multiphoton microscopy, also known as non-linear or two-photon microscopy, is an established technology used to study cells in live tissue for extended durations, providing researchers with three-dimensional imaging of whole organs, sections of organs, or specimens. It detects emitted light for visualization of cells, tagged with fluorescent markers such as proteins.
“This technology was created back in 1990 here at Cornell,” says Amanda Bares, a graduate student in the Meinig School of Biomedical Engineering. “These microscopes, however, are limited by the number of fluorescent label colors they can identify and therefore the cell types they can help us observe. If you have more than two or three cell types labeled with different colors, you can no longer tell them apart. And if you’re labeling neurons, blood vessels, and immune cells, it is hard to differentiate.”
Bares is a research team member in the Chris B. Schaffer-Nozomi Nishimura lab, whose specialty is creating new tools to optimally explore disease mechanisms. They build microscopes to visualize individual cells in live tissue and to study brain diseases such as Alzheimer’s. They also perfect methodologies such as laser ablation that allows for precision tissue extraction without damaging the surrounding tissue.
Seeing How Cells Interact in Living Tissue
In the Schaffer-Nishimura lab, Bares developed a hyperspectral multiphoton microscope. It provides about 50 different spectral channels and can therefore identify a very large number of color labels simultaneously in order to study complex disease mechanisms where several types of cells are implicated.
Although a few existing commercially viable systems may allow the detection of multiple color labels, they lose color resolution when used to observe areas deeper in the brain, for example. With Bares’ microscope, it’s possible to image cells almost a millimeter deep in the cortex. The mechanisms and structural details of Bares’ hyperspectral multiphoton microscope are complex, but the idea is to capture the color variance between the fluorescent labels.
One potential use for the technology is to employ it as a hypothesis testing tool. Researchers can use the microscope to visualize and test their hypotheses on how cells interact in living tissue. Because a large number of cell types can be seen with the hyperspectral microscope, they can also observe cell activity to generate further hypotheses for both normal and disease state biology.
The Hyperspectral Multiphoton Microscope in Action
Bares and her colleagues excite their sample, whatever they’re studying, with a short-pulsed femtosecond laser. This causes the labeled cells to glow, and the microscope collects the colored light and divides it into four broad color channels—the way most multiphoton microscopes work. The new hyperspectral multiphoton system, however, utilizes color-shifting filters that change the color of light allowed to pass through, based on the angle of the filter. With one of these filters in each broad color channel, the researchers can collect images for multiple filter angles, allowing them to pick up small differences in fluorescent label colors.
“So consider the standard blue, green, yellow and red channels,” Bares elaborates. “We collect an image from each of the color detectors in the microscope at the same time. Then we turn those filters, and collect a slightly redder shade, and then we turn and collect it again. Eventually we have 16 different color channels of information.”
They also use multiple colored lasers to excite the sample, because each fluorescent label changes how bright it glows, depending on the color of laser used to excite it. This additional information clearly differentiates fluorescent labels that are very similar in color. Collecting 16 channels of information for three lasers leads to 48 channels of color data. Once all data are collected, algorithms identify each color type, and the visualization is complete.
How a Passion for Lasers and Biology Led to Biomedical Engineering
Bares has always had a passion for lasers. Growing up in Bozeman, Montana, she inherited an appreciation for the devices from her father, an electrical engineer who designs power supplies for a laser company. As an undergraduate studying electrical engineering at Montana State University, she was also drawn to biology and managed to petition the department to replace many of her core course requirements with classes in anatomy, physiology, and organic chemistry.
When she was a sophomore, a professor recognized her technical aptitude and offered her the opportunity to conduct research with him, building laser instrumentation for carbon sequestration.
“There are programs that take carbon dioxide from the air, concentrate it and put it underground for long-term storage,” she explains. “And we created instruments that scanned lasers over an expansive test field just outside of campus to detect small concentrations of carbon dioxide to determine if any of the gas was leaking out. There was a buried pipe with controlled release holes. Although I wasn’t as interested in this particular application, I found the instrumentation exciting, and I got to play with lasers. So that was just fine with me.”
The following year she discovered biomedical engineering and realized immediately that she wanted to pursue a career in the field, since it would allow her to apply engineering principles to solve key problems in medicine. While applying to graduate school, initially Bares was not certain that Cornell’s biomedical engineering program was the ideal fit.
“I am trying to determine whether it should be offered as a service or if the device itself should be available for sale.”
“The deciding factor for me was the collaboration aspect here,” she recounts. “There are ample opportunities and active collaborations between labs. And there are so many professors doing very interesting and significant work. There’s Chris Schaffer of course, my adviser, but then there’s Warren Zipfel [Biomedical Engineering] who is also working on developing optimal microscopy for biomedical applications and another lab doing laser instrumentation in Applied Engineering and Physics. Also the biomedical engineering program here is very flexible. It allows you to explore the labs before you join.”
Bares enjoys the academic diversity at Cornell. Within her own lab there are neuroscientists, a veterinarian, and biologists who work alongside engineers. Their conversations are interdisciplinary. “I might ask to learn a bit about animal surgery, and in turn I’ll elucidate the imaging process,” she explains. Also, an incalculable number of research and research application opportunities are available, such as the grant proposal Bares co-wrote for which 10 faculty members proposed projects designed to utilize her hyperspectral multiphoton microscope.
Bares, a 2016 Engineering Commercialization Fellow
Bares is one of six recipients of the 2016 Commercialization Fellowship, an entrepreneurship initiative of the College of Engineering. Provided with a stipend and mentorship, Bares explored market applications of her microscope. She has had over 100 conversations with prospective customers to understand the best channels through which to deliver her technology.
“I am trying to determine whether it should be offered as a service or if the device itself should be available for sale. If I decide that clinical use is the best way to move forward with this, I will have to look into FDA approval. A part of this fellowship has been a series of workshops on intellectual property, and we’re working very closely with the Cornell Center for Technology Licensing.”
The pharmaceutical industry has expressed significant interest in her device to study pathologies and target specific areas of disease mechanisms precisely. After a talk Bares gave at an immunology symposium in Rochester, New York, the first question posed to her was “When can I buy this?” In academia immunologists could use the microscope to observe immune cells in real time. These applications do not have to be mutually exclusive. It’s possible, for example, to sell the device to key labs and structure a business model, based on the results of the applications.
Despite the clear technical advantages of the hyperspectral multiphoton microscope, regulatory challenges still exist for this type of microscope. The methodology has not yet been approved by the United States Food and Drug Administration for human use, but Bares is hopeful. She’d like to see two-photon microscopy employed in the surgical field to study tumor margins—investigating the healthy tissue around the site of a removed tumor to confirm that the entire growth is removed. In the meantime, Bares is working in the lab to fully characterize the capabilities of her technology for future, complex disease studies that involve multiple cell types.
Considering the microscope’s development thus far and the interest and curiosity it has generated in industry and academia, Bares’ technology has a credible opportunity for success.