Mary Ann Go

Simultaneous multi-site two-photon photostimulation in three dimensions.

We demonstrate simultaneous multi-site two-photon photolysis of caged neurotransmitters with close to diffraction-limited resolution in all three dimensions (3D). We use holographic projection of multiple focal spots, which allows full control over the 3D positions of uncaging sites with a high degree of localized excitation. Our system incorporates a two-photon imaging setup to visualize the 3D morphology of the neurons in order to accurately determine the photostimulation sites. We show its application to studies of synaptic integration by performing simultaneous and controlled glutamate delivery at multiple locations on dendritic trees.

Four-dimensional multi-site photolysis of caged neurotransmitters

Neurons receive thousands of synaptic inputs that are distributed in space and time. The systematic study of how neurons process these inputs requires a technique to stimulate multiple yet highly targeted points of interest along the neurons dendritic tree. Three-dimensional multi-focal patterns produced via holographic projection combined with two-photon photolysis of caged compounds can provide for highly localized release of neurotransmitters within each diffraction-limited focus, and in this way emulate simultaneous synaptic inputs to the neuron. However, this technique so far cannot achieve time-dependent stimulation patterns due to fundamental limitations of the hologram-encoding device and other factors that affect the consistency of controlled synaptic stimulation. Here, we report an advanced technique that enables the design and application of arbitrary spatio-temporal photostimulation patterns that resemble physiological synaptic inputs. By combining holographic projection with a programmable high-speed light-switching array, we have overcome temporal limitations with holographic projection, allowing us to mimic distributed activation of synaptic inputs leading to action potential generation. Our experiments uniquely demonstrate multi-site two-photon glutamate uncaging in three dimensions with submillisecond temporal resolution. Implementing this approach opens up new prospects for studying neuronal synaptic integration in four dimensions.

Simultaneous transfer of linear and orbital angular momentum to multiple low-index particles

We demonstrate simultaneous transfer of linear and orbital angular momentum (OAM) to hollow glass microbeads using a dynamic array of optical vortices. Previous reports have shown that the transfer of OAM is due to light scattering which creates a tangential force on a particle and causes it to move on a circular orbit around a vortex. In this paper we describe a case with reduced frictional force, as the low-index particle is pinned to the wall of the sample cell. This results in a more efficient transfer of OAM, which sets a hollow microbead into orbital motion around the optical vortex. We show that the localized OAM carried by each vortex in the array can be independently transferred to one microbead trapped per vortex. Finally, we present novel demonstrations showing simultaneous transfer of both orbital angular and linear momentum to multiple microbeads.

Optimal complex field holographic projection.

We describe a technique that uses complex field holograms to project three-dimensional light patterns. Holographic projection commonly uses phase-only encoding since accurately representing complex holograms using both amplitude and phase spatial light modulators reduces the optical throughput significantly. Here, we use a lossless projection via the generalized phase contrast method to produce the necessary amplitude pattern required for complex field holographic projection. We numerically evaluate the technique and demonstrate high optical throughput with reduced undesired high diffraction orders.

Targeted pruning of a neuron’s dendritic tree via femtosecond laser dendrotomy

Neurons are classified according to action potential firing in response to current injection. While such firing patterns are shaped by the composition and distribution of ion channels, modelling studies suggest that the geometry of dendritic branches also influences temporal firing patterns. Verifying this link is crucial to understanding how neurons transform their inputs to output but has so far been technically challenging. Here, we investigate branching-dependent firing by pruning the dendritic tree of pyramidal neurons. We use a focused ultrafast laser to achieve highly localized and minimally invasive cutting of dendrites, thus keeping the rest of the dendritic tree intact and the neuron functional. We verify successful dendrotomy via two-photon uncaging of neurotransmitters before and after dendrotomy at sites around the cut region and via biocytin staining. Our results show that significantly altering the dendritic arborisation, such as by severing the apical trunk, enhances excitability in layer V cortical pyramidal neurons as predicted by simulations. This method may be applied to the analysis of specific relationships between dendritic structure and neuronal function. The capacity to dynamically manipulate dendritic topology or isolate inputs from various dendritic domains can provide a fresh perspective on the roles they play in shaping neuronal output.

3-D Light Patterns for Spine-Targeted Probing of Neuronal Function

Information processing in brain circuits is achieved via synaptic transmission of neurotransmitters between neurons. Synaptic inputs to a neuron are distributed along its entire dendritic tree, which extends spatially in 3-D.

Patterned illumination for analysing neuronal function in 3D

We use patterned 3D multi-spot illumination to perform neuronal multi-site stimulation in rat brain tissue. Using a spatial light modulator, we holograpically project 3D light fields for multi-site two-photon photolysis of caged neurotransmitters to generate synaptic inputs to a neuron. Controlled photostimulation of multiple synapses from various locations in the dendritic tree provides a way to analyze how neurons integrate multiple inputs. Our holographic projection setup is incorporated into a two-photon 3D imaging microscope for visualization and for accurate positioning of specific uncaging sites along the neurons dendritic tree. We show two-photon images and the neurons response to holographic photostimulation of synapses along dendrites.

MODELING NEURONAL RESPONSE TO SIMULTANEOUS AND SEQUENTIAL MULTI-SITE SYNAPTIC STIMULATION

The flow of information in the brain theorizes that each neuron in a network receives synaptic inputs and sends off its processed signals to neighboring neurons. Here, we model these synaptic inputs to understand how each neuron processes these inputs and transmits neurotransmitters to neighboring neurons. We use the NEURON simulation package to stimulate a neuron at multiple synaptic locations along its dendritic tree. Accumulation of multiple synaptic inputs causes changes in the neurons membrane potential leading to firing of an action potential. Our simulations show that simultaneous synaptic stimulation approaches firing of an action potential at lesser inputs compared to sequential stimulation at multiple sites distributed along several dendritic branches.

From entanglement to neuroscience: new uses for laser beams shaped by spatial light modulators

The state of the art in spatial beam modulation is changing the way we can use laser beams. This talk will present several impressive new results in quite diverse areas of laser applications, all using the latest advances controlled spatial phase modulation.

Three-dimensional multi-site two-photon excitation for probing neuronal signal integration

Two-photon holographic microscopy (2PHM) offers the advantage of simultaneous multi-site excitation in three dimensions. This is useful for studies of neuronal signal integration, which require multiple controlled synaptic inputs delivered simultaneously onto dendritic trees. In this work, we holographically split a single femtosecond pulse-laser in order to project multiple foci onto the neuron. At each focus, two-photon photolysis of caged neurotransmitter molecules, which bind to receptors and mimic synaptic transmission, is performed, thereby allowing measurement of how neurons integrate multiple synaptic inputs.