Fluorescent Probes (Neuroscience)

Neuronal tracers are generally used for imaging neurons and neuronal circuits. Radioactive amino acids and peroxidase (HRP) were used in the past, but now fluorescent dyes are used as tracers. Neuronal tracers are classified into anterograde and retrograde types. As for anterograde tracers, neurons are labeled by injecting dyes into the neuronal cell bodies and letting the dyes travel through the anterograde axoplasmic flow from the cell body to the nerve terminal, while for retrograde tracers, neurons are labeled by injecting dyes into the nerve terminals and letting the dyes travel through the retrograde axoplasmic flow from the nerve terminal to the cell body.

Carbocyanine dyes are widely used as neuronal tracers, because of their stable and strong fluorescence in lipids such as cell membranes. Although they can be used as both anterograde and retrograde tracers in juvenile animals, anterograde labeling is known to be difficult in adults due to advanced myelination of the nervous system. Fujifilm Wako's DiIC18(3) is a type of carbocyanine dye with an excitation wavelength of 548 nm and a fluorescence wavelength of 565 nm. In addition, Fluoro-Gold (retrograde tracer) and labeled dextran (anterograde or retrograde tracer) are also available as neuronal tracers.

Lucifer yellow, a hydrazine derivative, is also a fluorescent dye used for imaging neurons. When strong blue excitation light is applied to neurons that have been injected with lucifer yellow in the presence of diaminobenzidine (DAB), DAB turns black through a photooxidation reaction. This method, called photo conversion, is known to visualize neurons and enables electron microscopic observation.

At synapses, exocytosis and endocytosis are frequently repeated for neurotransmitters release and vesicles recycling. Dyes that can label endocytic vesicles are therefore used for imaging synapses. Fluorescent cationic styryl dyes have been used for imaging synapses as fluorescent dyes that can label endocytic vesicles. They have a highly hydrophilic cationic charge head group at one end and a lipophilic tail at the other end. Although they are nonfluorescent in aqueous solutions, they exhibit strong fluorescence when bound to lipids such as cell membranes. As the dyes are released from vesicles along with neurotransmitters and cause a decrease in the fluorescent signal, the change in fluorescence intensity is thought to reflect the amount of endocytosis/exocytosis or synaptic activity.

A representative fluorescent cationic styryl dye is FM^®* dye, developed by Fei Mao et al. ViVidFluor from Fujifilm Wako, is equivalent to FM^® dye.

* FM^® is a registered trademark of Life Technologies.