Fluorescent probes have a wide range of uses in cellular and molecular biology. In my research, a fluorescent dye was injected into neocortical neurons in mice so that their morphology could be visualised.
Confocal microscopy is an imaging technique that uses pinhole apertures to remove out-of-focus light. This enables high resolution images to be taken from a specific focal plain within a sample. By capturing multiple images at different focal plains (moving along the Z-axis) a 3D image can be reconstructed and modelled.
Thick-Tufted Layer 5 Pyramidal Neurons
The neocortex is the most recently evolved part of the brain. It is involved in higher functions such as language, conscious thought, motor planning and spatial reasoning.
Pyramidal neurons in the neocortex are arranged into six layers; each layer having a distinct morphology and electrical properties. These layers are connected to each other in a microcircuit that recieves an input (typically in layer 4), processes that signal through the other layers and then sends it out to other areas of the brain.
Thick-tufted neurons in layer 5 (left & below) are the largest of the neocortex. They are important as they form the main output to subcortical areas such as the thalamus.
Detecting alpha-Synuclein in the brain
The potein alpha-Synuclein (aSyn) is known to aggregate during Parkinson's disease. Using fluorescent antibodies, it is possible to observe physiological levels of aSyn throughout the brain and also detect it inside neurons of interest.
Two neurons were filled with fluorescent dye (green). The top neuron was simultaneously filled with aSyn protein (red). The 3D confocal images were modelled using Imaris software.
Endogenous expression of αSyn throughout the mouse brain. A parasagittal section of mouse brain immunostained with anti-αSyn antibodies (red). Scale bar = 1mm.
Endogenous expression of αSyn in the mouse somatosensory neocortex. The layers of pyramidal neurons are labelled on each side. Layers 4 and 6 in particular show some expression of native αSyn within cell bodies while layers 1, 2/3 and 5 appear void of αSyn. Scale bar = 200 μm.