Two-photon microscopy of the brain
Mini-microscope conducts a "report" from the brain live
One of the main tasks of modern neurology is to understand how the functioning of the brain correlates with the work of different groups of its main cells – neurons. And thanks to the latest developments in the field of microscopy, we can now observe in real time how the "dialogue" of neurons gives rise to an intention that is realized in a specific behavioral action.
One of the modern technologies for studying the work of the brain is optical visualization using methods of genetics and microscopy. They are used, for obvious reasons, on laboratory animals, primarily mice and rats.
For example, the activity of individual neurons in the field of view of a microscope can be determined by changes in the concentration of intracellular calcium. In this imaging method, a special label binds to the free calcium of the cytoplasm and begins to fluoresce. And in order to make brain cells glow while working, a gene borrowed from a jellyfish is embedded in their genome, encoding the enzyme luciferase, which, when a specific substrate is added, triggers the bioluminescence process. In addition, populations of functionally different neurons can be "colored" in different ways due to their molecular specificity, and by interneuronal interaction it is possible to judge how certain cognitive functions are carried out.
As for microscopic research methods, at first stationary two-photon laser scanning microscopes were used to visualize the behavior of rodents, while the head of an individual with a removed skull fragment was fixed under the lens. But in this case, the animal is very limited in movements, and in order to track the work of neurons in complex behavior, it must behave naturally.
Two-photon microscopy is a method of studying micro–objects using an optical microscope, in which the fluorescence of an object is initiated by the simultaneous absorption of two photons, the energy of each of which is insufficient to excite fluorescence. The high localization of excitation and the use of low-energy photons makes it possible to study samples at a large focal depth (up to 1 mm) with high contrast and protects them from destruction under the influence of light.
Two-photon microscopy of the mouse brain. On the left are neurons stained with a calcium–sensitive green dye, on the right are neurons of a transgenic mouse producing a green fluorescent protein. The vessels are painted "Texas red".
Miniature devices were needed that could be fixed on the head of freely moving rodents. But the first 2P(e) two-photon miniscopes had many disadvantages, such as heavy weight and inflexible optical cables, and because of slow scanning, the image was distorted. The breakthrough came with the appearance of small and light single-photon (1P) miniscopes. However, it was impossible to obtain a three-dimensional image with their help.
Recently, scientists from the Institute of Systemic Neurobiology named after Kavli and the Center for Neural Computing of the Norwegian University of Science and Technology (Trondheim, Norway) we tested a sample of a miniature 2P device of a new generation, called Mini2P, which is devoid of many disadvantages of previous versions.
Article by Moser et al. Large-scale two-photon calcium imaging in freely moving mice is published in the journal Cell.
An image of the blood vessels of the mouse brain obtained using two-photon fluorescence microscopy. Different colors reflect the depth from the surface of the cerebral cortex up to 1 mm.
Mini2P can simultaneously record the work of thousands of cells and provides stable visualization of the mouse brain. It weighs less than 3 g, so it does not prevent the rodent from running, jumping, climbing, i.e. leading a normal mouse life. Mounted on the animal's head, thanks to the hole made in the skull, Mini2P shows "live" the work of the brain of a free active animal.
Using Mini2P, scientists have already visualized the work of populations of neurons in the visual cortex, the hippocampus, which plays an important role in memory, including spatial, as well as the medial entorhinal cortex – the link between the hippocampus and the cerebral cortex.
Two-photon microscopy of the brain of a mouse model of Alzheimer's disease. Pathological amyloid protein is colored blue, transgenic mouse neurons expressing green fluorescent protein are green, blood vessels are red.
With such a mini-microscope at their disposal, scientists can advance in understanding the nature of brain diseases. Thus, Alzheimer's disease often begins with damage to the entorhinal cortex, the most important memory center of the brain, which is accompanied by problems with memorization and with the ability to orient. Using mice serving as an animal model of Alzheimer's disease, Mini2P can be used to track changes in the behavior of neurons and understand which of them are most vulnerable to the pathological process.
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