Abstract
Patterned two-photon (2P) photolysis via holographic illumination is a powerful method to investigate neuronal function because of its capability to emulate multiple synaptic inputs in three dimensions (3D) simultaneously. However, like any optical system, holographic projectors have a finite space-bandwidth product that restricts the spatial range of patterned illumination or field-of-view (FOV) for a desired resolution. Such trade-off between holographic FOV and resolution restricts the coverage within a limited domain of the neuron’s dendritic tree to perform highly resolved patterned 2P photolysis on individual spines. Here, we integrate a holographic projector into a commercial 2P galvanometer-based 2D scanning microscope with an uncaging unit and extend the accessible holographic FOV by using the galvanometer scanning mirrors to reposition the holographic FOV arbitrarily across the imaging FOV. The projector system utilizes the microscope’s built-in imaging functions. Stimulation positions can be selected from within an acquired 3D image stack (the volume-of-interest, VOI) and the holographic projector then generates 3D illumination patterns with multiple uncaging foci. The imaging FOV of our system is 800×800 μm2 within which a holographic VOI of 70×70×70 μm3 can be chosen at arbitrary positions and also moved during experiments without moving the sample. We describe the design and alignment protocol as well as the custom software plugin that controls the 3D positioning of stimulation sites. We demonstrate the neurobiological application of the system by simultaneously uncaging glutamate at multiple spines within dendritic domains and consequently observing summation of postsynaptic potentials at the soma, eventually resulting in action potentials. At the same time, it is possible to perform two-photon Ca2+ imaging in 2D in the dendrite and thus to monitor synaptic Ca2+ entry in selected spines and also local regenerative events such as dendritic action potentials.
Highlights
Two-photon (2P) microscopy is widely used in neuroscience [1]
The point spread function (PSF) of 2P excitation with the holographic module measured by imaging 100 nm fluorescent beads shows a full width at half maximum (FWHM) of 0.49 μm (60×) and 0.64 μm (20×) in the lateral direction and 2.7 μm in the axial direction for both lenses
We found our holographic system to be primarily affected by oblique astigmatism due to the oblique incidence of the laser on the spatial light modulator (SLM)
Summary
Its applications include structural and functional imaging [2,3,4], and neuronal stimulation by photolysis of caged chemical compounds and activation of light-gated ion channels [5, 6]. The nonlinear 2P excitation process allows highly localized photolysis of caged neurotransmitters, thereby enabling targeted activation of single spines and effectively mimicking synaptic inputs onto a neuron. By activating multiple synapses with this technique, we can study how neurons integrate synaptic inputs to generate action potentials. Because the dendritic arbor of a neuron extends beyond a single plane, it is highly desirable to have three-dimensional (3D) access to individual synaptic sites to provide experimental flexibility. 3D access to a sufficiently large volume with high spatial resolution will allow to investigate synaptic integration across dendritic bifurcations for any spatial arrangement of the dendrites of interest
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