The major benefit of imaging with X-rays is that they offer a far greater penetration depth than electrons or visible photons, enabling reconstruction of 3D images. Within biology, this allows visualization of the interior of cells, for example, or the inner structure of complex neuronal networks.
But can microradiology produce images of comparable quality to electron and visible microscopy? According to lead author Yeukuang Hwu, from Academia Sinica in Taipei, Taiwan, this is now possible, thanks to two key developments from the team: a new record in spatial resolution and optimization of the staining procedure.
Increased resolution was achieved via the use of a Fresnel zone plate that acts as a magnifying lens. "With new nanofabrication techniques, we were able to fabricate zone plates that reached the world record in lateral resolution for hard X-rays," explained Hwu. "The main challenge was to reconcile the need for very narrow structures in the zone plates, required for resolution, and very thick structures, required to absorb X-rays. Structures that are narrow and thick create huge stability problems, but we were able to solve these by using a special geometry of the zone plate design."
Experiments using X-rays emitted by beamlines at the Advanced Photon Source at Argonne National Laboratory (Argonne, IL), and the National Synchrotron Radiation Research Center in Taiwan, demonstrated that the imaging system with Fresnel zone plates could achieve a spatial resolution of better than 20 nm with 8 keV photons.
A quantitative assessment of image resolution showed a Rayleigh contrast resolution of about 16.5 nm – said to be the highest performance to date for hard X-rays. Repeated tests with different zone plates and test patterns consistently indicated sub-20 nm resolution.
Suitable stain
The team's second breakthrough was the development of a microradiology-appropriate staining procedure, a crucial factor in neurobiological microscopy. Stains developed for optical imaging are not suitable for radiology: the fluorophore penetration is inadequate for thick X-ray specimens and fluorescent ingredients don't effectively enhance X-ray contrast.
Thus they searched for suitable staining ingredients, and came up with a modified Golgi-Cox approach (a staining protocol for neurons) using mercury and silver to increase X-ray contrast. Extending the incubation time to one month or more increased perfusion and enabled staining through the entire specimen depth.
The microradiology technique was tested by imaging coronal section from mouse brains. The researchers recorded X-ray micrographs with 8 keV photons filtered by a double-crystal monochromator, typically using 0.5 s per frame. For tomography reconstruction, sets of 280 projections spaced by 0.5 degrees were used.
The resulting high-quality neuroimages exhibited a range of novel features. For example, details of the neuron dendritic spine could be seen, demonstrating the ability to detect fine details at a subcellular level. Tomographic 3D images enabled morphology-based identification of different neurons and neuron parts. Images showing individual neurons, not just in the first layer but also in deeper layers, demonstrated the importance of stain penetration when imaging complex networks.
The researchers envisage three initial applications for their microradiology technique. "First, imaging neuron systems, in which 3D features are quite important," explained co-author Giorgio Margaritondo, from Ecole Polytechnique Fédérale de Lausanne (EPFL) in Switzerland. "Second, our approach is excellent for detecting angiogenesis, including extremely small vessels. Third, for tracing nanoparticles in organic systems to assess their behaviour and possible applications, for example, as imaging contrast agents."