By Melissa Ryan, MPFI Postbac 2019-20
The brain is an amazing computation machine. Everything you sense, do, and think is processed or generated by the hundred billion neurons intricately connected to each other in the nervous system. How this incredibly sophisticated process works is still largely unknown. In order to better understand the human experience and improve therapies for individuals suffering from disease of the nervous system, we must gain a better understanding of the fundamental rules that govern how the brain functions. Our ability to uncover these rules is directly related to technological advances. For example, before the invention of the microscope investigations of brain function were only possible through philosophy and observations of behavior. As scientists gained the ability to see the building blocks of the brain, a more complete understanding emerged – to understand how something works it is extremely useful to know the function of the parts it is made out of. One prominent technological advancement that enables scientists to see the synapses, organelles, and proteins that make neural function possible is Electron Microscopy (EM).
To understand how EM works it is beneficial to understand Light Microscopy (LM). Many people are familiar with Light Microscopes, which they may have used in their science classes in school. Light microscopes work by using a set of glass lenses to bend, or change the trajectory, of light rays. The manipulation of beams of light is the same for the lenses in light microscope as it is in a magnifying glass or eyeglasses. With LM you can see objects as large as a millimeter and as small as a micron (e.g., mitochondria or bacteria). The best resolution* that can be achieved with conventional LM is 200 nm. LM is an extremely useful tool for Neuroscientists, but if you are interested in studying smaller components of a neuron, like inside synapses or particular proteins inside the cell, EM is necessary. **
EM is similar to LM in that there are lenses that bend beams to allow us to see small objects. However, instead of bending beams of light with glass lenses, electron microscopes bend a beam of electrons with electromagnetic lenses; coiled wires attract or repel the electron beam based on how much energy is flowing through them. Whereas with a light microscope you would change what lenses you let the light pass through, you can change the beam of electrons by adjusting the current supplied to the electromagnets. With EM the largest objects you can observe are 100 microns (e.g., a cell) and the smallest is approximately a nanometer (e.g., a single molecule or protein). Such small objects are visible in EM because the wavelength of the beam of electrons is much smaller than that of the beam of light (approx..1 nm vs 400-700 nm). Because you can only see things that the beam interacts with, having a smaller wavelength essentially increases the number of interactions that can occur resulting in better resolution. The physics of LM and EM are far more complicated, especially when discussing fluorescent LM, however, the general rule of thumb is you can only see objects larger than the wavelength you are using.
* Resolution – the smallest distance at which two objects can be distinguished from one another.
**Super-resolution LM techniques enable scientist to see smaller objects than conventional LM. However, EM is still the gold-standard for ultrastructural analysis.