The human brain is one of the most complex systems in biology. With its 100 trillion connections, the neuronal network controls motor responses and sensory information that determine our memory, identity, consciousness and creativity. Understanding how the brain functions at a single-molecule level is key for understanding the foundations of neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease. The mechanisms underlying brain degeneration onset remain largely unresolved; current efforts are focusing on understanding its molecular hallmarks to improve early diagnosis and develop more efficient drugs. This guide summarizes how single-molecule imaging is unlocking the understanding of brain function in health and disease.
Challenges in understanding synapses
Brain tissue complexity in neurodegeneration
Resolving single synapses in tissue: PALM array tomography
Labeling single molecules in brain tissues
Brain functions are operated through neural connections between dendritic spines and axon terminals, known as synapses. Synapses operate in a sophisticated and cooperative manner in order to maintain brain function and adapt/respond to stimuli.
The small size of synapses, under 200 nm in size, can hinder the study of the molecular mechanisms regulating neurotransmission. Each synapse is composed of over 200 proteins and a synaptic cleft, across which communication between cell terminals is mediated, that can be as small as 20-40nm (Figure 1).
Understanding the crucial mechanisms that determine neuronal connections in health and disease is limited by multiple factors. Firstly, culture cell lines for certain brain cell types can be difficult to obtain and maintain in vitro, making it challenging to find truly predictive neural models. Currently, the most accessible super-resolution imaging studies use primary cortical and hippocampal cell cultures from mice or rats. Yet these models can be considered artificial environments with low complexity brain architectures. An attractive alternative for neuroscientists is the use of neurons differentiated from iPSCs (induced Pluripotent Stem Cells, generated directly from adult cells), which allows research on human cell lines. However, the lack of disease models necessary for iPCS is limiting research using this approach.
Another limitation in neuroimaging is the availability of labeling tools; available antibodies target the most common and best researched proteins while other players remain unexplored, hindering the detailed analysis of neuronal synapse architecture.
Because synapses are under 200 nm in size, their intricate molecular details are ‘invisible’ to conventional, diffraction limited light microscopy techniques. Understanding the molecular hallmarks of brain plasticity with single-molecule sensitivity entails characterizing neuronal synapse architecture, and studying the dynamics of synaptic proteins, including molecular interactions and distributions of neurotransmitters, nucleic acids and other biomolecules. Single-molecule imaging is a robust tool for answering the fundamental questions on brain function and dysfunction, particularly when combined with research on the complex interactions within the neuronal ecosystem.
In the presented example, neuronal synapses were visualized in 3-color dSTORM imaging using two presynaptic proteins; Bassoon (synaptic active zone, in magenta) and VGluT1 (synaptic vesicles, in green), and the postsynaptic protein Homer1 (postsynaptic density, in orange). The data revealed the presence and organization of neurotransmitter vesicles in between pre- and postsynaptic protein clusters (Figure 2). To learn more read the ONI application note on revealing synapse architecture at a single-molecule level.
Our brain is composed of highly complex and densely packed tissues with specialized functions. An adult human brain contains an estimated 86 billion neurons and a similar number of supporting cells, including astrocytes and microglia. On average, each brain cell is connected to around 25,000 other cells, meaning that tissues are integrated by local intertwined cells that project to distant regions. This makes three-dimensional (3D) visualization of brain tissues a challenging job for neuroscientists.
Current neuroscience studies focus on characterizing the different brain cell populations and their roles within neuronal circuits to better understand disease onset and progression, both at tissue and molecular levels. Some researchers apply in vivoimaging techniques on live tissues or image fixed tissue sections.
There are several challenges facing research on brain tissues, which commonly include sample sourcing and preparation, specimen labeling, imaging of thick sections, or 3-dimensional data acquisition, often involving time-consuming and laborious protocols. Current efforts are also concentrating on using stem cell models and molecular tools to mimic brain physiology and better explore the origins of neurological disorders.
Current ground-breaking studies are focusing on the discovery and validation of early-disease biomarkers in human fluids, such as cerebrospinal fluid (CSF), which could help diagnose neurodegenerative diseases at early/pre-symptomatic stages, given that high-sensitivity detection of basal levels of biomarkers is achieved (attomolar concentrations in most cases). This would also contribute to the better understanding of disease mechanisms and allow a better patient stratification prior to therapy. Early clinical trials are currently underway to test how small drugs can block or stimulate key molecular interactions involved in neurodegenerative disease progression.
In recent years, extracellular vesicles (EVs) have also been studied as potential carriers of molecules for intercellular communication within tissues and throughout the body, with their unique ability to cross the blood-brain barrier and facilitate communication between peripheral tissues and the central nervous system (CNS). Read our EV guide for more details on single-molecule imaging solutions for studying extracellular vesicles.
In dense tissue environments, depth often affects light scattering. The brain’s high lipid content (more than half of the solid matter) contributes to the mismatch in refractive index that causes light scattering. Tissue clearing is an approach to counteract this issue, however, it requires specialized training and arduous processing, normally done on >1mm-thick tissue sections.
An approach to tissue imaging that allows molecular and ultrastructure analysis of neuronal circuits in tissues is array tomography. Recent advances in super-resolution microscopy have enabled the development of combined PALM array tomography as a way to image deep tissues with high sensitivity by using physical sectioning, and achieve isotropic high-resolution in all three dimensions.
After identifying a unique structure as the reference point in each of the sections (Figure 4), each of the positions can be imaged using PALM imaging, this allows the visualization of synaptic protein clusters (i.e. PSD95 clusters) to determine their structure and their heterogeneity within brain tissues at a single-molecule level. To learn more you can download the ONI application note on resolving single synapses with PALM array tomography.
In vivo labeling of the molecules of interest can also pose a significant bottleneck for researchers, especially when preparing tissue samples for single-molecule imaging. Different labeling approaches can be used for brain tissue in vivo imaging, including photo-activatable fluorescent proteins (FPs) or Halo-tags. Halo-tags combine the advantages of genetically encoded fluorescent proteins with the superior brightness of synthetic dyes and photostability.
The advantage of using an imaging system with simultaneous dual color acquisition is that different labeling methods can be directly compared. In the example, PALM imaging on whole mouse brain tissue slices was used to evaluate the efficiency of in vivo fluorescent labeling of single molecules using Halo tags. The comparison of the localizations between the photo-activated protein PSD95-mEos2 (blue) and the labeled PSD95-Halo tag (magenta) was used to validate the efficiency of Halo-tag labeling in vivo. You can read more about this experiment in the ONI application note on single-molecule in vivo imaging of brain tissues.
The Nanoimager platform enables single-molecule neuroimaging of cultured neuronal cells and brain tissue sections, with a resolution reaching 20 nm. Combining automation features with super-resolution imaging allows the quick and efficient identification of region of interests to then gather localizations from thousands of synaptic proteins. It also facilitates acquisition of data on single molecule distribution, co-localization, or postsynaptic density profiles.
Supporting both fixed and live sample imaging, researchers can develop their own assays to build a complete spatio-temporal portrait of neuronal pathways. Single-molecule sensitivity means potential for improved, accelerated diagnostics and disease therapies. Understanding how drugs alter neurotransmission mechanisms and kinetics with minimal sample requirements can also translate into robust, fast and efficient drug profiling, in a more cost-efficient manner.
Single-molecule imaging has the potential to help establish and characterize new disease models to enable robust testing of novel therapeutics and facilitate drug development. With its compact design, lack of need for a dark room or additional infrastructure, the Nanoimager is the most accessible super-resolution tool ever designed. To learn more about the microscope and ONI, please visit oni.bio/nanoimager.
Single-molecule imaging opens new possibilities in neuroimaging field. It gives researchers new tools to:
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