Quantitative Imaging of Serotonin Autofluorescence with Multiphoton Microscopy

Excerpt

In this article we describe a technique to directly image vesicular serotonin in live neurons. In the context of serotonergic signaling in the brain, it is important to map out serotonergic storage and release sites, together with the distribution of serotonin receptors. Receptor mapping is possible by using endogenously labeled receptors, but serotonin vesicle mapping is more difficult. Following the early success in mapping serotonin in mast cells with three-photon excitation microscopy [1], we have recently demonstrated that such mapping is possible also in live neurons [2]. We describe here the technique of serotonin imaging in detail, together with a few application examples. This three-photon excitation based imaging technique should also be useful in general for live cell microscopy of UV chromophores.

What we present here is intended to be a practical guide and not a review of three-photon microscopy. For the technical details described here, we have strictly relied on our own hands-on experience. The description is divided into three main sections. “Multiphoton Microscopy” presents a brief summary of the concepts of multiphoton excitation. This is followed by a description of the optical technique of serotonin imaging in “Optical Setup.” Finally, “Imaging Serotonin and Other Monoamines in Live Neurons” describes a few examples of serotonin imaging in neuronal cells and also describes how this technology can possibly be extended to image other monoamines.