New Microscopy Technique Reveals Whole Picture of Cellular Environments
Imagine a photo without its background. The subjects would remain, but the context would be lost, leaving the photo to tell a partial story.
This is a challenge researchers face when studying cells with in vivo fluorescence imaging, a tool for seeing inside a living organism. They can see cells in vivid colors but often miss the surrounding tissues, which means key information about health and disease goes unseen.
“It’s like only the cell is there,” said Yi Xue, an associate professor of biomedical engineering at the University of California, Davis, describing what she sees with current microscopy techniques. “But of course, the surrounding tissues are there in the background.”
To solve this problem, Xue and a team of UC Davis biomedical engineers have developed a multimodal imaging technique allowing researchers to see the full picture, from cells to their surrounding tissue. Their new method, called fluorescence-illuminated diffraction tomography, is described in a new paper published in Nature Communications.
All a Glow
Xue’s method combines fluorescent and phase imaging. Fluorescent imaging is great at pinpointing specific cells, while phase imaging excels at quantitatively revealing shapes of label-free structures in the body.
Combining the two imaging techniques for thin samples, such as a single layer of cells, has been well explored, but it has struggled with imaging deep tissue, such as the brain. The deeper the tissue, the more light is required, which makes the imaging more complicated and resource-intensive to produce.
Conventional techniques typically require the light source and camera to be positioned on opposite sides of the sample, with illumination strong enough to pass through the tissue. However, when the tissue is thick, transmitting sufficient light through the entire sample can be extremely difficult or even impossible.
The workaround for this shortcoming, Xue’s team discovered, is to use the cells. In fluorescent imaging, a fluorescent molecule stains a cell or tissue, causing it to glow.
“The cells can become the little light source inside of the sample,” Xue said.
In other words, the stained cell becomes like a bright candle in a dark room. The cell’s light reveals the shape of its surroundings, much like a candle might reveal the walls of its room. Because the light source is within the organism, there is no need for an external light source on the transmission side, making deep tissue imaging more achievable than ever before.
Setting the Scene
In order to see the whole scene, the researchers paired their cellular candle technique with a new computational method. The computational method can run through the complex physics and math of deep, bulky tissues refracting (or bending) light.
Xue’s team designed the model to require little computational power, making the technology accessible to most labs. In one controlled comparison using the same computer and measurements, they showed that their computational method reduced reconstruction time from three hours to 50 minutes. Their model also improved resolution for deep tissue imaging despite requiring less memory.
Her team demonstrated the model and imaging technique in three proof-of-concept experiments. In one, with support from Professor of Biomedical Engineering Soichiro Yamada, the team used cultured label-free cells with an external fluorescence substrate. In another, with support from Associate Professor of Chemical Engineering Jiandi Wan, the team used cultured bovine muscle tissue with an external fluorescent substrate. To investigate the cellular candle approach, Assistant Professor of Biomedical Engineering Aidan Gilchrist helped prepare fluorescently labeled cells.
Through these experiments, the technique’s reconstruction distinguished individual cells and revealed shapes associated with stages of muscle development, suggesting the technique could help researchers investigate how cellular structure and composition relate to cellular health.
The Big Picture
Xue’s fluorescence-illuminated diffraction tomography aims to provide unparalleled insights into cells and their surrounding structures. For example, researchers could use the multimodal imaging technique to study the interaction between neurons and blood vessels in the brain, which is essential for understanding the cause of neurodegenerative disease and cerebrovascular diseases, such as dementia and stroke.
A key piece of the technology’s design is ease of implementation. Xue designed the method so that researchers can easily adapt it to standard two-photon microscopy hardware. Additionally, the efficient computational model means most labs will not need to upgrade their equipment to start seeing improvements in their in vivo imaging.
This research was supported by Xue’s CAREER Award from the National Science Foundation and the MIRA Award from the National Institutes of Health.
