Multiple fluorescence 2PE imaging. Wikimedia Commons

Multiphoton fluorescence microscopy (MPM) uses pulsed near-infrared lasers to excite fluorophores with non-linear excitation, providing deeper tissue penetration than single photon fluorescence techniques, with less photobleaching and damage to biological tissues. The technique is based on the simultaneous absorption of two or more long wavelength photons to cause excitation, which occurs only at a point where the light intensity is large enough to allow absorption of more than one photon. This excitation state normally decays to the lowest energy level of the conventional single photon state and from there transitions into the ground state, accompanied by fluorescence.

Fluorescence correlation spectroscopy (FCS) is a computational tool that performs statistical analysis of fluorescence fluctuations in a system to study molecular dynamics and diffusion. Equilibrium fluctuations corresponding to Brownian motion generate subtle variations in fluorescence. The strength and duration of fluctuations are autocorrelated to provide information about the time scale by which these fluctuations occur. FCS can provide molecular concentrations, diffusion coefficients, particle radii, and chemical reaction rates, making it an extremely valuable tool for neuroscientists, if it can be utilized along with live synaptic imaging.

FCS Setup, Mllyjn. Wikimedia Commons/a>

In this project, three-photon excitation at 740 nm will be employed to image serotonin autofluorescence using a Bio-rad Radiance 2100 confocal microscope. This system uses a ti:sapphire pulsed coherent laser which can be tuned between 700 and 1000 nm. Experiments will be performed using a 60X/ 1.4 NA water-immersion objective lens, with a 360 nm emission filter. If this filter is not available, a 380 nm filter will be used instead. Samples will be prepared and viewed in a steel chamber slide. First, serotonin will be examined in vitro at concentrations of 100 nM, 500 nM, 1 μM and 100 μM. Stock solutions of serotonin hydrochloride will be prepared in 0.1 M perchloric acid and diluted to their respective concentrations in phosphate buffer (pH 7.4) on the day of the experiment. These concentrations will be pipetted onto the slide and imaged with the Bio-Rad system. Once optimal imaging parameters have been achieved with this method, serotonin autofluorescence will be studied in a rat brain slice. First, the dorsal striatum region of a brain slice from a male Sprague-Dawley rat will be imaged to see if serotonin can be distinguished. Next, a selected concentration of serotonin will be injected into this region, to mimic the effect of neurotransmitter release. The slice will be maintained in oxygenated artificial cerebral spinal fluid (aCSF) at pH 7.4, before being transferred to the imaging equipment. It will then be seen if the autofluorescent serotonin signal can be distinguished from any background fluorescent signals in the brain slice. If this is successful, the method will be applied to look at an endogenous serotonin release event. For this experiment, a brain slice will be prepared as before, but this time will be perfused with a .1 M potassium chloride solution right before transfer to the imaging center, in order to induce membrane depolarization and synaptic release of serotonin. This will test the feasibility of this technique in characterizing a real neurotransmission event.

If these experiments prove successful, they could prove to be of use for real-time, spatially resolved characterization of chemical neurotransmission. With the resolution capable with confocal fluorescent microscopy combined with the decreased phototoxicity of multiphoton excitation, the dynamic processes of chemical release, diffusion and reuptake can be observed within a single synapse. Combined with FCS, actual concentrations and kinetic rates can be calculated for these neurotransmission events, providing essential data for neurological research and understanding.