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In JoVE (1)

Other Publications (6)

Articles by John Keyser in JoVE

 JoVE Bioengineering

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

1Department of Computer Science and Engineering, Texas A&M University, 2Beckman Institute for Advanced Science and Technology, University of Illinois, 3Department of Electrical and Computer Engineering, Kettering University, 43Scan, 5Department of Veterinary Integrative Biosciences, Texas A&M University


JoVE 3248

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

Other articles by John Keyser on PubMed

Visualization of Fibrous and Thread-like Data

Thread-like structures are becoming more common in modern volumetric data sets as our ability to image vascular and neural tissue at higher resolutions improves. The thread-like structures of neurons and micro-vessels pose a unique problem in visualization since they tend to be densely packed in small volumes of tissue. This makes it difficult for an observer to interpret useful patterns from the data or trace individual fibers. In this paper we describe several methods for dealing with large amounts of thread-like data, such as data sets collected using Knife-Edge Scanning Microscopy (KESM) and Serial Block-Face Scanning Electron Microscopy (SBF-SEM). These methods allow us to collect volumetric data from embedded samples of whole-brain tissue. The neuronal and microvascular data that we acquire consists of thin, branching structures extending over very large regions. Traditional visualization schemes are not sufficient to make sense of the large, dense, complex structures encountered. In this paper, we address three methods to allow a user to explore a fiber network effectively. We describe interactive techniques for rendering large sets of neurons using self-orienting surfaces implemented on the GPU. We also present techniques for rendering fiber networks in a way that provides useful information about flow and orientation. Third, a global illumination framework is used to create high-quality visualizations that emphasize the underlying fiber structure. Implementation details, performance, and advantages and disadvantages of each approach are discussed.

Visualization of Cellular and Microvascular Relationships

Understanding the structure of microvasculature structures and their relationship to cells in biological tissue is an important and complex problem. Brain microvasculature in particular is known to play an important role in chronic diseases. However, these networks are only visible at the microscopic level and can span large volumes of tissue. Due to recent advances in microscopy, large volumes of data can be imaged at the resolution necessary to reconstruct these structures. Due to the dense and complex nature of microscopy data sets, it is important to limit the amount of information displayed. In this paper, we describe methods for encoding the unique structure of microvascular data, allowing researchers to selectively explore microvascular anatomy. We also identify the queries most useful to researchers studying microvascular and cellular relationships. By associating cellular structures with our microvascular framework, we allow researchers to explore interesting anatomical relationships in dense and complex data sets.

Hardware Accelerated Segmentation of Complex Volumetric Filament Networks

We present a framework for segmenting and storing filament networks from scalar volume data. Filament networks are encountered more and more commonly in biomedical imaging due to advances in high-throughput microscopy. These data sets are characterized by a complex volumetric network of thin filaments embedded in a scalar volume field. High-throughput microscopy volumes are also difficult to manage since they can require several terabytes of storage, even though the total volume of the embedded structure is much smaller. Filaments in microscopy data sets are difficult to segment because their diameter is often near the sampling resolution of the microscope, yet these networks can span large regions of the data set. We describe a novel method to trace filaments through scalar volume data sets that is robust to both noisy and undersampled data. We use graphics hardware to accelerate the tracing algorithm, making it more useful for large data sets. After the initial network is traced, we use an efficient encoding scheme to store volumetric data pertaining to the network.

Inference-Based Surface Reconstruction of Cluttered Environments

We present an inference-based surface reconstruction algorithm that is capable of identifying objects of interest amongst a cluttered scene, and reconstructing solid model representations even in the presence of occluded surfaces. Our proposed approach incorporates a predictive modeling framework that uses a set of user provided models for prior knowledge, and applies this knowledge to the iterative identification and construction process. Our approach uses a local to global construction process guided by rules for fitting high quality surface patches obtained from these prior models. We demonstrate the application of this algorithm on several example datasets containing heavy clutter and occlusion.

Fast Macro-scale Transmission Imaging of Microvascular Networks Using KESM

Accurate microvascular morphometric information has significant implications in several fields, including the quantification of angiogenesis in cancer research, understanding the immune response for neural prosthetics, and predicting the nature of blood flow as it relates to stroke. We report imaging of the whole mouse brain microvascular system at resolutions sufficient to perform accurate morphometry. Imaging was performed using Knife-Edge Scanning Microscopy (KESM) and is the first example of this technique that can be directly applied to clinical research. We are able to achieve ≈ 0.7μm resolution laterally with 1μm depth resolution using serial sectioning. No alignment was necessary and contrast was sufficient to allow segmentation and measurement of vessels.

Multiscale Exploration of Mouse Brain Microstructures Using the Knife-edge Scanning Microscope Brain Atlas

Connectomics is the study of the full connection matrix of the brain. Recent advances in high-throughput, high-resolution 3D microscopy methods have enabled the imaging of whole small animal brains at a sub-micrometer resolution, potentially opening the road to full-blown connectomics research. One of the first such instruments to achieve whole-brain-scale imaging at sub-micrometer resolution is the Knife-Edge Scanning Microscope (KESM). KESM whole-brain data sets now include Golgi (neuronal circuits), Nissl (soma distribution), and India ink (vascular networks). KESM data can contribute greatly to connectomics research, since they fill the gap between lower resolution, large volume imaging methods (such as diffusion MRI) and higher resolution, small volume methods (e.g., serial sectioning electron microscopy). Furthermore, KESM data are by their nature multiscale, ranging from the subcellular to the whole organ scale. Due to this, visualization alone is a huge challenge, before we even start worrying about quantitative connectivity analysis. To solve this issue, we developed a web-based neuroinformatics framework for efficient visualization and analysis of the multiscale KESM data sets. In this paper, we will first provide an overview of KESM, then discuss in detail the KESM data sets and the web-based neuroinformatics framework, which is called the KESM brain atlas (KESMBA). Finally, we will discuss the relevance of the KESMBA to connectomics research, and identify challenges and future directions.

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