The Frontiers in Neurophotonics Symposium is a biennial event that brings together neurobiologists and physicists/engineers who share interest in the development of leading-edge photonics-based approaches to understand and manipulate the nervous system, from its individual molecular components to complex networks in the intact brain. In this Community paper, we highlight several topics that have been featured at the symposium that took place in October 2022 in Québec City, Canada.
SignificanceTypical light sheet microscopes suffer from artifacts related to the geometry of the light sheet. One main inconvenience is the non-uniform thickness of the light sheet obtained with a Gaussian laser beam.AimWe developed a two-photon light sheet microscope that takes advantage of a thin and long Bessel-Gauss beam illumination to increase the sheet extent without compromising the resolution.ApproachWe use an axicon lens placed directly at the output of an amplified femtosecond laser to produce a long Bessel-Gauss beam on the sample. We studied the dopaminergic system and its projections in a whole cleared mouse brain.ResultsOur light sheet microscope allows an isotropic resolution of 2.4 μm in all three axes of the scanned volume while keeping a millimetric-sized field of view, and a fast acquisition rate of up to 34 mm2 / s. With slight modifications to the optical setup, the sheet extent can be increased to 6 mm.ConclusionThe proposed system’s sheet extent and resolution surpass currently available systems, enabling the fast imaging of large specimens.
Damon DePaoli, Alicja Gasecka, Mohamed Bahdine, Jean Deschenes, Laurent Goetz, Jimena Perez-Sanchez, Robert Bonin, Yves De Koninck, Martin Parent, Daniel Côté
Optogenetics has become an integral tool for studying and dissecting the neural circuitries of the brain using optical control. Recently, it has also begun to be used in the investigation of the spinal cord and peripheral nervous system. However, information on these regions’ optical properties is sparse. Moreover, there is a lack of data on the dependence of light propagation with respect to neural tissue organization and orientation. This information is important for effective simulations and optogenetic planning, particularly in the spinal cord where the myelinated axons are highly organized. To this end, we report experimental measurements for the scattering coefficient, validated with three different methods in both the longitudinal and radial directions of multiple mammalian spinal cords. In our analysis, we find that there is indeed a directional dependence of photon propagation when interacting with organized myelinated axons. Specifically, light propagating perpendicular to myelinated axons in the white matter of the spinal cord produced a measured reduced scattering coefficient (μs′) of 3.52 ± 0.1 mm − 1, and light that was propagated along the myelinated axons in the white matter produced a measured μs′ of 1.57 ± 0.03 mm − 1, across the various species considered. This 50% decrease in scattering power along the myelinated axons is observed with three different measurement strategies (integrating spheres, observed transmittance, and punch-through method). Furthermore, this directional dependence in scattering power and overall light attenuation did not occur in the gray matter regions where the myelin organization is nearly random. The acquired information will be integral in preparing future light-transport simulations and in overall optogenetic planning in both the spinal cord and the brain.
KEYWORDS: Spinal cord, Optical fibers, Optogenetics, Tissue optics, Light scattering, Signal attenuation, Monte Carlo methods, 3D modeling, Neuroscience, Brain
Optogenetics is currently one of the most popular technique in neuroscience. It enables cell-selective and temporally-precise control of neuronal activity. Good spatial control of the stimulated area and minimized tissue damage requires a specific knowledge about light scattering properties. Light propagation in cell cultures and brain tissue is relatively well documented and allows for a precise and reliable delivery of light to the neurons. In spinal cord, light must pass through highly organized white matter before reaching cell bodies present in grey matter, this heterogenous structure makes it difficult to predict the propagation pattern. In this work we investigate the light distribution properties through mouse and monkey spinal cord. The light propagation depends on a fibers orientation, leading to less deep penetration profile in the direction perpendicular to the fibers and lower attenuation in the direction parallel to the fibers. Additionally, the use of different illumination wavelengths results in variations of the attenuation coefficient. Next, we use Monte-Carlo simulation to study light transport. The model gives a full 3-D simulation of light distribution in spinal cord and takes into account different scattering properties related to the fibers orientation. These studies are important to estimate the minimum optical irradiance required at the fiber tip to effectively excite the optogenetic proteins in a desired region of spinal cord.
Optical tissue properties limit visible light depth penetration in tissue. Because of this, the recent development of optogenetic tools was quickly followed by the development of light delivery devices for in vivo optogenetics applications. We summarize the efforts made in the last decade to design neural probes that combine conventional electrophysiological recordings and optical channel(s) for optogenetic activation, often referred to as optodes or optrodes. Several aspects including challenges for light delivery in living brain tissue, the combination of light delivery with electrophysiological recordings, probe designs, multimodality, wireless implantable system, and practical considerations guiding the choice of configuration depending on the questions one seeks to address are presented.
KEYWORDS: Axons, Spinal cord, Objectives, CARS tomography, Optical filters, In vivo imaging, Bandpass filters, Luminescence, Microscopes, Signal to noise ratio
In vivo imaging of cellular dynamics can be dramatically enabling to understand the pathophysiology of nervous system diseases. To fully exploit the power of this approach, the main challenges have been to minimize invasiveness and maximize the number of concurrent optical signals that can be combined to probe the interplay between multiple cellular processes. Label-free coherent anti-Stokes Raman scattering (CARS) microscopy, for example, can be used to follow demyelination in neurodegenerative diseases or after trauma, but myelin imaging alone is not sufficient to understand the complex sequence of events that leads to the appearance of lesions in the white matter. A commercially available microendoscope is used here to achieve minimally invasive, video-rate multimodal nonlinear imaging of cellular processes in live mouse spinal cord. The system allows for simultaneous CARS imaging of myelin sheaths and two-photon excitation fluorescence microendoscopy of microglial cells and axons. Morphometric data extraction at high spatial resolution is also described, with a technique for reducing motion-related imaging artifacts. Despite its small diameter, the microendoscope enables high speed multimodal imaging over wide areas of tissue, yet at resolution sufficient to quantify subtle differences in myelin thickness and microglial motility.
Using the vectorial diffraction theory established by Richards and Wolf, we demonstrate that the resolution of a two-photon microscope can be improved with a radially polarized TM01 laser beam and an interface between dielectrics, instead of the linearly polarized Gaussian beam already used in laser scanning microscopy. To verify the theoretical results, we developed a mode converter producing radially polarized beams and we have integrated it in a commercial two-photon microscope.
We present two optical systems that can transform a Gaussian beam into the lowest-order transverse magnetic laser
beam (the so-called TM01 laser beam). When a TM01 laser beam is focused by an objective with a high numerical aperture (NA > 0.9), theory predicts the appearance of a bright central feature at focus; the width of this feature is
smaller than that of the focal spot obtained with a Gaussian beam [1,2]. Our goal is to improve the lateral resolution of
two-photon microscopy when a TM01 laser beam is used.
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