Location
Philadelphia, PA
Start Date
9-5-2018 1:00 PM
Description
Introduction: Technology is a powerful analytic tool. This abstract outlines the use of a custom macro to analyze rat sciatic nerve histology sections as part of a larger study to evaluate functional and histologic outcomes after segmental nerve injury. The problem facing analysis of nerve sections is that the microscopic features of a nerve, such as the individual axon, cannot be viewed on the same scale as the entire nerve bundle. The goal of this analysis was to extrapolate the average total values of axon count, axon density, fiber diameter, and myelin thickness on a nerve section image taken at 400x magnification to the total area captured at 40x magnification.
Methods: Images of nerve sections stained with toluidine blue were captured using a microscope with a mounted camera and image acquisition software (Nikon Eclipse E800; Nikon DS-Ri1; NIS Elements BR 3.10 SP3 Hotfix5). Multiple images at different light intensities were used to determine a contrast and saturation that would grant the best accuracy. From these images, a custom macro using Image-Pro Premier (Version 9.1.5262.28) was developed to compute the desired measurements (Media Cybernetics, Rockville MD).
Results: At 400x magnification, circular shape recognition was used to determine each axon myelin sheath pair in the selected area. Area analyzed was within the “circle of good definition”. After an axon myelin pair was recognized, multiple diameter measurements were taken, and these values were the key to calculations of axon area, fiber diameter, and myelin thickness. Two images taken at 400x magnification for each sample were used to calculate an average for each measurement. An image of the entire nerve slice at 40x was taken, and this calculated area was used extrapolate measurements to the entire nerve section.
Discussion:The utility of this software and macro is exciting due to its reproducibility, accuracy, and efficiency. Not only does it take human processing and potential error out of the equation, but it solves the scale discrepancy when trying to analyze microscopic parameters on a larger scale. This procedure is versatile and can be implemented in future histologic analysis of nerve sections.
Embargo Period
5-30-2018
Use of a Custom Macro in Analysis of Rat Sciatic Nerve Sections
Philadelphia, PA
Introduction: Technology is a powerful analytic tool. This abstract outlines the use of a custom macro to analyze rat sciatic nerve histology sections as part of a larger study to evaluate functional and histologic outcomes after segmental nerve injury. The problem facing analysis of nerve sections is that the microscopic features of a nerve, such as the individual axon, cannot be viewed on the same scale as the entire nerve bundle. The goal of this analysis was to extrapolate the average total values of axon count, axon density, fiber diameter, and myelin thickness on a nerve section image taken at 400x magnification to the total area captured at 40x magnification.
Methods: Images of nerve sections stained with toluidine blue were captured using a microscope with a mounted camera and image acquisition software (Nikon Eclipse E800; Nikon DS-Ri1; NIS Elements BR 3.10 SP3 Hotfix5). Multiple images at different light intensities were used to determine a contrast and saturation that would grant the best accuracy. From these images, a custom macro using Image-Pro Premier (Version 9.1.5262.28) was developed to compute the desired measurements (Media Cybernetics, Rockville MD).
Results: At 400x magnification, circular shape recognition was used to determine each axon myelin sheath pair in the selected area. Area analyzed was within the “circle of good definition”. After an axon myelin pair was recognized, multiple diameter measurements were taken, and these values were the key to calculations of axon area, fiber diameter, and myelin thickness. Two images taken at 400x magnification for each sample were used to calculate an average for each measurement. An image of the entire nerve slice at 40x was taken, and this calculated area was used extrapolate measurements to the entire nerve section.
Discussion:The utility of this software and macro is exciting due to its reproducibility, accuracy, and efficiency. Not only does it take human processing and potential error out of the equation, but it solves the scale discrepancy when trying to analyze microscopic parameters on a larger scale. This procedure is versatile and can be implemented in future histologic analysis of nerve sections.