3-dimensional morphological analysis of microglia in the sheep brain

Area of Science:

• Neuroscience
• Immunology
• Microscopy and Imaging

Background:

• Activated microglia exhibit distinct morphological changes, transitioning from ramified to amoeboid shapes.
• Accurate morphometric assessment is crucial for identifying subtle shifts in microglia activation states.
• While 3D analysis with Imaris software is common, standardized protocols and parameter rationales are lacking.

Purpose of the Study:

• To investigate the impact of microscope type, magnification, and investigator variability on microglia morphometric analysis using Imaris.
• To establish optimal parameters for accurate and reproducible 3D microglia tracing.
• To provide a guide for researchers new to microglia analysis, highlighting potential pitfalls and best practices.

Main Methods:

• Exploration of Imaris software capabilities and limitations for microglia analysis.
• Comparative analysis of microglia tracing from widefield versus confocal microscopy images.
• Assessment of image acquisition parameters, including objective magnification (20x vs. 40x).
• Evaluation of microglia morphology in sheep under naïve and activated (cardiopulmonary bypass) conditions.

Main Results:

• Confocal microscopy yielded more accurate microglia tracings than widefield microscopy.
• Highest inter-analyst consistency was achieved with confocal images using a 20x objective.
• Optimal tracing accuracy was obtained with confocal images acquired using a 40x objective.
• 3D microglial analysis detected more activation markers compared to traditional 2D methods.

Conclusions:

• Confocal microscopy with a 40x objective is recommended for precise microglia morphometric analysis.
• Standardized step-by-step instructions and cautionary notes are provided to enhance accuracy and consistency.
• This work aims to mitigate common pitfalls in microglia analysis, improving research reproducibility.