From Confocal to Volume Electron Microscopy: Investigating Microglia Structure and Function in Brain Disorders
Leveraging different microscopy methods to better understand microglial activities in brain remodeling and disease research
Microglia are immune cells within the brain that function to regulate brain development, repair injury, and maintain and remodel neuronal networks. Dr. Marie-Ève Tremblay is a leading neuroscientist in her field. In 2010, she was first author on the paper that revealed that microglia actively remodel neuronal circuits during normal physiological conditions.
Today, she is a Professor and leads a research team at the University of Victoria, Canada, focused on exploring the significance of microglial remodeling of neuronal circuits and elimination of synapses in the pathogenesis of brain disorders such as major depressive disorder, schizophrenia, and neurodegenerative diseases that include Parkinson’s and Alzheimer’s. She champions use of a broad variety of microscopy techniques, including confocal microscopy, high magnification scanning electron microscopy (SEM) chip mapping, and 3D volume electron microscopy using a ZEISS Crossbeam FIB-SEM, to continually make new discoveries pertaining to microglial structure and function in brain disease research.
Our goal is to design novel therapeutic strategies that specifically target microglia to promote brain resilience and healthy cognitive functions during aging. Microscopy provides key insights into microglial activities in brain development and remodeling. We combine many different microscopy technologies – from confocal to high magnification SEM and 3D electron microscopy and more – to give us the most complete picture possible of microglial structure and function.
Measuring Microglial Function
One core function of microglial cells is removal of tissue debris by phagocytosis. This activity can be evaluated using confocal microscopy. In the example shown on the right, microglial phagolysosomal activity is quantified through the count of cluster of differentiation 68 (CD68) puncta inside ionized calcium binding adaptor molecule 1 (IBA1)-positive (+) cells.
Dr. Tremblay and her team have used confocal microscopy for this application in many publications, including a mouse model of maternal immune activation and a macaque monkey model of Parkinson's disease. In a mouse model of Huntington's disease, they observed an increased microglial phagolysosomal activity in their striatum at presymptomatic stages of pathology compared to healthy controls.
Intra- and Intercellular Microglial Characteristics
Analyzing Cell Health & Function - or Dysfunction
In the Tremblay lab, chip mapping methodologies are used to analyze differences in intra- and inter-cellular characteristics of cellular health and function - or dysfunction. These 5 nm resolution images allow analysis of changes in the nuclear heterochromatin pattern and various organelles within microglia (e.g., endoplasmic reticulum, Golgi apparatus dilation, and endosomes with or without contents, such as synaptic elements). These analyses have also revealed how these changes in microglia impact the surrounding microenvironment by quantifying interactions with other parenchymal features (e.g., pre- and post-synaptic elements).
This image exemplifies the visualization of IBA1+ microglia in the brain parenchyma and demonstrates microglial direct contacts with surrounding neural structures as well as phagolysosomal inclusions. This workflow was used in their publication where a microglial state, dark microglia, that can currently only be visualized using electron microscopy, was analyzed in a mouse model of Alzheimer’s disease pathology as well as in post-mortem human brain samples. Their findings suggest dark microglia involvement in pathological remodeling of the brain, notably in neurodegenerative diseases.
3D Modeling of Cellular Processes and Whole Cells
An intimate understanding of cellular interactions
Three-dimensional modeling of cellular processes and whole cells allows for a unique and comprehensive understanding of the morphological features of these cells at up to a 3 nm x 3 nm x 3 nm resolution.
In this example, volume electron microscopy (vEM) using the ZEISS Crossbeam FIB-SEM was used to create comprehensible 3D models of dendritic spine density in the striatum of a mouse model of Huntington’s disease pathology where microglia exhibited a distinct role in the synaptic alteration and loss. A model of brain structures is built from numerous, sequential SEM images at nanometric resolution and provides an intimate understanding of cellular interactions. Specifically, visualizing the spines builds an understanding of the consequences of microglial synaptic pruning and interactions with synapses in disease conditions and how they directly impact behavior.
The insight we gain combining different microscopy techniques is essential for the understanding of complex phenomena. Microglial research has relied on the use and combination of different modalities to study these cells in a non-invasive way, without altering their structure and function. Having access to different microscopy methods with complementary capabilities in our own lab has been crucial to the success of our research team.