The combination of optogenetics and two-photon microscopy (2PM), in which pulsed IR lasers are used to image and manipulate neuronal activity, has started to unveil the links between neuronal circuits and behaviour in mice. However, standard 2PM is performed in head-fixed mice that cannot perform free behaviour. To overcome this, few groups have started the ‘miniaturised microscope revolution’ and developed portable 2P microscopes to image neuronal activity in freely moving mice. Our group has been in the frontline of this revolution, with the demonstration of the first 2P flexible fiberscope for optogenetic photostimulation (2P-FENDO). In this project, by combining ultrafast lasers, fiber optics and micro-optics engineering we will develop an improved version of 2P-FENDO, with a larger field of view and higher signal to noise ratio. In collaboration with an established international group in neuroscience, we will use the fiberscope to study the motor system in freely moving mice.

Optical microscopy, and especially two-photon microscopy (2PM), is rapidly becoming irreplaceable for the study of neurons and neural circuits, thanks to two important advances. First, genetically encoded calcium indicators, fluorescent proteins that report on changes in calcium ions concentration, enable to image neurons firing. Second, optogenetics provide the key to switch on or off neuronal activity with light. The combination of 2P imaging and 2P optogenetic photostimulation, often referred to as an all-optical approach, has shown that it is indeed possible to relate neuronal activity to perception and behaviour in mice [1–3]. However, until now, all-optical studies have been performed in mice that are head-fixed under the microscope and cannot perform free behaviour.

To extend these studies to freely moving mice, different groups have developed light-weighted miniaturized 2P (or even 3P) microscopes. They are typically based on individual optical fibers and compact scanning units attached to the animal head, and are capable of high resolution 2P activity imaging in freely moving mice [4,5]. Despite their impressive imaging performances, because of the single fiber delivery approach, these systems are not capable of precise 2P optogenetic photostimulation of single or multiple neurons. Very recently, we have solved this limitation by replacing a single fiber with a fiber bundle (composed of ~ 10 000 individual cores) and by using wavefront shaping approaches to direct the photostimulation laser to the targeted neurons. With this, we have developed 2P-FENDO, the first 2P fiberscope capable of all-optical studies in freely moving mice [6]. Starting from this first design, in this project the candidate will develop the new generation 2P-FENDO, with the objective of greatly enlarging the accessible field of view (FOV) and improving the optical signal to noise ratio (SNR).

The candidate will work on three main axes:
1) The design, with an optical simulation software (Zemax), and assemble of a new micro-optical system to relay the distal end of the fiber bundle to the specimen. The relay optics will enlarge the FOV, allow 3D operation with a tuneable element and minimize aberrations;
2) The development of new pulse compression and diagnostic strategies to transmit the shortest and highest energy pulses through the fiber and maximize the SNR for 2P fluorescence.
3) The development of new spatial beam shaping approaches to maximize the transmission of the laser light through the fiber bundle and the excitation efficiency of the desired neurons.

The candidate will be trained in the theory and experimental implementation of advanced and nonlinear optical techniques (wavefront shaping, 2P microscopy, pulse shaping). In collaboration with neurophysiologists of the wavefront engineering group, she/he will test the improved 2P-FENDO in proof of principle experiments in mice. After this first validation, the candidate will participate in an international collaboration with the group of Ian Oldenburg (Rutgers University), to study the mouse motor system. During a short stay in the Oldenburg lab, the candidate will duplicate 2P-FENDO and participate in preliminary experiments. The final aim of the collaboration is to understand how neurons, in the mouse motor cortex, choose a specific action when multiple alternatives are possible. 2P-FENDO, with its unique capabilities to image and manipulate neuronal activity in freely moving mice, will help to shed light onto these processes.


1. Carrillo-Reid et al. (2019), Cell, 178, 447, DOI: 10.1016/j.cell.2019.05.045
2. Jennings et al. (2019), Nat. 2019, 1, DOI: 10.1038/s41586-018-0866-8
3. Buetfering et al. (2022), Nat. Neurosci., 25, 1225, DOI: 10.1038/s41593-022-01151-0
4. Zong et al. (2022), Cell, 185, 1240, DOI: 10.1016/j.cell.2022.02.017
5. Klioutchnikov et al. (2022), Nat. Methods, , DOI: 10.1038/s41592-022-01688-9
6. Accanto et al. (2023), Neuron, 1, DOI: doi.org/10.1016/j.neuron.20

The aim of this project will be to conceive and realize three-photon excitation (3PE) strategies for all-optical investigation of neuronal circuits in-depth. 3PE uses longer wavelengths that undergo less scattering comparing to two-photon excitation strategies, whilst the cubic power dependence of the excited fluorescence signal to the excitation light intensity further suppresses out-of-focus excitation, enhancing contrast and penetration depth. 3PE microscopy is increasingly used in neurosciences for imaging deep brain layers (down to ~1 mm). Here, we will use it combined to optogenetics for in-depth control of neuronal activity.
The candidate will design, develop and validate a 3PE microscope based on spatiotemporal light-targeting methods developed by our group. With this system, we aim to demonstrate for the first time 3P manipulation of neuronal circuits via optogenetics with the long term prospective of using the system for circuit investigation in primates.

A complete exploration of the brain to reveal the concepts involved in its computation requires the development of joint strategies incorporating physics, chemistry, neurobiology, molecular and computer engineering, aiming to monitor and probe neuronal activity in vivo during behavior [1].

About 20 years ago, neuroscientists conceived a revolutionary approach to reach this goal by proposing optogenetics [2], a genetic strategy to make neurons sensitive to light. Thanks to optogenetics combined with functional imaging of genetically encoded activity reporters, the neuronal function can be all-optically controlled by delivering light into the brain. This new research framework inevitably called for new optical methods to control light distribution across a network of neurons entwined in a scattering medium, as the intact brain. The lack of axial confinement and limited penetration depth of visible light, established two-photon excitation (2PE) as the gold standard for in vivo functional imaging [3]. Our group has pioneered over the last 15 years illumination methods for efficient, scanless 2P light-targeted photostimulation [4] as well, like computer-generated holography in combination with temporal focusing [5], that optimize the excitation volume at high temporal resolution, overcoming the intrinsic limitations of optogenetic tools. Novel strategies for functional volume imaging and holographic-based light shaping [5] enabled the extension of neuronal 2PE investigation to a large population of neurons across a 3D volume and all-optical neural circuit interrogation at near single-cell resolution in living mice [6]. Yet, light scattering limits 2PE investigations to superficial cortical areas few hundred of micrometers deep in the brain.

The aim of this PhD project will be to study and implement strategies for in-depth all-optical brain interrogation by integrating current approaches with three-photon excitation (3PE) [8]. 3PE is advantageous over 2PE, showing reduced scattering due to longer excitation wavelengths used, and higher excitation localization, since fluorescence signal axially falls off as ~1/z^4 (with z being the distance from the focal plane), compared to 1/z^2 in 2PE. Out-of-focus excitation is suppressed, contributing to higher signal-to-noise ratio.
The candidate will be trained on advanced non-linear microscopy techniques to design, develop and validate a holographic 3PE microscope based on ad hoc laser sources for 3P excitation featuring low-duty-cycle, high-energy pulses. She/he will work in close collaboration with biologists in the group, for the proof-of-principle validation of the microscope, in both in vitro and in vivo experiments on mouse cortex.

The candidate will also have the chance to validate the system on a vision restoration project aiming to extend for the first-time optogenetic manipulation to deep cortical layers of the primate visual cortex, in collaboration with G. Gauvain in the group of O. Marre at the Vision Institute. In primates, the primary visual cortex receives sensory inputs from relay cells of the thalamus in its layer IV, at 800-1000 µm from the brain surface. The goal of that project is to optically manipulate axon terminals in the layer IV of the primary visual cortex, to mimic and replace activity generated by natural visual stimuli.

References
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