What Is Synapse Electron Microscopy and How Does It Work?

The human brain operates through communication between its billions of neurons at junctions known as synapses. These gaps, where signals are transmitted, are fundamental to how the brain processes information and learns. Since synapses are too small to be seen with a conventional light microscope, scientists use the power of electron microscopes to visualize them. Synapse electron microscopy allows for the direct observation of the brain’s physical wiring, revealing the architecture of these connections.

Peering into the Nanoscale: Principles of Synapse Electron Microscopy

The primary reason electron microscopy is required for studying synapses is scale. Synaptic structures are measured in nanometers, a scale light microscopes cannot resolve. A microscope’s resolving power is limited by the wavelength of its illumination source; since an electron’s wavelength can be up to 100,000 times shorter than that of visible light, electron microscopes achieve magnifications of over 1 million times.

An electron microscope works by emitting a beam of electrons within a vacuum chamber, which is necessary because air molecules would deflect the electrons. Instead of glass lenses, electromagnetic lenses focus this electron beam onto the specimen. As electrons pass through or scatter off the sample, they generate a highly magnified image.

Obtaining clear images requires careful preparation of neural tissue. The first step is chemical fixation, where chemicals lock all cellular structures in place. This is followed by staining with osmium tetroxide, which helps make cell membranes more visible.

Because water would evaporate in the microscope’s vacuum, it is removed through dehydration and replaced with a liquid resin that hardens. Using an instrument called an ultramicrotome, this hardened block is cut into ultrathin sections, often 40 to 90 nanometers thick, thin enough for an electron beam to pass through.

Revealing Synaptic Ultrastructure: What We Can See

An electron microscope reveals the synapse not as a single point, but as a complex and highly organized structure. The images clearly show the presynaptic terminal, the part of the neuron that sends the signal. This terminal is filled with small, spherical structures called synaptic vesicles, each containing neurotransmitter molecules. Also visible are mitochondria, which supply the energy required for synaptic transmission.

The images also delineate the synaptic cleft, the physical gap that separates the presynaptic neuron from the postsynaptic neuron. This space is typically only 20 to 40 nanometers wide. On the other side of this cleft is the postsynaptic element, the part of the receiving neuron.

A prominent feature of the postsynaptic element is the postsynaptic density (PSD), which appears as a dark, electron-dense thickening on the membrane. The PSD is a complex scaffold of proteins, including the receptors that bind to neurotransmitters and the signaling molecules that carry the message into the receiving cell. The size and complexity of the PSD are often correlated with the strength of the synapse.

Electron microscopy also allows neuroscientists to classify synapses based on their appearance. Asymmetric synapses, where the postsynaptic density is visibly thicker than the presynaptic membrane, are excitatory, meaning they increase the likelihood of the receiving neuron firing. Symmetric synapses, with pre- and postsynaptic membranes of similar thickness, are inhibitory.

Technological Approaches in Synaptic Imaging

The field of synapse electron microscopy encompasses a variety of techniques, each offering unique advantages for studying neural connections.

• Transmission Electron Microscopy (TEM): This foundational method creates high-resolution, two-dimensional images by passing a beam of electrons through an ultrathin slice of tissue. It was the first technique to reveal the detailed ultrastructure of the synapse.
• Serial Block-Face Scanning Electron Microscopy (SBF-SEM): This Volume EM technique uses an ultramicrotome inside the microscope to shave off a thin layer of tissue after it has been imaged. Repeating this process generates a stack of images that can be computationally reconstructed into a 3D model.
• Focused Ion Beam Scanning Electron Microscopy (FIB-SEM): This method uses a focused ion beam to mill away the tissue surface layer by layer while a scanning electron beam images the new face. It provides higher resolution in the vertical dimension than SBF-SEM, ideal for reconstructing fine details.
• Electron Tomography (ET): For the highest possible 3D resolution, a sample is tilted at various angles relative to the electron beam, and an image is captured at each angle. These images are then computationally combined to create a 3D reconstruction, or tomogram, of a single synapse.
• Correlative Light and Electron Microscopy (CLEM): This approach first uses light microscopy to identify specific neurons or events, such as a recently active neuron. The same sample is then processed for electron microscopy to visualize the ultrastructure of that exact location, linking a synapse’s function directly to its form.

Landmark Discoveries and Understanding Brain Function

The application of electron microscopy to neural tissue has driven fundamental advances in neuroscience. In the 1950s, the first clear images of the synapse provided by Sanford Palay and George Palade confirmed the “neuron doctrine.” These images showed definitively that neurons are distinct cells separated by a physical gap, not a continuous network.

Electron microscopy also provided visual evidence for the quantal theory of neurotransmitter release. Scientists had hypothesized that neurotransmitters were released in discrete packets, and EM images visualized the synaptic vesicles. Seeing them clustered at the presynaptic terminal provided a clear structural basis for how these chemical packets are discharged.

The ability to see synapses in detail has been important for understanding synaptic plasticity, the process by which connections between neurons strengthen or weaken over time as a basis for learning and memory. EM studies have shown that learning can lead to physical changes in synapses, such as an increase in the size of the postsynaptic density or changes in the shape of dendritic spines.

Large-scale Volume EM projects are at the heart of the emerging field of connectomics, which aims to create complete “wiring diagrams” of brain regions. By meticulously tracing every neuronal connection in a given volume of tissue, researchers can map the intricate circuits that give rise to specific behaviors and cognitive functions.

Finally, synapse electron microscopy provides a window into brain disorders. Studies have revealed synaptic loss is a feature of Alzheimer’s disease, and more subtle alterations in synaptic structure have been linked to conditions such as schizophrenia and autism spectrum disorders. By identifying these synaptic pathologies, EM helps guide research into the underlying mechanisms of these diseases.