Engineering Blood Vessels to Image Bioluminescence Deep Within the Brain

Researchers frequently tag cells with fluorescent proteins, which enable them to monitor tumor growth or quantify alterations in gene expression that transpire during cell differentiation.

Although this method is effective in imaging cells and some bodily tissues, imaging structures located deep within the brain has proven challenging due to excessive light scattering before detection.

Now, researchers at MIT have developed a cutting-edge method for identifying this kind of light, or bioluminescence, in the brain: They manipulated the brain's blood vessels to express a protein that makes them enlarge when exposed to light. Then, the dilatation can be seen using magnetic resonance imaging (MRI), enabling researchers to identify the light source.

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. 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.”

Alan Jasanoff, Professor, Department of Biological Engineering, Massachusetts Institute of Technology 

Researchers may be able to delve deeper into the brain's inner workings, thanks to the novel method that Jasanoff and his associates have devised.

The study's principal author, Jasanoff, is also an associate investigator at MIT's McGovern Institute for Brain Research. The research was published in the journal Nature Biomedical Engineering. The principal authors of the work are Robert Ohlendorf and Nan Li, both former postdocs at MIT.

Detecting Light

Many creatures, such as fireflies and jellyfish, contain bioluminescent proteins. These proteins are used by researchers to mark particular proteins or cells whose luminosity can be measured with a luminometer. The protein luciferase, which glows in various colors, is one of the proteins that is frequently utilized for this purpose.

Deep brain luciferase detection was a goal for Jasanoff's lab, which specialized in creating novel MRI imaging techniques. They devised a technique to turn the brain's blood vessels into light detectors to accomplish that.

The researchers created light-responsive blood vessels because a common type of magnetic resonance imaging (MRI) uses blood flow variations in the brain to image changes in blood flow.

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.”

Alan Jasanoff, Professor, Department of Biological Engineering, Massachusetts Institute of Technology

The scientists designed the blood arteries to contain Beggiatoa photoactivated adenylate cyclase (bPAC), a bacterial protein, to make them light-sensitive. This enzyme creates a chemical known as cAMP in response to light, which stimulates blood vessels to widen.

The balance between oxygenated and deoxygenated hemoglobin, which has distinct magnetic characteristics, is altered when blood arteries widen. MRI is capable of detecting this change in magnetic characteristics.

BPAC responds especially to blue light, which has a short wavelength, hence it detects light created within close range. To transfer the bPAC gene selectively to the smooth muscle cells that comprise blood arteries, the researchers employed a viral vector. Rats receiving this viral injection showed light sensitivity in many of the brain's blood vessels.

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, the way I like to describe our approach is that we essentially turn the vasculature of the brain into a three-dimensional camera.

Alan Jasanoff, Professor, Department of Biological Engineering, Massachusetts Institute of Technology

The researchers next implanted cells that were designed to express luciferase in the presence of a substrate known as CZT, after the blood vessels had been made sensitive to light. The MRI scan of the rats' brains showed dilated blood vessels, which allowed the researchers to identify luciferase in the brains of the animals.

Tracking Changes in the Brain

Subsequently, the researchers investigated if their method could identify light generated by brain cells, provided that the cells were modified to express luciferase. They introduced the GLuc luciferase gene into cells located in the striatum, a deep brain region. 

The locations where the light had been released were visible on MRI imaging after the animals had received an injection of the CZT substrate.

According to Jasanoff, there are several potential applications for this method, which the researchers named bioluminescence imaging utilizing hemodynamics, or BLUsH, to aid in the advancement of scientific knowledge regarding the brain.

One such application is in mapping gene expression variations by associating luciferase expression with a particular gene. This may make it easier for researchers to see how gene expression varies during the formation of new memories or throughout embryonic development and cell differentiation.

Moreover, luciferase may be utilized to map the anatomical relationships between cells or to disclose the mechanisms of cell-to-cell communication.

In addition to modifying the method for usage in mice and other animal models, the researchers now intend to investigate a few of those applications.