Better imaging of biological tissues often leads to a deeper understanding. Now, scientists have developed an approach that combines seven imaging methods, including in vivo imaging, synchrotron X-ray, and volume electron microscopy. The method, called synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT), was used to image two different areas of the brain in mice—the olfactory bulb and the hippocampus. SXRT provided context at the subcellular level while also capturing information about the surrounding environment. The technique could be applied to other areas of the brain or parts of the body, providing scientists with a more detailed understanding of many different biological structures and tissues.

This work is published in Nature Communications in the article, “Functional and multiscale 3D structural investigation of brain tissue through correlative in vivo physiology, synchrotron microtomography, and volume electron microscopy.

“Our approach provides a reliable way to overcome the challenge of imaging structures at vastly different scales,” noted Carles Bosch, PhD, principal laboratory research scientist in the Sensory Circuits and Neurotechnology Laboratory at the Francis Crick Institute. “We believe it will be a powerful tool to investigate neuronal circuits in the brains of mammals as well as the structure and function of other tissues.”

The researchers used in vivo calcium imaging to visualize neurons in specific regions of the brain and see which neurons were active when the mice were exposed to odors. After the mice were euthanized, brain tissue samples were imaged using various methods including synchrotron X-ray tomography, which captures samples up to several millimeters in length. This scale is sufficient for scientists to see whole neural networks and also where particular cells or other structures sit within the wider context of the sample. Importantly, this method does not damage the sample so it can be imaged again using another technique.

The researchers then selected areas of particular interest to be imaged with electron microscopy, capturing intricate detail at a high resolution. At some target areas, this could map details as small as 10 nm, allowing researchers to see tiny structures like the individual synapses that connect neurons.

More specifically, they wrote that, in the olfactory bulb, “combining SXRT and SBEM enabled disambiguation of in vivo-assigned regions of interest.” And, in the hippocampus, “superficial pyramidal neurons in CA1a displayed a larger density of spine apparati than deeper ones.”

Using computer algorithms, they combined the results to create a complete map of the structure and function of the section of the brain they were studying, up to a few cubic millimeters.

“We’re interested in applying this approach to the brain, where it is important to gather information about whole neural networks which are several millimeters in length alongside information about specific neurons and synapses,” said Andreas Schaefer, PhD, head of the Sensory Circuits and Neurotechnology Laboratory at the Crick. “But it also has great potential to be useful in other settings, like cancer biology where researchers aim to understand the activity of particular cells in the context of the wider tumor.”