DS0401 - Etude des systèmes biologiques, de leur dynamique, des interactions et inter-conversions au niveau moléculaire

Three-dimensional Holography for Parallel Neuronal Activation – 3DHoloPAc

Three-dimensional holography for parallel neuronal activation

3DHoloPAc is an attempt to further develop methods that we proposed in the recent past in Wavefront-Engineering Microscopy group at Paris Descartes University, such as computer generated holography (CGH) and generalized phase contrast (GPC), for efficient optical stimulation of neurons. The aim is to provide the proper tool to better follow the 3D complexity of the brain at a high temporal and spatial resolution, by using optical wavefront engineering in two-photon excitation.

Three-dimensional simultaneous multi-cell activation, at single-cell precision and sub-millisecond temporal resolution

The primary goal of this project is to enable parallel three-dimensional (3D) activation of neurons via the use of optogenetic approaches and light-patterning methods used for the generation of extended laser patterns that illuminate neurons’ surface simultaneously. We use Computer-Generated Holography (CGH), as the light-patterning method, a pure phase modulation technique using a liquid-crystal based spatial light modulator (LC-SLM). This project targets the extension of this method in 3Ds, aiming to project patterns in different axial planes in the whole excitation volume achievable through high-Numerical Aperture (NA) objectives, in a microscope using two-photon excitation. The initial specific aims of the project are: i) to develop new tools for 3D holographic stimulation with extended patterns, restricted at the time of the submission to 3D diffraction-limited spots, ii) to modulate the intensity of the excitation patterns, such as to adapt to the expression level of the targeted optogenetic molecule, and to intensity inhomogeneity arising from the scattering length of the tissue and uneven diffraction efficiency of the SLM in the excitation field. iii) To prove 3D multicell optogenetic activation in brain slices and in the retina.

CGH is a method using a liquid crystal based spatial light modulator (LC-SLM) device to rearrange light in multiple spots of different sizes (ranging from diffraction-limited spots up to large excitation areas covering the whole field of excitation), or arbitrary excitation patterns. The ability of 3D arbitrary pattern projection, although very common for projecting 3D diffraction-limited spots, for instance in applications like optical tweezers, has never been rigorously demonstrated or used for extended area patterns, until the submission of this project, an approach that can be extremely beneficial in neurobiology, where networks are developed in three dimensions. Moreover, combination of 3D light patterning in two-photon excitation with temporal focusing of the laser pulses enables maintaining very good axial confinement despite the lateral extent of the illumination patterns.
On the other hand, neuronal activate through optogenetics has initialized a revolution in the field of neuroscience during the last decade toward reversible and selective stimulation of specific neuronal populations in intact brain preparations. Neurons can now be genetically manipulated to express proteins that, upon photon absorption, generate transmembrane electrical currents.
3DHoloPAc uses those cutting-edge techniques in optics, molecular engineering and neuroscience, in an effort to offer to scientists a flexible tool for complex interrogation and manipulation of neural circuits through optical stimulation of neurons.

During the first 18 months, we developed a more accurate algorithm for projecting 3D extended arbitrary CGH patterns in a microscope using high-NA objectives. Remarkably, we extended this method for use with temporal focusing, for which we proved in the past, that, in combination with light-patterning methods, could improve the axial resolution of large-area patterns, thus also improving the spatial resolution of neuronal stimulation. Because of the way temporal focusing is implemented experimentally, the technique is limited in projecting patterns only in 2Ds. Here, we proposed a unique way of overcoming this problem: our optical system enables remote axial displacement of temporally focused holographic patterns, as well as generation of multiple temporally focused holographic targets occupying separate axial planes. In our two-step system, a first LC-SLM is addressed with phase holograms controlling the transverse target light distribution, and a second LC-SLM, positioned after the temporal-focusing grating, is addressed with Fresnel lens-phase functions and controls targets’ axial position. This configuration, called 3D CGH-TF, can jointly translate single or multiple spatio-temporally focused patterns across the sample volume.
We also developed LC-SLM alignment protocols for 3D CGH-TF that consider diffraction-efficiency, and the position of the phase profile projection on the LC-SLM for each pattern at each plane.
As a first proof-of-principle application of the 3D CGH-TF system we performed simultaneous multiplane two-photon single-cell resolution, photoconversion of Kaede-expressing neurons and multi-cell optogenetic activation of neurons co-expressing ChR2-H134R-mCherry and GCaMP5G in the spinal cord of zebrafish (see Hernandez et al. Nature Commun. 2016).

The main results so far concern the demonstration of the first optical system reporting 3D depth-resolved patterned illumination with temporal focusing developed here for neuronal excitation. This method can, however, be applied to any other 3D light-patterning application, requiring a superior axial resolution.
For the next half of the project, we will use the system, or variants of it, in two directions: i) for performing 2P multi-cell activation of neurons expressing optogenetic tools, lying in different axial planes in the mouse visual cortex, and ii) performing 2P multi-cell activation of bipolar cells expressing optogenetic tools, while monitoring the responses of the ganglion cells in the retina, the latter in collaboration with people from the Vision Institute (Paris). Both studies require preliminary work for the biological preparation in the molecular level for choosing the right combination of optogenetic actuator and calcium sensor, since multi-cell responses detection will be through calcium imaging. We are currently working toward this direction testing different opsins in terms of expression level, efficiency and kinetic properties, for finding the ideal one to combine with two-photon calcium imaging. We also work on the optimization of injection protocols for efficient co-expression in neurons of the opsin and calcium indicator.
The experiments proposed here conducted successfully, they will give us the thrust to extend the experimentations first in anesthetized animals and then to exploit and further develop the acquired expertise to experiments in freely moving animals. Results will be extremely important and useful in the future for the use of 3D holographic schemes to other collaborative projects of our group.

Publication in peer-reviewed journal:
Hernandez O, Papagiakoumou E, Tanese D, Fidelin K, Wyart C and Emiliani V (2016) Three-dimensional spatiotemporal focusing of holographic patterns Nat. Commun. 7 11928

Invited conference:
E. Papagiakoumou, Multi-plane excitation with depth-resolved holographic patterns, The Brain in Focus: New Approaches to Imaging Neurons and Neural Circuits, Rungsted - North Copenhagen, Denmark, April 17-20, 2016.

Patent submitted:
V. Emiliani, E. Papagiakoumou, D. Tanese, N. Accanto, E. Ronzitti, Optical system for spatiotemporal shaping the wavefront of the electric field of an input light beam to create three-dimensional illumination, Date of submission: April 7, 2017.

Optical means for stimulating and monitoring neuronal activity in intact brain preparations have provided a lot of insight in neurophysiology during the last decade, towards a better understanding on how brain works. Optogenetic actuators, calcium or voltage imaging probes and other molecular tools that generate localized and precisely timed activity in living systems have contributed a lot to the adoption of light as the alternative way to electrodes for exciting or following neuronal responses. Despite the precision in molecular targeting of all these tools, specificity of light illumination is still crucial for successfully studying communication of neurons in three-dimensional (3D) networks. Thus, novelties for optical methods that ameliorate spatiotemporal resolution are expected to expand, in order to better simulate neurons’ physiological processes.
3DHoloPAc is an attempt to further develop methods that we proposed in the recent past in Wavefront-Engineering Microscopy group at Paris Descartes University, such as computer generated holography (CGH) and generalized phase contrast (GPC), for efficient optical stimulation of neurons. The aim is to provide the proper tool to better follow the 3D complexity of the brain at a high temporal and spatial resolution, by using optical wavefront engineering in two-photon excitation. Specifically, the project concerns the adaptation of CGH, for simultaneous pattern projection in different axial planes in the sample volume. CGH is a method using a liquid crystal based spatial light modulator (SLM) device to rearrange light in multiple spots of different sizes (ranging from diffraction-limited spots up to large excitation areas covering the whole field of excitation), or arbitrary excitation patterns. The ability of 3D arbitrary pattern projection, although very common for projecting 3D diffraction-limited spots, for instance in applications like optical tweezers, has never been rigorously demonstrated or used for extended area patterns, an approach that can be extremely beneficial in neurobiology, where networks are developed in three dimensions. Moreover, combination of 3D light patterning with temporal focusing of the excitation pulses will enable maintaining very good axial confinement despite the lateral extent of the illumination patterns.
In addition we will use CGH to modulate the intensity that is attributed to each pattern in order to correct intensity inhomogeneity arising from scattering inside the tissue and reduced diffraction efficiency of the SLM at the borders of the excitation field. The same feature will be explored as a means to homogenize the response of cells expressing optogenetic actuators in different levels. The ultimate goal is to develop a system able to adapt and respond to the needs of any kind of biological preparation. The realization of a two-photon excitation 3D holographic microscope will enable simultaneous 3D multi-cell stimulation that we propose to test in photoactivation of neurons expressing optogenetic molecules in mouse brain cortical slices for proof-of-principle experiments, and then in the mouse retina, an ideal system for testing 3D stimulation approaches due to its layered structure, for understanding how the visual information is transmitted from the bipolar to the ganglion cells. The latter application will be held in the framework of collaboration with the Vision Institute in Paris.
My expertise acquired in the field of light patterning with liquid crystal spatial light modulators and two-photon excitation of neurons by working within the Wavefront-Engineering Microscopy group for eight years assures the feasibility of the project and its success. Positive results will motivate us to extend the application of our methods in the future to ‘in vivo’ experiments.

Project coordination

Eirini PAPAGIAKOUMOU (Laboratoire de Neurophotonique)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CNRS Laboratoire de Neurophotonique

Help of the ANR 305,859 euros
Beginning and duration of the scientific project: September 2015 - 36 Months

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