3D tissue analysis offers critical benefits for neuroscience research
The imaging and analysis of tissues is vital across a number of biological fields, particularly neuroscience, where the accurate characterization of neural tissue plays an important role in our understanding of the nervous system. Historically, these samples were imaged as thin slices, providing highly detailed, but ultimately limited and two dimensional, views of the brain. Technological improvements in both hardware and software have led to the development of volumetric techniques such as serial sectioning, where thin slices of a sample are cut and analyzed in sequence to produce a series of 2D images, which can then be reconstructed into a 3D representation of the tissue. Biology ultimately functions in three dimensions, and therefore, such 3D histology has given us a range of truly unprecedented insights into the structural and functional organization of nervous tissue.
It can, however, be difficult to determine how biologically representative these serial sectioning results are, as the mechanical process of sectioning can crush, skew, and otherwise distort the sample. This approach is also destructive, so further analysis of the sample is, at the very least, complicated by the fact that it is converted into a series of 2D slices. Further advancements in 3D tissue histology would, therefore, benefit from novel methodologies that can obtain volumetric information while keeping the sample intact.
Nondestructive tissue histology with light-sheet microscopy
Light-sheet microscopy (LSFM) is a fluorescence imaging technique that uses a laser sheet to illuminate and obtain fluorescence data for thin sample slices without mechanical sectioning. Previously limited by the opaque nature of most biological tissues, improvements in tissue clearing have made this a viable approach for high-quality tissue histology, even for thicker samples. Researchers at the Chinese Academy of Medical Sciences leveraged this method, along with tissue expansion, for the nondestructive analysis of microglia in the whole hippocampus of mice.
In short, the tissue clearing leverages a delipidation solution to selectively remove lipids in the sample, thereby reducing its opacity; fluorescent immunolabeling was also performed after this step. One of the aspects that this study hoped to capture is the immune response of microglia. To observe this, Liu and Xiu treated some samples with a lipopolysaccharide (LPS) after tissue clearing; this is a common method for inducing immune response.
Tissue expansion fills a sample with a polymerizing hydrogel that isotropically swells the specimen; this increases the resolution of biomolecules of interest without changing the overall structure of the sample. Liu and Xiu were able to achieve a 4x linear expansion of the hippocampus (i.e., a 64x volumetric expansion).
Using this combination of tissue clearance and expansion, detailed 3D reconstructions of the hippocampus could be created. The authors note that the size of the expanded samples was too great for whole hippocampus LSFM; regions of interest containing individual microglia were instead imaged within the samples.
Facilitating histological data analysis with Amira Software
Many cutting edge imaging techniques, including light-sheet microscopy, produce large quantities of data, which will only increase as higher resolution instrumentation continues to improve. In these contexts, automated reconstruction and segmentation algorithms become more and more vital as they substantially speed up data analysis and reduce the strain and human bias of manual processing.
Amira Software is designed specifically to improve the efficiency, accuracy, and clarity of this kind of data analysis. Built-in data processing workflows, along with optional, easy-to-install modules, facilitate reconstruction and segmentation, enabling a standardized approach that strengthens reliability and reproducibility. Liu and Xiu utilized Amira Software not only for reconstruction and segmentation, but also to extract a number of critical parameters from the hippocampus dataset, including whole cell and cell body volume, aspect ratio, as well as the total number of branches, their intersections, and more.
Comparing these parameters before and after LPS treatment clearly showed that the microglia were activated and exhibited a marked immune response (Figure 2). LPS-treated microglia appear to be rounder than the control sample, with shorter, sparser branches. Quantitative analysis confirms these observations.
Conclusions
Light-sheet microscopy, together with tissue clearing and expansion, provide a novel, nondestructive method for the 3D analysis of biological tissues. The authors note that their methodology can likely be adapted to a number of other neurological samples, including neurons and astrocytes, highlighting the exciting potential of light-sheet microscopy for 3D tissue histology in neuroscience. With the support of Amira Software, data processing and visualization become streamlined and reliable, avoiding the length and subjectivity of manual approaches. We are eager to see how this methodology will continue to evolve, and the insights that it will provide, expanding our understanding of neurological behavior and immune response.
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Read the full details of the study highlighted in this blog in the Journal of Neuroimmunology >
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