Scientists can label cells with bioluminescent probes—proteins that glow—making it possible to track the growth of a tumor, or measure changes in gene expression that occur as cells differentiate. While this technique works well in cells and some tissues of the body, it is more difficult to apply to image structures deep within the brain, because the light scatters too much before it can be detected.
MIT engineers have now developed a novel way to detect this bioluminescence in the brain. Testing their technique in rat models, they engineered blood vessels of the brain to express a protein that causes them to dilate in the presence of light. That dilation can then be observed using magnetic resonance imaging (MRI), allowing researchers to pinpoint the source of light. The technique, which the team dubbed bioluminescence imaging using hemodynamics, or BLUsH, could enable researchers to explore the inner workings of the brain in more detail than has previously been possible.
“A well-known problem that we face in neuroscience, as well as other fields, is that it’s very difficult to use optical tools in deep tissue,” said Alan Jasanoff, PhD, an MIT professor of biological engineering, brain and cognitive sciences, and nuclear science and engineering. “One of the core objectives of our study was to come up with a way to image bioluminescent molecules in deep tissue with reasonably high resolution.”
Jasanoff, who is also an associate investigator at MIT’s McGovern Institute for Brain Research, is the senior author of the team’s report in Nature Biomedical Engineering, titled “Imaging bioluminescence by detecting localized haemodynamic contrast from photosensitized vasculature.” Former MIT postdocs Robert Ohlendorf, PhD, and Nan Li, PhD, are the lead authors of the paper.
Bioluminescent proteins are found in many organisms, including jellyfish and fireflies. Scientists use these proteins to label specific proteins or cells, whose glow can be detected by a luminometer. “Bioluminescent probes are widely used to monitor biomedically relevant processes and cellular targets in living animals,” the authors wrote. One of the proteins often used for this purpose is luciferase, which comes in a variety of forms that glow in different colors. While bioluminescent imaging (BLI) can be used to investigate a broad range of molecular and cellular processes using optical detection, “Localization of bioluminescent probes in vivo is nevertheless severely compromised by scattering and attenuation of emitted light in bone and soft tissue,” the team continued. “This is particularly a problem in the brain, where the skull impedes photon propagation, especially at short wavelengths.”
Jasanoff’s lab, which specializes in developing new ways to image the brain using MRI, wanted to find a way to detect luciferase deep within the brain. To achieve that, they came up with a method for transforming the blood vessels of the brain into light detectors. One form of MRI works by imaging changes in blood flow in the brain, so the researchers engineered the blood vessels themselves to respond to light by dilating. “We recently showed that engineered proteins and peptides could be used to convert molecular signals into changes in blood flow that can be sensitively detected using MRI or other hemodynamic imaging techniques,” they wrote. “In this study, we implement the strategy of BLI using hemodynamics (BLUsH).”
“Blood vessels are a dominant source of imaging contrast in functional MRI and other non-invasive imaging techniques, so we thought we could convert the intrinsic ability of these techniques to image blood vessels into a means for imaging light, by photosensitizing the blood vessels themselves,” Jasanoff noted.
To make the blood vessels sensitive to light, the researchers engineered them to express a bacterial protein called Beggiatoa photoactivated adenylate cyclase (bPAC). When exposed to light, this enzyme produces a molecule called cAMP, which causes blood vessels to dilate. When blood vessels dilate, it alters the balance of oxygenated and deoxygenated hemoglobin, which have different magnetic properties. This shift in magnetic properties can be detected by MRI. “In the BLUsH paradigm, bioluminescent reporters activate light-dependent photoreceptor proteins expressed in vascular cells and stimulate downstream signalling cascades,” the team further explained. “These signaling events, in turn, cause local dilation of blood vessels and haemodynamic contrast detectable by MRI or other imaging modalities.”
bPAC responds specifically to blue light, which has a short wavelength, so it detects light generated within close range. The researchers used a viral vector to deliver the gene for bPAC specifically to the smooth muscle cells that make up blood vessels. When this vector was injected in rats, blood vessels throughout a large area of the brain became light-sensitive. “Blood vessels form a network in the brain that is extremely dense. Every cell in the brain is within a couple dozen microns of a blood vessel,” Jasanoff said. “The way I like to describe our approach is that we essentially turn the vasculature of the brain into a three-dimensional camera.”
Once the blood vessels were sensitized to light, the researchers implanted cells that had been engineered to express luciferase if a substrate called CZT is present. In the rats, the researchers were able to detect luciferase by imaging the brain with MRI, which revealed dilated blood vessels.
The researchers then tested whether their technique could detect light produced by the brain’s own cells, if they were engineered to express luciferase. They delivered the gene for a type of luciferase called GLuc to cells in a deep brain region known as the striatum. When the CZT substrate was injected into the animals, MRI imaging revealed the sites where light had been emitted.
The authors envisage that their BLUsH platform could be used in a variety of ways to help scientists learn more about the brain. For one, it could be used to map changes in gene expression, by linking the expression of luciferase to a specific gene. This could help researchers observe how gene expression changes during embryonic development and cell differentiation, or when new memories form. Luciferase could also be used to map anatomical connections between cells or to reveal how cells communicate with each other.
The researchers now plan to explore some of those applications, as well as adapting the technique for use in mice and other animal models. “Extensions of the BLUsH technique can include its generalization to detection of additional luminescent reporters, including chemiluminescent probes and alternative luciferases that sense cell signalling or other biological events,” the investigators concluded. Suggesting some potential uses, the team wrote, “Future applications could employ luciferin analogues engineered for better blood–brain barrier permeability or for multiplexed parallel detection of luciferase variants with different substrate selectivity…BLUsH thus provides a basis for connecting powerful optical reporter technology to state-of-the-art haemodynamic imaging in deep tissue. We anticipate that this synthesis will enable a diversity of molecular and cellular process to be studied over a range of spatiotemporal scales in living animals of any size.”
