Creating High-Resolution Multiscale Maps of Human Tissue Using Multi-beam SEM

Abstract

Multi-beam scanning electron microscopy (mSEM) enables high-throughput, nano-resolution imaging of macroscopic tissue samples, providing an unprecedented means for structure-function characterization of biological tissues and their cellular inhabitants, seamlessly across multiple length scales. Here we describe computational methods to reconstruct and navigate a multitude of high-resolution mSEM images of the human hip. We calculated cross-correlation shift vectors between overlapping images and used a mass-spring-damper model for optimal global registration. We utilized the Google Maps API to create an interactive map and provide open access to our reconstructed mSEM datasets to both the public and scientific communities via our website www.mechbio.org. The nano- to macro-scale map reveals the tissue’s biological and material constituents. Living inhabitants of the hip bone (e.g. osteocytes) are visible in their local extracellular matrix milieu (comprising collagen and mineral) and embedded in bone’s structural tissue architecture, i.e. the osteonal structures in which layers of mineralized tissue are organized in lamellae around a central blood vessel. Multi-beam SEM and our presented methodology enable an unprecedented, comprehensive understanding of health and disease from the molecular to organ length scale.

Sample preparation protocol enabling nano-to-mesoscopic mapping of cellular connectomes and their habitats in human tissues and organs

“…in organs and organ systems [1][2][3] . Seamless, multiscale and multimodal imaging provides a platform for interdisciplinary research and the trans-dimensional linking of biological structure and function [1][2][3][4][5][6][7][8][9][10][11] . The U.S. National Institutes of Health's (NIH) Connectome Project, launched in 2009, aimed to map "the human brain...to connect its structure to function and behavior" 10 .…”

“…The status of these organ connectomes provides a powerful early indicator of degenerative changes and disease states over the lifespan of the organism. Three-dimensional multiscale imaging methods allow unprecedented study of structure and function across length scales; from organ to tissue, cells to molecules [7][8][9]14 .…”

“…The emergence of multibeam scanning electron microscopy, with 61 or 91 parallel beams, is heralded by a new age of organ connectomics and presents a suitable platform for imaging large areas at nanometer resolution 1,[5][6][7][8][9] . Despite such pivotal advances in imaging technologies, biological tissue specimen preparation has remained unchanged and presents a barrier to translation of cutting-edge imaging modalities as well as to unleashing the potential of multimodal imaging.…”

“…1), details of the protocol have been requested by the medical and scientific community alike, providing impetus for its publication. [5][6][7]9,15 Presented in this protocol is a detailed workflow for sample preparation of sizeable tissue specimens (up to 10 cm in diameter, Fig. 2) of human and large mammalian origin.…”

Prospective Design, Rapid Prototyping, and Testing of Smart Dressings, Drug Delivery Patches, and Replacement Body Parts Using Microscopy Aided Design and ManufacturE (MADAME)

“…This algorithm can be used to co-register images and their collages from imaging modalities as diverse as confocal laser imaging (yielding e.g., porosity gradients) (22, 23), second harmonic imaging (yielding e.g., collagen and elastin fiber gradients) (4), atomic force and electron microscopy (24, 25), multibeam scanning electron microscopy (26), computed tomography, magnetic resonance imaging (27), etc. These data sets, when encoded in computer aided design and computer aided manufacture file formats, serve as inputs for combined weaving of fiber patterns and multidimensional advanced manufacture (e.g., 3D printing or laser sintering) of porous structures.…”