A Correlative Method for Imaging Identical Regions of Samples by Micro-CT, Light Microscopy, and Electron Microscopy

Abstract

We present a method in which a precise region of interest within an intact organism is spatially mapped in three dimensions by non-invasive micro-computed X-ray tomography (micro-CT), then further evaluated by light microscopy (LM) and transmission electron microscopy (TEM). Tissues are prepared as if for TEM including osmium fixation, which imparts soft tissue contrast in the micro-CT due to its strong X-ray attenuation. This method may therefore be applied to embedded, archived TEM samples. Upon selection of a two-dimensional (2-D) projection from a region of interest (ROI) within the three-dimensional volume, the epoxy-embedded sample is oriented for microtomy so that the sectioning plane is aligned with the micro-CT projection. Registration is verified by overlaying LM images with 2-D micro-CT projections. Structures that are poorly resolved in the micro-CT may be evaluated at TEM resolution by observing the next serial ultrathin section, thereby accessing the same ROI by all three imaging techniques. We compare white adipose tissue within the forelimbs of mice harboring a lipid-altering mutation with their littermate controls. We demonstrate that individual osmium-stained lipid droplets as small as 15 µm and separated by as little as 35 µm may be discerned as separate entities in the micro-CT, validating this to be a high-resolution, non-destructive technique for evaluation of fat content.

Combining micro-computed tomography with histology to analyze biomedical implants for peripheral nerve repair

“…2012). But an advantage of osmium it has been used to combine micro-CT imaging with post-imaging histology at both the light and electron microscopic levels (Handschuh et al 2013; Sengle et al 2013; Scheller et al 2014). With iodine, post-imaging histological studies have been limited, except for very superficial analyses of tissue structure, because iodine treatment renders soft tissues dense and brittle and imparts a yellow color (Jeffery et al 2011).…”

The Role of Micro-CT in 3D Histology Imaging

“…While some of our initial results were surprising, the data that we collected begin to elucidate elements of the 3D histology workflow that are seldom examined and facilitate the understanding of a pipeline to correspond in vivo and ex vivo imaging modalities [2,3,4,5,6]. Our results also indicate that micro-CT imaging technology is a very useful addition to the current workflow through its capacity to provide information on tissue morphology, volume of interest, tissue block quality and reference data for 3D histology models.…”

“…High- resolution 3D histology imaging is particularly advantageous to the discovery of diagnostic patterns in its capacity to improve correlation between imaging modalities, such as MRI, conventional CT and glass histology [2,3,4,5,6]. We are interested in using multimodality 3D imaging and 3D histology image data to accurately model, analyze, understand and test disease at high resolution in tissue samples imaged at ×20 to ×40 (0.46-0.23 μm/pixel).…”

X-Ray Microscopy as an Approach to Increasing Accuracy and Efficiency of Serial Block-Face Imaging for Correlated Light and Electron Microscopy of Biological Specimens

“…This suggests that XRM may be capable of imaging samples prepared for SBEM. There have been initial efforts to use XRM as an intermediate step in a multiscale, correlated imaging workflow (Burnett, et al, 2014; Sengle, et al, 2013). In this report, we describe our efforts to use XRM as part of an improved SBEM correlative imaging workflow.…”