The spread of microtomography as a tool for visualization of soft tissues has had a significant impact on a better understanding of complex biological systems. This technique allows a detailed three-dimensional quantitative view of the specimen to be obtained, correlating its morphological organization with its function, providing valuable insights on the functionality of the tissue. Regularly overlooked, but of great importance, proper sample mounting and preparation are fundamental for achieving the highest possible image quality even for the high-resolution imaging systems currently under development. Here, a quantitative analysis compares some of the most common sample-mounting strategies used for synchrotron-based X-ray microtomography of soft tissues: alcoholic-immersion, paraffin-embedding and critical-point drying. These three distinct sample-mounting strategies were performed on the same specimen in order to investigate their impact on sample morphology regardless of individual sample variation. In that sense, the alcoholic-immersion strategy, although causing less shrinkage to the tissue, proved to be the most unsuitable approach for a high-throughput high-resolution imaging experiment due to sample drifting. Also, critical-point drying may present some interesting advantages regarding image quality but is also incompatible with a high-throughput experiment. Lastly, paraffin-embedding is shown to be the most suitable strategy for current soft tissue microtomography experiments. Such detailed analysis of biological sample-mounting strategies for synchrotron-based X-ray microtomography are expected to offer valuable insights on the best approach for using this technique for 3D imaging of soft tissues and following morphometric analysis.

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Commonly used methods to visualize the biological structure of brain tissues at subcellular resolution are confocal microscopy and two-photon microscopy. Both require slicing the sample into sections of a few tens of micrometers. The recent developments in X-ray microtomography enable three-dimensional imaging at sub-micrometer and isotropic resolution with larger biological samples. In this work, we developed and compared original microtomography methods and staining protocols to improve the contrast for in vitro mouse neuron imaging. Using Golgi's method to stain neurons randomly, we imaged the whole set of mouse brain structures. For specific and nonrandom neuron labeling, we conjugated 20 nm gold nanoparticles to antibodies used in the immunohistochemistry (IHC) method, using anti-NeuN to label specifically neuronal nuclei. We applied an original subtraction dualenergy method for microtomography in the vicinity of the Au L-III absorption edge and compared image reconstructions to confocal microscopy images acquired on the same samples. The results show the possibility to characterize the 3D entire brain structure of mice. They demonstrated a high contrast and neuron detection improvement by applying the dual-energy method coupled to IHC staining.

Cateretê, the coherent X-ray scattering beamline at the new Brazilian synchrotron 5bent-achromat source, Sirius [1] is dedicated to coherent diffraction imaging (CDI) as well as X-ray photon correlation spectroscopy (XPCS) studies. Making the most of the coherence properties of the ultra-low emittance of the Sirius accelerator, will enable to perform 3D imaging of micrometer sized specimen down to few nanometers spatial resolution.

Described here are image reconstruction optimizations for ptychographic coherent X-ray scattering data and X-ray fluorescence, which have been developed for the new fourth-generation synchrotron light source, Sirius, at the Brazilian Synchrotron Light Laboratory. The optimization strategy has been applied to the standard experimental strategy for ptychographic and fluorescence experiments on the Carnaúba beamline which involves the use of high-speed continuous scans (fly scans) for a fast acquisition time over large areas through the use of a newly proposed trajectory named the alternating linear trajectory. The scientific computing developments presented here target an efficient use of graphical processing units (GPUs) to the point where large fly-scan acquisitions can be processed in real time on a local high-performance computer. Some optimizations involving a custom fast Fourier transform implementation and use of mixed precision can be applied to other algorithms and phase-retrieval techniques, and therefore this work provides a general optimization scheme. Finally, the optimization strategy presented here has improved performance by a factor of ∼2.5 times faster when compared with non-optimized GPU implementations.