Biological matter may change shape via water absorption or loss. For example, brain tissue shows non-uniform shrinkage during formalin fixation and paraffin embedding, which is the most common tissue preparation for conventional histological analysis. Local deformations can be analyzed with non-rigid registration of non-destructive three-dimensional imaging datasets. We utilized synchrotron radiation microtomography at the ANATOMIX beamline of Synchrotron SOLEIL to image a mouse brain with 3 micron voxel length after formalin fixation, immersion in ascending alcohol series and xylene, and after paraffin embedding. We created a pipeline for non- rigid registration to align the volumes and extract volumetric strain fields. In this way, we could visualize the swelling/shrinkage of anatomical features. This method avoids time-consuming segmentation of brain regions, however it is sensitive to the registration parameters. In this proceedings paper, we discuss the selection of registration parameters in order to generate plausible volumetric strain fields. This protocol can be deployed to any type of shape change of biological matter and allows for the quantification of the related processes.
Successful tomographic imaging of soft tissues with micrometer resolution includes preparation, acquisition, re- construction, and data evaluation. Tissue preparation is essential for hard X-ray microtomography, because staining- and embedding materials can substantially alter the biological tissue post mortem. We performed to- mographic imaging of zebrafish embryos in alcohol and after paraffin embedding with a conventional X-ray source and at a synchrotron radiation facility. The resulting multi-modal, three-dimensional data were registered for direct comparison. Single-cell precision was reached for the synchrotron radiation-based approach, which allows for segmentation of full organs such as the embryonic kidneys. While this approach offers an order of magnitude higher spatial resolution, many of the anatomical features can be readily recognized with the more accessible laboratory system. Propagation-based data acquisition enabled us to demonstrate the complementary nature of the edge-enhanced absorption contrast- and the phase contrast-based modality for visualizing multiple microanatomical features. While ethanol and paraffin embeddings allowed for identification of the same anatomical structures, paraffin-embedding, however, led to more artefacts and shrinkage. The presented multi-modal imaging approaches can be further extended to visualize three to four orders of magnitude larger volumes such as adult zebrafish or complete organs of larger animals such as mouse brains. Going towards even larger volumes, such as the human brain, presents further challenges related to paraffin embedding, data acquisition and handling of the peta-byte scale data volumes. This study provided a multi-modal imaging strategy by the combination of X-ray sources and sample embeddings which can play a role in addressing these challenges.
X-ray grating interferometry (XGI) is a phase-contrast imaging technique that allows for a quantitative measurement of the refractive index with high density resolution in a model-independent manner—i.e. without a priori knowledge of the specimen composition. However, the retrieval of the X-ray wavefront phase shift relies on the accurate measurement of the interference pattern phase shift, making XGI vulnerable to phase wrapping when the interference pattern phase shift, related to the derivative of the wavefront phase shift, is large. Standard procedure for avoiding phase wrapping involves submerging the specimen in a water bath to reduce the mismatch of the index of refraction at the boundaries, but this requires a top-down rotation stage and is susceptible to gas bubble formation inside the water bath. Our team has presented an algorithm to remove phase wrapping artifacts for cylindrically shaped specimens that is applied to the phase-retrieved sinogram. This algorithm models and replaces phase-wrapped data to prevent the spread of “cupping” artifacts due to the integration of the differential phase during reconstruction. We give a criterion for selecting the modeling parameters so that the resulting measurement of the index of refraction matches the results of measurements without phase wrapping. We also apply this technique to cases where phase wrapping occurs at multiple interfaces. This algorithm allows for XGI measurements without a water bath and top-down rotation stage at synchrotron and laboratory facilities, especially as sensitivity increases.