![]() ![]() DETAILS ON THE SAMPLE NEURON IMAGES USED IN THIS TUTORIAL With a 2D view, this could just seem like a shorter branch, but looking at the 3D view, if the branch ends at the edge of the z plane, it’s possible that the branch is actually longer but was just included in a different section of tissue. For example, sometimes branches that are part of the neuron are cut off the slice. When tracing neurons imaged using a confocal, it helps to keep the z plane and the overall 3D shape in mind as it could interfere with some measurements like overall dendritic length. ![]() With this, the series of 2D photos can be combined, allowing for reconstruction and 3D visualization and analyses of the specimen in question. If the 2-dimensional slices are made of the x and y planes, the thickness that is available as a third-dimension is the z plane. A series of clear 2D images can then be taken across the z plane. ![]() Using fluorescent imaging technique (*) and an extra spatial filter, this confocal can selectively collect light at a specific z-plane, filtering out any out-of-focus illumination. The image files used in this tutorial were taken using a Leica confocal microscope (model SP8). Howard’s lab hopes that better understanding of branching at the cellular level will also shed light on these processes at the molecular and tissue levels.This post will show you how you can get one of these (dendrite tracing) using FIJI: a how-to FIJI on neuron tracing CONFOCAL IMAGING There is a whole world of branching in biology, from the veins and arteries of the circulatory system to the bronchioles of the lung. “This means that now we can focus on the tips, because if we can understand how they work, then we can understand how the whole shape of the cell comes about,” says Howard. “We found that we can completely explain neuronal growth and the overall morphology in terms of just what the tips of the cells are doing,” says Sabyasachi Sutradhar, PhD, associate research scientist and joint lead author of the study. “And we found that no, they’re not inflating like a balloon, but rather growing and branching their tips.” “Before our study, there was a theory that neurons may be dilating and deflating like a balloon,” says Sonal Shree, PhD, associate research scientist and lead author of the study. Analysis of dendritic tips revealed their dynamic and stochastic (randomly determined) growth, which fluctuated among growing, shrinking, and paused states. But in as little as five days, they blossomed into big, tree-like structures with thousands of branches. In the earliest stages of development, the sensory neurons began with only two or three dendrites. After imaging the neurons at different stages of development, the team was able to create time-lapse movies of the growth. Because neurons reside just under the cuticle, the researchers were able to observe this process in real time in live larvae. To visualize this process, they tagged neurons with fluorescent markers and imaged them on a spinning disk microscope. The team studied neuronal growth in fruit flies as they matured from embryos into larvae. “We’re working on this branching process-how do branches form and grow? That is what’s underlying the whole way the nervous system works.” “Neurons are highly branched cells, and they’re like this because each neuron makes a connection with thousands of other neurons,” says Joe Howard, PhD, Eugene Higgins Professor of Molecular Biophysics and Biochemistry and professor of physics, and senior researcher of the study. Their findings are published in Science Advances. And now, Yale researchers have discovered the molecular mechanism behind the growth of this complex system. When the human brain develops, these structures branch out in a beautifully intricate, yet poorly understood, way that allows nerve cells to form connections and send messages throughout the body. Our nervous system is composed of billions of neurons that speak to each other through their axons and dendrites. ![]()
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