Two-Photon Microscopy for Chemical Neuroscience

Abstract

Microscopes using non-linear excitation of chromophores with pulsed near-IR light can generate highly localized foci of molecules in the electronic singlet state that are concentrated in volumes of less than one femtoliter. The three-dimensional confinement of excitation arises from the simultaneous absorption of two IR photons of approximately half the energy required for linear excitation. Two-photon microscopy is especially useful for two types of interrogation of neural processes. First, uncaging of signaling molecules such as glutamate, as stimulation is so refined it can be used to mimic normal unitary synaptic levels. In addition, uncaging allows complete control of the timing and position of stimulation, so the two-photon light beam provides the chemical neuroscientist with an “optical conductor's baton” which can command synaptic activity at will. A second powerful feature of two-photon microscopy is that when used for fluorescence imaging it enables the visualization of cellular structure and function in living animals at depths far beyond that possible with normal confocal microscopes. In this review I provide a survey of the many important applications of two-photon microscopy in these two fields of neuroscience, and suggest some areas for future technical development.

Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics

“…In Table 1, we compare the brightness of L-MAG0 and L-MAG0 460 with two popular types of caged glutamate (14). MNI-glutamate is still the most widely used compound in 2P applications, despite low brightness, due to its high specificity at high concentrations (2).…”

“…Brightness, defined for fluorophores as the product of absorption cross-section and fluorescence quantum yield, gives the experimenter an objective metric to assess fluorescent reporters and identify appropriate optical parameters, such as the optimal excitation wavelength and range of light intensities (18). By comparison, relatively little information is available on the 2P properties of optogenetic and photopharmacological tools (12,14). Wider adoption of 2P optical manipulations should follow from quantitative characterization of 2P brightness for optogenetic and photopharmacological reagents.…”

“…In particular, the inherent spatial confinement of 2P absorption is critical for optical manipulation of individual cells (10) in the intact mammalian brain, where it is difficult to control the spatial extent of gene expression or confine soluble reagents. However, 2P-excited optogenetics is not yet as widespread in adoption as 2PM, being widely perceived to require sophisticated optical techniques (11)(12)(13) or reagent concentrations that compromise pharmacological specificity (2,14).…”

Cloaked Caged Compounds: Chemical Probes for Two‐Photon Optoneurobiology

“…While initial reports of two-photon uncaging used probes [4,5] originally designed for linear actuation, more recently caged compounds designed for 2P excitation have been introduced for Glu, GABA, IP 3 , calcium, etc [6] . Some of these have even proved useful for independent 2P physiological studies [2] . It is striking that caged Glu probes, in particular, have proved very useful but caged GABA probes much less so.…”

“…However, the photon is more precise and versatile than the electron, so the former is not merely starting to "replace" the latter, but light, in combination with chemistry, is being used to open completely new avenues of physiological discovery [1] . To deliver their potential, the new chemical technologies require novel light sources and so pulsed lasers have been vital for time-resolved chemical biology [2] . Enabled by ultra-fast, pseudocontinuous Ti:sapphire lasers, neurobiology now routinely uses two-photon (2P) microscopy for live cell imaging in complex tissue such as acutely isolated brain slices and living animals.…”