1) The document discusses preliminary studies using two-photon microscopy to image brain areas of zebra finches through their thin skin and hollow skull structure for non-invasive monitoring of brain activity.
2) Experiments were conducted imaging hollow fibers filled with Rhodamine B passed through fixed zebra finch skin and skull samples to evaluate spatial resolution and distortion. Reflectance confocal measurements were also taken to determine scattering properties of fresh and fixed skin and skull.
3) The goal is to determine if two-photon microscopy can provide sufficient resolution for in vivo brain imaging and metabolism monitoring of zebra finches as a model for studying vocal recognition, without requiring craniotomy as in other small animal studies.
This document describes an experiment that used near-infrared spectroscopy (NIRS) to noninvasively measure the optical properties of a songbird's brain. Researchers placed optical fibers on the head of anesthetized zebra finches to transmit laser light and collect the light after it passed through the brain tissue. They were able to measure the absorption and scattering coefficients of the caudal nidopallium region of the brain in vivo. This technique could help monitor brain activity and oxygenation levels in songbirds.
In this application, Cellvizio was used to study the neuronal degeneration and regeneration processes in live, anaesthetized, adult Thy1-YFP transgenic mice.
This document discusses various techniques for studying the brain, including:
- Diffusion Spectrum Imaging (DSI) allows mapping of axonal trajectories but not individual neurons due to MRI resolution limitations.
- Two-photon microscopy can image live mouse brains up to 1mm depth. Confocal laser scanning microscopy provides high-resolution images of brain structures in vitro.
- Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) provide the highest magnifications up to 1 million times but require thin samples and only work in vitro.
Charting the human thalamus - basic contepts and recent developmentsDr. Jakab András
This document summarizes a study on developing a probabilistic tractography and segmentation method to chart the human thalamus. The study used diffusion tensor imaging and probabilistic tractography to visualize cortico-thalamic connections. It then developed a statistical shape model of the mean thalamus atlas incorporating these connectivity maps. The method was able to align the atlas to individual subjects' geometry with sub-millimeter accuracy, outperforming conventional alignment methods. This individualized target mapping method could help guide image-guided neurosurgery of the thalamus.
This document discusses research on using upconversion nanoparticles (UCNPs) to enable mice to see near-infrared light. UCNPs were created that convert 980nm infrared light to visible green light. The UCNPs were injected subretinally in mice and bound to photoreceptors, acting as nanoantennas. Behavioral experiments showed the UCNP-injected mice could distinguish infrared light from darkness via their pupillary response, demonstrating they gained the ability to "see" infrared wavelengths thanks to the UCNPs. The researchers aim to expand the visible light spectrum that can be detected by the eye.
Understanding of light sensing organs in biology creates opportunities for the development of novel optic systems that cannot be available with existing technologies. The insect's eyes, i.e., compound eyes, are particularly notable for their exceptional interesting optical characteristics, such as wide fields of view and infinite depth-of-field. While the construction of man-made imaging systems with these characteristics is of interest due to potential for applications in micro air vehicles (MVAs) and clinical endoscopes, currently available devices offer only limited capabilities due to their use of compound lens systems in planar geometries. In this presentation, I discuss a complete set of materials, design layouts and integration schemes for digital cameras that mimic fully hemispherical compound eyes. Certain of the concepts extend recent advances in ‘stretchable electronics’ that provide previously unavailable options in design. I also discuss another interesting hierarchical micro- and nanostructures that can be found in eyes of night-active insects such as moth and mosquito. I present research trends on fabrication methods, optical characteristics, and various applications for artificial micro-/nanostructures that resemble ‘moth eye’ structure.
The document presents a method called ThinBlinDe for blind deconvolution of confocal microscopy images. It aims to address limitations of optical sectioning microscopy including blurring from diffraction and aberrations, out-of-focus light when using a pinhole, and shot noise. The method breaks through computational limits to perform blind deconvolution, improving image contrast and resolution. Results are shown applying the method to phantom, microsphere, and plant cell data.
This document discusses vision and visual navigation in nocturnal insects. It begins by explaining how nocturnal insects have modified their visual systems and behavior to operate under low light conditions, which can be up to 11 orders of magnitude lower than daylight. Specifically, it discusses the optical designs of compound eyes that have evolved for high sensitivity, including superposition eyes. It also covers retinal adaptations like large photoreceptor responses, as well as strategies for enhancing vision like temporal and spatial summation in the brain. Finally, it examines how nocturnal insects navigate using celestial cues like the moon and polarized light, as well as terrestrial visual cues like tree canopies and landmarks.
This document describes an experiment that used near-infrared spectroscopy (NIRS) to noninvasively measure the optical properties of a songbird's brain. Researchers placed optical fibers on the head of anesthetized zebra finches to transmit laser light and collect the light after it passed through the brain tissue. They were able to measure the absorption and scattering coefficients of the caudal nidopallium region of the brain in vivo. This technique could help monitor brain activity and oxygenation levels in songbirds.
In this application, Cellvizio was used to study the neuronal degeneration and regeneration processes in live, anaesthetized, adult Thy1-YFP transgenic mice.
This document discusses various techniques for studying the brain, including:
- Diffusion Spectrum Imaging (DSI) allows mapping of axonal trajectories but not individual neurons due to MRI resolution limitations.
- Two-photon microscopy can image live mouse brains up to 1mm depth. Confocal laser scanning microscopy provides high-resolution images of brain structures in vitro.
- Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) provide the highest magnifications up to 1 million times but require thin samples and only work in vitro.
Charting the human thalamus - basic contepts and recent developmentsDr. Jakab András
This document summarizes a study on developing a probabilistic tractography and segmentation method to chart the human thalamus. The study used diffusion tensor imaging and probabilistic tractography to visualize cortico-thalamic connections. It then developed a statistical shape model of the mean thalamus atlas incorporating these connectivity maps. The method was able to align the atlas to individual subjects' geometry with sub-millimeter accuracy, outperforming conventional alignment methods. This individualized target mapping method could help guide image-guided neurosurgery of the thalamus.
This document discusses research on using upconversion nanoparticles (UCNPs) to enable mice to see near-infrared light. UCNPs were created that convert 980nm infrared light to visible green light. The UCNPs were injected subretinally in mice and bound to photoreceptors, acting as nanoantennas. Behavioral experiments showed the UCNP-injected mice could distinguish infrared light from darkness via their pupillary response, demonstrating they gained the ability to "see" infrared wavelengths thanks to the UCNPs. The researchers aim to expand the visible light spectrum that can be detected by the eye.
Understanding of light sensing organs in biology creates opportunities for the development of novel optic systems that cannot be available with existing technologies. The insect's eyes, i.e., compound eyes, are particularly notable for their exceptional interesting optical characteristics, such as wide fields of view and infinite depth-of-field. While the construction of man-made imaging systems with these characteristics is of interest due to potential for applications in micro air vehicles (MVAs) and clinical endoscopes, currently available devices offer only limited capabilities due to their use of compound lens systems in planar geometries. In this presentation, I discuss a complete set of materials, design layouts and integration schemes for digital cameras that mimic fully hemispherical compound eyes. Certain of the concepts extend recent advances in ‘stretchable electronics’ that provide previously unavailable options in design. I also discuss another interesting hierarchical micro- and nanostructures that can be found in eyes of night-active insects such as moth and mosquito. I present research trends on fabrication methods, optical characteristics, and various applications for artificial micro-/nanostructures that resemble ‘moth eye’ structure.
The document presents a method called ThinBlinDe for blind deconvolution of confocal microscopy images. It aims to address limitations of optical sectioning microscopy including blurring from diffraction and aberrations, out-of-focus light when using a pinhole, and shot noise. The method breaks through computational limits to perform blind deconvolution, improving image contrast and resolution. Results are shown applying the method to phantom, microsphere, and plant cell data.
This document discusses vision and visual navigation in nocturnal insects. It begins by explaining how nocturnal insects have modified their visual systems and behavior to operate under low light conditions, which can be up to 11 orders of magnitude lower than daylight. Specifically, it discusses the optical designs of compound eyes that have evolved for high sensitivity, including superposition eyes. It also covers retinal adaptations like large photoreceptor responses, as well as strategies for enhancing vision like temporal and spatial summation in the brain. Finally, it examines how nocturnal insects navigate using celestial cues like the moon and polarized light, as well as terrestrial visual cues like tree canopies and landmarks.
Hippocampal Place Cells in Echolocating Bats stanfordneuro
1) Hippocampal place cells in bats rapidly update their spatial firing based on incoming sensory information from echolocation calls. Place cell spikes occurring later after an echolocation call show reduced spatial selectivity compared to earlier spikes.
2) This demonstrates that place cells can integrate new sensory information on a fast time scale of hundreds of milliseconds to tune their spatial firing based on the acuity of available sensory information.
3) Previous studies in rodents found slower dynamics of place cell remapping in response to changes in sensory information, but bats provide a system to study rapid updating of spatial signals.
This document provides an overview of cell structures and microscopy techniques used to study cells. It discusses the different types of microscopes like light microscopes, electron microscopes, and their uses and limitations. Key differences between prokaryotic and eukaryotic cells are outlined. The document also explains why cells need to be small, noting that diffusion works best over short distances and the nucleus can only handle a limited amount of information. Organelles allow eukaryotic cells to compartmentalize functions within internal membranes.
Positionig system of the brain(noble prize in medicine 2014) amir mahmodzadeh
The document discusses the hippocampus, which is a structure in the medial temporal lobe involved in consolidating short-term to long-term memory and spatial navigation. It contains several regions including the dentate gyrus, Cornu Ammonis fields (CA1-CA3), and subiculum. Place cells and grid cells encode spatial information and are involved in cognitive mapping. Recording techniques can monitor the activity of these cells in rodents as they move through environments. The hippocampus and entorhinal cortex work together to form episodic memories and support functions like memory, learning, emotion processing, and navigation.
Measuring visual acuity and contrast sensitivity by optomotor reflex in rodentsInsideScientific
There is a growing need for behavioral readouts to monitor disease progression and to assess the success of a potential therapy. In vision research, observing the optomotor reflex (OMR) is an important and widely established method for assessing visual acuity and contrast sensitivity in rodents. These tests can be performed with freely moving animals without any need for anaesthesia or restraints. In addition, since OMR is a reflex-based behavior, observing it does not require any training of the animal.
In this webinar, sponsored by Striatech and supported in part by Stoelting, researchers will present objective and bias-free results obtained using a newly developed automated optomotor system. For more information, please visit: https://insidescientific.com/webinar/measuring-visual-acuity-contrast-sensitivity-optomotor-reflex-striatech
Night vision allows vision in low light conditions using biological or technological means. Biologically, many animals can see better than humans at night due to traits like more light-sensitive cells in the retina or a tapetum lucidum layer. Technologically, night vision is achieved through three main categories - image intensification, active illumination using infrared light, and thermal imaging which detects temperature differences. Image intensification magnifies available light while active infrared uses infrared cameras and illuminators.
BAAI Conference 2021: The Thousand Brains Theory - A Roadmap for Creating Mac...Numenta
Jeff Hawkins presented a talk on "The Thousand Brains Theory: A Roadmap to Machine Intelligence" at the Beijing Academy of Artificial Intelligence Conference on 1st June 2021. In this talk, he discussed the key components of The Thousand Brains Theory and Numenta's recent work.
The document discusses neural correlates of higher level brain functions. It covers several topics:
1) Experience arises at the quantum level in ion channel proteins, with quantum properties like coherence and entanglement playing a role.
2) Construction of perception involves transitions from quantum to classical domains in the brain, mediated by ion channel proteins. Top-down processes and long-range connections in large brains are important for conscious perception.
3) Perception emerges from complex interactions between ascending and recurrent signaling in the brain, with feedback thought to be crucial for awareness. Receptive field properties evolve along synaptic distances in hierarchical cortical networks.
This document summarizes research on the visual system from the retina to primary visual cortex (V1). Key points include:
- The retina contains two types of ganglion cells (midget and parasol) that project to different layers in the lateral geniculate nucleus (LGN).
- Hubel and Wiesel discovered that V1 neurons have receptive fields tuned to stimulus orientation, forming the basis of the hierarchical model of visual processing.
- V1 contains two main cell types, simple cells with discrete receptive fields and complex cells without. Retinotopic maps and ocular dominance columns organize V1 architecture.
This document discusses various soft computing techniques for iris recognition, specifically focusing on two neural network approaches: Competitive neural network Learning Vector Quantization (LVQ) and Adaptive Resonance Associative Map (ARAM). It provides an overview of iris recognition as a biometric method, summarizes preprocessing steps like localization, segmentation, and normalization of iris images. It also describes feature extraction and matching steps. Finally, it defines artificial neural networks and discusses how LVQ and ARAM can be used for pattern matching in iris recognition applications.
Can we extract a mind from a plastic-embedded brain? - Kenneth Hayworth - H+ ...Humanity Plus
Ken Hayworth
Can we extract a mind from a plastic-embedded brain?
We now have a good working theory of consciousness – the phenomenal self model (Metzinger 2009), and we have a good understanding of the human cognitive architecture (Anderson 2007) within which this self model is implemented. The key components of this cognitive architecture are declarative memory chunks and productions – thought to be implemented as stable attractors in the neural networks of the cortex and basal ganglia. According to neural network theory, such stable attractors are robustly defined by the synaptic connectivity between neurons. In small pieces of tissue such synaptic connectivity is easily preserved using chemical fixation and embedding in plastic, and it should be relatively easy to adapt these protocols into a surgical procedure performed in hospitals to preserve whole human brains. Such plastic embedded brain tissue can be imaged at the nanometer level using new automated techniques (SBFSEM, FIBSEM, Tape-to-SEM), and we can directly extrapolate these techniques to future ones that will enable all the synaptic connections within a human brain to be mapped allowing a fully accurate simulation of the original preserved mind. In short, we have a complete sketch of how mind uploading will work and we have a mandate to implement emergency brain preservation in hospitals for all who desire access to this future technology.
Kenneth Hayworth, a postdoctoral fellow at Harvard University, is the inventor of several technologies for high-throughput volume imaging of neural circuits at the nanometer scale. He received a PhD in Neuroscience from the University of Southern California for research into how the human visual system encodes spatial relations among objects. Hayworth is a vocal advocate for brain preservation and mind uploading, and runs a website (www.brainpreservation.org) calling for the implementation of an Emergency Glutaraldehyde Perfusion procedure in hospitals, and for the development of a Whole Brain Plastic Embedding procedure which can demonstrate perfect ultrastructure preservation across an entire human brain.
This document contains descriptions of 14 art submissions to the Art of Neuroscience 2017. The submissions include jewelry and artworks inspired by neuroscience concepts, research on gender stereotyping and brain responses, paintings exploring connections between neuroscience and art, images of brain structures and cells created using various microscopy techniques, and more. The submissions are from researchers in multiple countries and cover a wide range of neuroscience topics communicated through different artistic mediums and styles.
Optical clearing is a new technique that improves 3D imaging of the enteric nervous system (ENS). Researchers used optical clearing and confocal microscopy to image the human ileum in 3D. Optical clearing reduced light scattering in the tissue, allowing deeper photon penetration and higher definition images. This generated a panoramic 3D view of gut wall structures like nerves, muscles, and crypts. The researchers plan to use this method to study gastrointestinal disease mechanisms by observing microstructures, vasculature, and nerve networks in animal models and comparing healthy and diseased human tissues.
Night vision works by detecting small amounts of light, such as moonlight or starlight, that are invisible to the human eye. There are two main types of night vision technologies: image intensification and thermal imaging. Image intensification uses an image intensifier tube to amplify available light so that more light enters the eye, allowing the user to see in low light conditions. Thermal imaging detects infrared radiation emitted or reflected from objects to create a thermal image without needing visible light. Within image intensification there are four recognized generations that reflect increasing technological sophistication, with Generation 3 being the most advanced currently available night vision.
Blindness is a serious condition that is feared by many. Researchers are working on developing artificial vision technologies to help restore sight for the blind. One such technology is a bionic eye, which uses a camera and implant to stimulate the retina and optic nerve to generate images in the brain. The retina plays a key role in vision, containing rods, cones and ganglion cells that transmit light signals to the brain. Retinal diseases can lead to blindness by damaging these cells. Researchers are working to bypass damaged areas and provide artificial stimulation to restore some level of sight.
Trauma management during tragic years of lebanon part iiAnis Baraka
The document discusses a SomaSensor device that uses near infrared spectroscopy (NIRS) to non-invasively measure oxygen saturation levels in the brain. It works by emitting light through the skull from an LED light source and detecting the light at different depths using photodetectors on the surface of the head, allowing it to isolate changes in the brain by subtracting the shallow measurement from the deep one. This technique provides depth resolution to minimize contamination from superficial signals.
Technological advancements in science visualization are described. X-ray crystallography allows scientists to see molecules in 3D by converting diffraction patterns from x-rays hitting crystals into visual maps. Microscopy techniques like light and electron microscopes allow magnification of small objects. Telescopes collect light or radio waves to view distant images. DNA fingerprinting identifies individuals by comparing DNA fragments. Computers process large data amounts and automate tools. Medical imaging techniques like X-rays and MRI produce enhanced soft tissue images. Remote sensing uses GPS, radar, sonar and satellites to detect location and environment. Virtual reality and simulations mimic real-world activities. Holograms produce 3D images using lasers.
The document describes several submissions to the Art of Neuroscience 2017 competition. The submissions include:
1. An electron microscope image showing a microglial lysosome and autophagosome, capturing the process of autophagy.
2. A description and calcium imaging data from cortical neurons in a mouse brain, showing patterns of neuronal activity.
3. A sample of hippocampal neurons labeled for microtubules and actin, imaged with a fluorescent microscope.
4. An artwork inspired by Michelangelo's "The Creation of Adam", depicting interacting pyramidal cells in the mouse brain.
5. Diffusion MRI data quantifying whole-brain axonal connections in the rat brain, clearly
Vicarious Systems at Singularity Summit 2011Scott Brown
A makes more sense than B. Using a shirt to dry one's feet after getting them wet is a common and sensible thing to do, while using glasses to dry one's feet does not really make logical sense and would not be an effective way to dry one's feet.
Integrated Optofluidic device to study Interaction of particle or solution wi...prathul Nath
Microfluidics and integrated optics combined to study the interaction of particles or solution with light flowing through a microfluidic channel. Involves dye excitation through evanescent wave coupling.
This document summarizes a study that used picosecond optical tomography with a white laser and streak camera to measure changes in oxyhemoglobin and deoxyhemoglobin concentration in the brains of zebra finches in response to auditory stimulation. The technique showed submicromolar sensitivity and was able to resolve fast changes in the hippocampus and auditory forebrain with 250 μm resolution. Stimulation resulted in an early decrease in hemoglobin and oxyhemoglobin levels, followed by an increase in blood oxygen availability and pronounced vasodilation after stimulus end. The findings provide direct evidence linking blood oxygen level-dependent signals to changes in oxygen transport in birds.
This document discusses using an optic fiber sensor to measure levels of NADH (reduced form of nicotinamide adenine dinucleotide) in the rat brain during different sleep-wake states. The sensor allows for non-invasive, continuous measurements of NADH fluorescence in freely moving rats. Preliminary findings show NADH levels increase moderately in the periaqueductal gray-nucleus raphe dorsalis region during slow-wave sleep compared to waking, and increase more during paradoxical sleep. NADH levels in the cortex also increase moderately during slow-wave and paradoxical sleep compared to waking. Variations in NADH levels may reflect changes in brain oxidative energy metabolism across sleep-wake
Hippocampal Place Cells in Echolocating Bats stanfordneuro
1) Hippocampal place cells in bats rapidly update their spatial firing based on incoming sensory information from echolocation calls. Place cell spikes occurring later after an echolocation call show reduced spatial selectivity compared to earlier spikes.
2) This demonstrates that place cells can integrate new sensory information on a fast time scale of hundreds of milliseconds to tune their spatial firing based on the acuity of available sensory information.
3) Previous studies in rodents found slower dynamics of place cell remapping in response to changes in sensory information, but bats provide a system to study rapid updating of spatial signals.
This document provides an overview of cell structures and microscopy techniques used to study cells. It discusses the different types of microscopes like light microscopes, electron microscopes, and their uses and limitations. Key differences between prokaryotic and eukaryotic cells are outlined. The document also explains why cells need to be small, noting that diffusion works best over short distances and the nucleus can only handle a limited amount of information. Organelles allow eukaryotic cells to compartmentalize functions within internal membranes.
Positionig system of the brain(noble prize in medicine 2014) amir mahmodzadeh
The document discusses the hippocampus, which is a structure in the medial temporal lobe involved in consolidating short-term to long-term memory and spatial navigation. It contains several regions including the dentate gyrus, Cornu Ammonis fields (CA1-CA3), and subiculum. Place cells and grid cells encode spatial information and are involved in cognitive mapping. Recording techniques can monitor the activity of these cells in rodents as they move through environments. The hippocampus and entorhinal cortex work together to form episodic memories and support functions like memory, learning, emotion processing, and navigation.
Measuring visual acuity and contrast sensitivity by optomotor reflex in rodentsInsideScientific
There is a growing need for behavioral readouts to monitor disease progression and to assess the success of a potential therapy. In vision research, observing the optomotor reflex (OMR) is an important and widely established method for assessing visual acuity and contrast sensitivity in rodents. These tests can be performed with freely moving animals without any need for anaesthesia or restraints. In addition, since OMR is a reflex-based behavior, observing it does not require any training of the animal.
In this webinar, sponsored by Striatech and supported in part by Stoelting, researchers will present objective and bias-free results obtained using a newly developed automated optomotor system. For more information, please visit: https://insidescientific.com/webinar/measuring-visual-acuity-contrast-sensitivity-optomotor-reflex-striatech
Night vision allows vision in low light conditions using biological or technological means. Biologically, many animals can see better than humans at night due to traits like more light-sensitive cells in the retina or a tapetum lucidum layer. Technologically, night vision is achieved through three main categories - image intensification, active illumination using infrared light, and thermal imaging which detects temperature differences. Image intensification magnifies available light while active infrared uses infrared cameras and illuminators.
BAAI Conference 2021: The Thousand Brains Theory - A Roadmap for Creating Mac...Numenta
Jeff Hawkins presented a talk on "The Thousand Brains Theory: A Roadmap to Machine Intelligence" at the Beijing Academy of Artificial Intelligence Conference on 1st June 2021. In this talk, he discussed the key components of The Thousand Brains Theory and Numenta's recent work.
The document discusses neural correlates of higher level brain functions. It covers several topics:
1) Experience arises at the quantum level in ion channel proteins, with quantum properties like coherence and entanglement playing a role.
2) Construction of perception involves transitions from quantum to classical domains in the brain, mediated by ion channel proteins. Top-down processes and long-range connections in large brains are important for conscious perception.
3) Perception emerges from complex interactions between ascending and recurrent signaling in the brain, with feedback thought to be crucial for awareness. Receptive field properties evolve along synaptic distances in hierarchical cortical networks.
This document summarizes research on the visual system from the retina to primary visual cortex (V1). Key points include:
- The retina contains two types of ganglion cells (midget and parasol) that project to different layers in the lateral geniculate nucleus (LGN).
- Hubel and Wiesel discovered that V1 neurons have receptive fields tuned to stimulus orientation, forming the basis of the hierarchical model of visual processing.
- V1 contains two main cell types, simple cells with discrete receptive fields and complex cells without. Retinotopic maps and ocular dominance columns organize V1 architecture.
This document discusses various soft computing techniques for iris recognition, specifically focusing on two neural network approaches: Competitive neural network Learning Vector Quantization (LVQ) and Adaptive Resonance Associative Map (ARAM). It provides an overview of iris recognition as a biometric method, summarizes preprocessing steps like localization, segmentation, and normalization of iris images. It also describes feature extraction and matching steps. Finally, it defines artificial neural networks and discusses how LVQ and ARAM can be used for pattern matching in iris recognition applications.
Can we extract a mind from a plastic-embedded brain? - Kenneth Hayworth - H+ ...Humanity Plus
Ken Hayworth
Can we extract a mind from a plastic-embedded brain?
We now have a good working theory of consciousness – the phenomenal self model (Metzinger 2009), and we have a good understanding of the human cognitive architecture (Anderson 2007) within which this self model is implemented. The key components of this cognitive architecture are declarative memory chunks and productions – thought to be implemented as stable attractors in the neural networks of the cortex and basal ganglia. According to neural network theory, such stable attractors are robustly defined by the synaptic connectivity between neurons. In small pieces of tissue such synaptic connectivity is easily preserved using chemical fixation and embedding in plastic, and it should be relatively easy to adapt these protocols into a surgical procedure performed in hospitals to preserve whole human brains. Such plastic embedded brain tissue can be imaged at the nanometer level using new automated techniques (SBFSEM, FIBSEM, Tape-to-SEM), and we can directly extrapolate these techniques to future ones that will enable all the synaptic connections within a human brain to be mapped allowing a fully accurate simulation of the original preserved mind. In short, we have a complete sketch of how mind uploading will work and we have a mandate to implement emergency brain preservation in hospitals for all who desire access to this future technology.
Kenneth Hayworth, a postdoctoral fellow at Harvard University, is the inventor of several technologies for high-throughput volume imaging of neural circuits at the nanometer scale. He received a PhD in Neuroscience from the University of Southern California for research into how the human visual system encodes spatial relations among objects. Hayworth is a vocal advocate for brain preservation and mind uploading, and runs a website (www.brainpreservation.org) calling for the implementation of an Emergency Glutaraldehyde Perfusion procedure in hospitals, and for the development of a Whole Brain Plastic Embedding procedure which can demonstrate perfect ultrastructure preservation across an entire human brain.
This document contains descriptions of 14 art submissions to the Art of Neuroscience 2017. The submissions include jewelry and artworks inspired by neuroscience concepts, research on gender stereotyping and brain responses, paintings exploring connections between neuroscience and art, images of brain structures and cells created using various microscopy techniques, and more. The submissions are from researchers in multiple countries and cover a wide range of neuroscience topics communicated through different artistic mediums and styles.
Optical clearing is a new technique that improves 3D imaging of the enteric nervous system (ENS). Researchers used optical clearing and confocal microscopy to image the human ileum in 3D. Optical clearing reduced light scattering in the tissue, allowing deeper photon penetration and higher definition images. This generated a panoramic 3D view of gut wall structures like nerves, muscles, and crypts. The researchers plan to use this method to study gastrointestinal disease mechanisms by observing microstructures, vasculature, and nerve networks in animal models and comparing healthy and diseased human tissues.
Night vision works by detecting small amounts of light, such as moonlight or starlight, that are invisible to the human eye. There are two main types of night vision technologies: image intensification and thermal imaging. Image intensification uses an image intensifier tube to amplify available light so that more light enters the eye, allowing the user to see in low light conditions. Thermal imaging detects infrared radiation emitted or reflected from objects to create a thermal image without needing visible light. Within image intensification there are four recognized generations that reflect increasing technological sophistication, with Generation 3 being the most advanced currently available night vision.
Blindness is a serious condition that is feared by many. Researchers are working on developing artificial vision technologies to help restore sight for the blind. One such technology is a bionic eye, which uses a camera and implant to stimulate the retina and optic nerve to generate images in the brain. The retina plays a key role in vision, containing rods, cones and ganglion cells that transmit light signals to the brain. Retinal diseases can lead to blindness by damaging these cells. Researchers are working to bypass damaged areas and provide artificial stimulation to restore some level of sight.
Trauma management during tragic years of lebanon part iiAnis Baraka
The document discusses a SomaSensor device that uses near infrared spectroscopy (NIRS) to non-invasively measure oxygen saturation levels in the brain. It works by emitting light through the skull from an LED light source and detecting the light at different depths using photodetectors on the surface of the head, allowing it to isolate changes in the brain by subtracting the shallow measurement from the deep one. This technique provides depth resolution to minimize contamination from superficial signals.
Technological advancements in science visualization are described. X-ray crystallography allows scientists to see molecules in 3D by converting diffraction patterns from x-rays hitting crystals into visual maps. Microscopy techniques like light and electron microscopes allow magnification of small objects. Telescopes collect light or radio waves to view distant images. DNA fingerprinting identifies individuals by comparing DNA fragments. Computers process large data amounts and automate tools. Medical imaging techniques like X-rays and MRI produce enhanced soft tissue images. Remote sensing uses GPS, radar, sonar and satellites to detect location and environment. Virtual reality and simulations mimic real-world activities. Holograms produce 3D images using lasers.
The document describes several submissions to the Art of Neuroscience 2017 competition. The submissions include:
1. An electron microscope image showing a microglial lysosome and autophagosome, capturing the process of autophagy.
2. A description and calcium imaging data from cortical neurons in a mouse brain, showing patterns of neuronal activity.
3. A sample of hippocampal neurons labeled for microtubules and actin, imaged with a fluorescent microscope.
4. An artwork inspired by Michelangelo's "The Creation of Adam", depicting interacting pyramidal cells in the mouse brain.
5. Diffusion MRI data quantifying whole-brain axonal connections in the rat brain, clearly
Vicarious Systems at Singularity Summit 2011Scott Brown
A makes more sense than B. Using a shirt to dry one's feet after getting them wet is a common and sensible thing to do, while using glasses to dry one's feet does not really make logical sense and would not be an effective way to dry one's feet.
Integrated Optofluidic device to study Interaction of particle or solution wi...prathul Nath
Microfluidics and integrated optics combined to study the interaction of particles or solution with light flowing through a microfluidic channel. Involves dye excitation through evanescent wave coupling.
This document summarizes a study that used picosecond optical tomography with a white laser and streak camera to measure changes in oxyhemoglobin and deoxyhemoglobin concentration in the brains of zebra finches in response to auditory stimulation. The technique showed submicromolar sensitivity and was able to resolve fast changes in the hippocampus and auditory forebrain with 250 μm resolution. Stimulation resulted in an early decrease in hemoglobin and oxyhemoglobin levels, followed by an increase in blood oxygen availability and pronounced vasodilation after stimulus end. The findings provide direct evidence linking blood oxygen level-dependent signals to changes in oxygen transport in birds.
This document discusses using an optic fiber sensor to measure levels of NADH (reduced form of nicotinamide adenine dinucleotide) in the rat brain during different sleep-wake states. The sensor allows for non-invasive, continuous measurements of NADH fluorescence in freely moving rats. Preliminary findings show NADH levels increase moderately in the periaqueductal gray-nucleus raphe dorsalis region during slow-wave sleep compared to waking, and increase more during paradoxical sleep. NADH levels in the cortex also increase moderately during slow-wave and paradoxical sleep compared to waking. Variations in NADH levels may reflect changes in brain oxidative energy metabolism across sleep-wake
1) Male zebra finches were found to respond differently to calls from their mate versus other females depending on their social context. Specifically, males responded much more to their mate's call when with a mated pair compared to when alone or with other unmated males.
2) Previous studies found male zebra finches did not show mate recognition from female calls. However, the new study found female calls contain identifiable acoustic features allowing for individual recognition.
3) The results suggest social context can influence mate recognition in birds, challenging the view that complex social assessments are unique to primates.
This document summarizes a study that used functional magnetic resonance imaging (fMRI) and near-infrared spectroscopy (NIRS) to measure hemodynamic changes in the brain of zebra finches in response to hypercapnia. Hypercapnia induces vasodilation and is often used to model hemodynamic responses. Both fMRI, which detects blood oxygen level-dependent (BOLD) signals, and NIRS, which measures concentrations of oxyhemoglobin and deoxyhemoglobin, clearly showed increases in blood oxygen saturation in the brain during hypercapnia. The results provide the first correlation in songbirds between hemodynamic parameter variations measured by NIRS and local BOLD signal variations measured by fMRI.
This document discusses limitations of the classical homogenization approach for modeling light absorption in tissues. It considers a two-dimensional model where light absorption occurs only within parallel, thin blood vessels and not in the surrounding tissue. The model contains three small parameters: E is the ratio of vessel spacing to tissue size, d is the ratio of vessel thickness to spacing, and v is a large parameter related to absorption strength within vessels. The paper shows that classical homogenization is valid when E→0, v→∞, d→0, and E2vd→0, but is invalid when E→0, v→∞, d→0, and E2vd2→∞, requiring non-classical asymptotic expans
This document describes the construction and characterization of recombinant viruses containing the HIV-1 env gene from seminal strains. The researchers amplified the V1-V3 region of env from seminal plasma samples and used it to construct chimeric viruses by co-transfecting the V1-V3 fragment into a V1-V3 deleted vector. Four chimeric viruses were able to replicate and were characterized as using CXCR4 as a coreceptor. Confocal microscopy was used to observe the interaction of the cell-free viral particles with reporter cell lines. The recombinant viruses representing seminal strains are useful tools for studying heterosexual HIV transmission and testing microbicides.
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1. Two-photon deep imaging through skin and skull of Zebra
finches : Preliminary studies for in vivo brain metabolism
monitoring
D. Abi-Haidara , T. Oliviera , S. Mottina , C. Vignalb and N. Mathevonb
a Laboratoire ´
Hubert Curien (ex-LTSI), UMR5516 CNRS & University of Saint-Etienne,
´
Saint-Etienne, France;
´ ´
b Laboratoire ENES, EA3988, University of Saint-Etienne, Saint-Etienne, France
ABSTRACT
Zebra Finches are songbirds which constitute a model for neuro-ethologists to study the neuro-mechanisms of
vocal recognition. For this purpose, in vivo and non invasive monitoring of brain activity is required during
acoustical stimulation. MRI (Magnetic Resonance Imaging) or NIRS (Near InfraRed Spectroscopy) are suitable
methods for these measurements, even though MRI is difficult to link quantitatively with neural activity and
NIRS suffers from a poor resolution. In the particular case of songbirds (whose skin is thin and quite transparent
and whose skull structure is hollow), two-photon microscopy enables a quite deep penetration in tissues and
could be an alternative. We present here preliminary studies on the feasability of two-photon microscopy in
these conditions. To do so, we chose to image hollow fibers, filled with Rhodamine B, through the skin of Zebra
finches in order to evaluate the spatial resolution we may expect in future in vivo experiments. Moreover, we used
the reflectance-mode confocal configuration to evaluate the exponential decrease of backreflected light in skin
and in skull samples. Following this procedure recently proposed by S.L. Jacques and co-workers, we planned
to determine the scattering coefficient µs and the anisotropy g of these tissues and make a comparison between
fixed and fresh skin and skull samples for future Monte Carlo simulations of the scattering in our particular
multi-layered structure.
Keywords: two-photon fluorescence, microscopy, neuro-imaging, scattering media, songbird
1. INTRODUCTION
In order to understand vocal recognition and social behaviors of animals, it is quite obvious that in vivo ex-
periments are crucial. Moreover, analyses of the neuro-mechanisms underlying vocal recognition created a need
for reliable and non invasive tools to probe activations of brain area involved in vocal processes. Amongst all
animals, songbirds, and particularly Zebra Finches (Taeniopygia guttata) are interesting to study because they
happen to be specialists in vocal emission and recognition, which are deeply involved in their social interactions.1
In order to monitor neuro-activations, functional MRI and NIRS (Near Infra-Red Spectroscopy) have been used
and have demonstrated their efficiency.2–4 Unfortunately, the BOLD (Blood Oxygen Level Dependent) signal
measured in fMRI experiments remains difficult to link quantitatively with parameters expressing neural activ-
ity. On the contrary, NIRS gives quantitative information about cerebral blood volume (CBV) and hemoglobin
oxygen saturation level which reflects cerebral activity, but it is not an imaging method and it suffers from a
poor millimetric resolution. In other terms, we have to admit that the study of neuro-mechanisms requires the
use of different and complementary monitoring methods.
For more than ten years now, two-photon microscopy has been widely adopted for in vivo imaging because
of its ability to keep a good resolution (a few micrometers), even in the depth (1mm) of scattering biological
samples.5–7 In more recent years, two-photon microscopy has been used for in vivo brain monitoring.8–13 Many
of the studies have been performed on rats or mice and proved very efficient. Unfortunately, the thickness (1.6-
2.2mm) and the highly scattering properties of rats and mice skull requires to remove a part of the skull and
Further author information: (Send correspondence to T.O.)
T.O.: E-mail: thomas.olivier@univ-st-etienne.fr, Telephone: +33 (0)4 77 91 58 20
2. replace it with a glass window. Other approaches tend to reduce many problems associated with craniotomy
by reducing the thickness of the skull9 to about 200-250µm. Miniature head-mounted elements and optical
fibers have been successfully also employed on freely moving rats, but still requires craniotomy.8 More recently,
two-photon micro-endoscopy experiments have been tested with very thin gradient index lenses, allowing to keep
micrometer resolutions, even 1.5mm deep inside the brain.11, 12 In most of these various multiphoton in vivo brain
studies, the monitoring relies on the study of the dynamics of red blood cells in brain capillaries.10 This gives
a reliable information about neural activations in quite small brain areas. By staining the plasma with various
fluorophores, contrast can be obtained between the fluorescent plasma and the absorbing blood cells. These
studies, based on red blood cells counting seem to require quite a good resolution which necessitates craniotomy.
However, the feasability of such a measurement has been demonstrated through 250µm thinned skull. Another
way to monitor brain activity can be the use of endogenous fluorescence of NAD(P)H or flavoproteins involved
in the energy metabolism of neurons.14 Even if this endogenous fluorescence is quite weak, it seems to be an
interesting way as it does not seem to require as much resolution as the counting of red blood cells.
In the case of songbirds like Zebra Finches, craniotomy is problematic as the birds are quite fragile. Moreover,
Zebra Finches have a pneumatic skull structure composed of 25µm-thick internal and external cortical bones
linked by approximately 250µm-high trabeculae. Indeed, the skull of Zebra Finches is much thinner than the
skull or rats or mice, and mainly composed of air. Our goal is to evaluate the possibility to perform two-photon
imaging in vivo through the skull, and maybe the skin, of Zebra Finches. Particularly, the brain area we would
like to investigate is the NCM (caudomedial neostriatum) which is highly involved in song processing. This brain
area is located between the caudal and the dorsal part of the bird’s head and 1.2mm to 2.7mm below the external
surface of the head. The hippocampus could also be an area of interest and is located above the NCM, between
0.6 and 1mm below the surface of the head.
At first sight, this particular skull structure is more transparent than the skull structure of small mammalians,
however, as the refractive index and scattering properties are very heterogeneous in all directions, it is important
to evaluate the effect of this structure on the image quality and the axial and lateral resolutions of multiphoton
microscopy. This heterogeneous structure is likely to distort the image. As a first step before setting up in
vivo procedures and experiments we decided to work on a simplified structure (i.e. only on fixed skull samples)
and to image a clearly identified object. The object we chose to image is a 10µm-hollow core fiber filled with
Rhodamine B, which possesses a high two-photon absorption cross section. In this work, we will present various
images and processed XYZ stacks performed on fixed skull samples, and particularly we will try to quantify the
distorsion and loss of resolution induced by the scattering of the incident beam while passing through the skull
sample.
Besides, it could be interesting to use simulations of light scattering inside the skin and skull of Zebra Finches
in order to better evaluate the feasability of in vivo monitoring of the neuro-activations. This requires accurate
knowledge of the scattering coefficients of fresh skin and skull samples. We used the recent experimental procedure
described by S. L. Jacques et al.,15, 16 based on reflectance-mode confocal measurements of the exponential decay
of reflectivity as the focus is moved along the optical axis inside the scattering sample. This measurement
allows the determination of both the scattering coefficient µs and the anisotropy g from the parameters ρ and µ
describing the exponential decay of reflectivity. This determination requires the performing of Monte Carlo
simulations that we have not performed yet. However, comparisons between the exponential decay coefficients ρ
and µ of fresh and fixed samples are presented, allowing us to evaluate the effect of fixing tissues to perform
more convenient experiments.
2. MATERIALS AND METHODS
In order to perform two-photon imaging as well as reflectance mode confocal imaging of skin or skull samples,
we used a TCS-SP2 confocal microscope from LEICA Microsystems. The laser system was a Ti:Sapphire MIRA
900 oscillator from COHERENT pumped with a 5W VERDI Nd:YAG laser at 532nm. The laser system delivers
typically 800mW, 200fs pulses with a 76MHz repetition rate. The wavelength can be adjusted between 710 and
900nm with a maximum power around 800nm. The experimental setup is represented on Figure 1. Only the
elements that were relevant during our experiments are represented.
3. 2.1. Two-photon imaging experiments
In the first set of experiments, i.e. for two-photon imaging, two-photon fluorescence is collected just after the
microscope objective via a dichroic beam splitter which is transparent to wavelengths greater than 715nm. The
collected visible radiation spectrum is split by another dichroic beamsplitter in two channels (non-descanned pho-
tomultipliers), one collecting blue/green radiations up to 550nm and the other one collecting yellow/orange/red
radiations starting from 550nm. As the confocal pinhole is useless for two-photon microscopy, the use of such
non-descanned detectors enables to collect the maximum fraction of fluorescence from the focus. Microscope
objectives that were used are long working distance water-immersion LEICA objectives (HCX APO L U-V-I
20× NA-0.5 and HCX APO L U-V-I 40× NA-0.8). An electro-optical modulator (EOM) was used to adjust the
laser power at the entrance of the confocal system. This was particularly useful to automatically compensate for
energy losses due to scattering as the focus moves inside the samples.
(a) (b)
Figure 1. Schematic representation of the microscopy configurations used in this work. (a) Main elements of the LEICA
TCS-SP2 microscopy system used in this work for confocal reflectance mode measurements of the diffusion coefficients or
two-photon imaging of the hollow fiber filled with Rhodamine B. For two-photon imaging: BS: dichroic mirror transparent
to visible radiations, DBS1: dichroic beam splitter transparent to NIR radiations starting from 715nm, DBS2: dichroic
beam splitter separating the visible radiation reflected by DBS1 into two channels (λ > 550nm and λ < 550nm), NDD1
and NDD2: non-descanned detectors (photomultipliers). (b) Details of the sample used in two-photon imaging: IM:
immersion medium, Sk: skull, HF: hollow fiber, RhB: Rhodamine B, St: stand. For confocal reflectance mode: BS: 30/70
beam splitter, Sc: XY-scanner, MO: microscope objective, S: sample, TS: translation stage, P: confocal pinhole, PMT1:
photomultiplier.
The hollow fibre we used had a 120µm total diameter and a 10µm hollow core, which simulates a single
rectilinear blood capillary. We used roughly 2cm-long fibers, the core of which was filled with Rhodamine
by capillarity and sealed with glue at both extremities. In order to have a maximum fluorescence, without
any quenching problems, various tests were performed with Rhodamine 6G and Rhodamine B in ethanol with
concentration varying from 10−5 to 10−1 mol/l. The highest fluorescence was recorded with Rhodamine B
for 10−2 mol/l and for a wavelength equal to 850nm. This concentration and wavelength were kept for all the
two-photons experiments presented in this work.
A couple of Zebra Finches with an average weight of 12g were sacrificed with a lethal intra-muscular injection
of pentobarbital. Their head were plucked a couple of days days before the death. The blood was removed from
the vascular system by cutting aorta just after the death of the animal in order to avoid staining of the bone
or skin tissues with blood during the dissection. Approximately 4mm×4mm pieces of skull were taken between
4. the dorsal and the caudal parts of the bird’s head and along the sagittal suture. These skull samples were fixed
with a 4% paraformaldehyde solution.
As described on figure 1-(b), the fiber was set on a 100µm deep rectilinear furrow carved on a plexiglas
half-cylinder whose curvature radius is equal to 7mm, which is the average curvature of the Zebra Finches head.
In order to prevent the filling of the skull’s pneumatic cavity with water, the immersion medium we used for the
microscope objective was a transparent gel, mainly composed of water (echography gel). This gel also held the
skull sample and prevented it from sliding during the recording of the image stacks.
2.2. Reflectance mode confocal experiments
In the second set of experiments, i.e. for reflectance mode confocal measurements, the dichroic beam splitter
DBS1 is removed and the BS beam splitter is replaced with a 30/70 beam splitter (30% of reflection and
70% of transmission). The microscope objective that was used is a long working distance water-immersion
LEICA objective (HCX APO L U-V-I 40× NA-0.8). The pinhole was set to 21.27µm which corresponds to
the 40× magnified size of the inner Airy pattern at the focus. In these conditions, the light collected by the
PMT1 photomultiplier is proportionnal to the light backreflected by the sample. For safety reasons, our LEICA
confocal system is designed to prevent NIR radiations from entering the photomultipliers of the confocal head.
Thus, these experiments were performed with a wavelength of 710nm in order to be as close as possible to the
two-photon experimental conditions described in part 2.1.
In reflectance mode confocal configuration, the amount of reflected light collected by the microscope objective
mainly comes from the focus and is linked to the backscattering properties of a thin slice in the sample. This
reflectivity decreases exponentially as the focus penetrates inside a scattering samples.15 The reflectivity decrease
as a function of depth inside the sample can be represented by the following equation:
R(z) = ρ exp(−µz) (1)
where R(z) represents the reflectivity of the sample at a distance z from the sample surface. ρ represents
then the reflectivity on the surface of the sample and µ the attenuation coefficient. These coefficients are
linked to the scattering coefficient µs and the anisotropy g, which would then lead to the reduced scattering
coefficient µ′ = µs (1 − g). The coefficients ρ and µ are not independently linked to the scattering coefficients µs
s
and g. Indeed, for example, when the anisotropy g decreases, there is less light that is backscattered, which
reduces ρ, as well as the attenuation coefficient µ. Monte Carlo simulations of the scattering of light inside
the sample are needed to find the couple (µs , g) corresponding to a couple (ρ, µ). We have not performed such
simulations for the moment. However, as a first step, our goal here is to compare the reflectivity and attenuation
coefficient of fresh tissues with the paraformaldehyde-fixed tissues we used in the previous experiments with
Rhodamine B filled hollow fiber.
We realized 512×512×100 image stacks. The total transversal field was 750×750µm-wide and the height of
a stack varied between 100 and 400µm depending on the sample roughness. We then plotted as a function of z
(depth in the sample) the average reflectivity in a 20×20µm region of interest (ROI), which corresponds roughly
to an average on 14×14 pixels. The typical z-profiles that were obtained are represented on Figure 2 for various
ROI sizes. This figure shows the profile we obtained on two different parts of the sample (A and B) and with
ROI sizes varying from 10×10µm to 40×40µm. As it can be seen on B, the decrease can be dependent on the
ROI size. This was observed when the sample is locally not perpendicular to the optical axis. The reflectivity
variations are then smoothed and the maximum reflectivity is not representative of the theoretical exponential
decay. For this reason, the ROI were selected on part of the stacks where the sample was quite perpendicular to
the optical axis. According to these measurements, the 20×20µm size of the ROI was chosen as it seemed to be
the best compromise to have a representative position of the reflectivity maximum as well as enough pixels to
make a significant averaging that prevents the experimental curves from oscillating to much.
These measurements and exploitations were performed on skull and skin samples. A first set of measurement
were carried out on samples freshly taken just after the death of the animal (see procedure in 2.1) and kept in a
petri dish filled with a Phosphate Buffer Saline (PBS) solution, which was then used as the immersion medium
for the microscope objective. In these conditions, five image stacks were recorded on various parts of one sample
6. Figure 3. Two-photon images of Zebra Finch skull (projected stacks). On the left: 1mm slice seen from the side. On
the right: projected stack seen from above. In both images trabeculae repartition and dimensions can be observed.
Figure 4. Two-photon fluorescence image sequence taken from one stack. First, the endogenous fluorescence of the
external cortical bone surface and the trabeculae is observed and then the fluorescence coming from the Rhodamine B
filled hollow fiber is observed, giving a distorted and enlarged image of the fiber core due to scattering in cortical bones
and trabeculae.
as described previously while (c) and (d) experiments were performed with thinner skull samples with only one
cortical layer, roughly 100µm-thick, that were taken from younger birds. In (a), (b) and (c), the ×20 objective
was used, while the ×40 objective was used in (d).
In order to evaluate the lateral and axial extent of the fiber image, x-projections of the previous stacks are
presented on Figure 6 (x represents then the axial direction of the fiber). Each of these stacks was obtained by
selecting only 200µm-thick slices along the x axis. These slices were selected in particular parts of the image
where the fiber image was not too distorted by the scattering in trabeculae.
Lateral and axial profiles of the fluorescence radiated from the Rhodamine B filled hollow core of the fiber
are represented on Figure 7. As it can be seen, the image of the fiber core is enlarged by the scattering. The
axial and lateral full width at half maximum (FWHM) of the fluorescence spatial repartition is summed up in
table 1, as well as the position of the maximum of fluorescence from the skull external cortical part.
As a conclusion, these two-photon imaging studies indicate that it is possible to collect fluorescence at depths
close to 1mm through the skull of Zebra Finches. However, from experiments (a) and (b), it is clear that this
fluorescence was collected mainly between trabeculae. As these trabeculae cover only 10% of the skull area
(seen from above), it remains possible to collect an important part of the fluorescence. The distorsion and the
enlargement of the image of the fiber remains problematic, especially in the axial direction of the beam, with
7. (a) (b) (c) (d)
Figure 5. Two-photon images of the hollow core fiber through various skull samples. Each image is an average z-
projection of the stack. The first line contains the images obtained with red/orange/yellow fluorescence (λ 550nm)
while the second line represents the images obtained with blue/green fluorescence (λ 550nm). As it can be seen, (c) and
(d) stacks were performed with skull samples having very few or no trabeculae. These samples were taken from younger
birds and were approximately 100µm-thick and only composed of one single cortical bone.
Figure 6. x-projected stacks of the previously presented stacks (where x is the axial direction of the fiber).
an axial FWHM of the fluorescence repartition greater than 1mm in case of experiment (a). As it can be seen
from experiment (c) and (d), a more moderate enlargement of 50-70µm in the lateral direction and 400µm in
the axial direction can be achieved at depths greater than 700µm if working with younger Zebra Finches.
8. 1.0 (a)
Normalized Fluorescence (b) (a)
Normalized Fluorescence
(c) 1.0 (b)
(c)
(d)
(d)
0.5
0.5
0.0
0.0
−0.3 −0.2 −0.1 0.0 0.1 0.2 0.3 0.0 0.5 1.0 1.5
y (mm) z (mm)
Figure 7. Lateral (left) and axial (right) profiles of the fluorescence radiated by the Rhodamine B filled hollow core fiber.
Table 1. Summary of the data extracted from the x-projection represented on Figure 6 (The position column is the
position of the maximum from the skull surface).
Experiment Lateral FWHM Axial FWHM Position
(a) 141µm 1.2mm 0.71mm
(b) 205µm 0.98mm 0.36mm
(c) 53µm 0.41mm 0.74mm
(d) 76µm 0.36mm 0.78mm
3.2. Reflectance mode confocal measurements
As described in section 2.2, reflectance mode confocal measurements were performed on fresh and fixed skull and
skin samples. Each reflectivity curve has been fitted with the theoretical expression R(z) = ρ exp(−µz). The
least squared fitting method was used to find the values of ρ and µ. Figure 8 represents the experimental curves
and the theoretical fits for ten ROI taken from one stack recorded on a fixed skin sample. All the points were
taken into account for the fit, starting from the maximum of the reflectivity curves.
rho = 0.00315 rho = 0.00290 rho = 0.00283 rho = 0.00178 rho = 0.00255
mu = 73.8 mu = 49.9 mu = 56.4 mu = 49.4 mu = 49.9
R² = 0.998 R² = 0.99 R² = 0.997 R² = 0.997 R² = 0.996
rho = 0.00209 rho = 0.00314 rho = 0.00377 rho = 0.0025 rho = 0.0029
mu = 51.5 mu = 56.6 mu = 55.6 mu = 50.6 mu = 58.1
R² = 0.997 R² = 0.996 R² = 0.995 R² = 0.996 R² = 0.997
Figure 8. Reflectivity curves measured as a function of the depth in a fixed skin sample. We present here 10 measurements
carried out on 20×20µm zones spread on various parts of one 512×512×100-stack. Experimental points (circles) are fitted
with the theoretical reflectivity function R(z) = ρ exp(−µz) (lines). The fitted values of ρ and µ (in mm−1 ) are given
here as well as the correlation coefficient R2 .
All the fitted values of ρ and µ were compiled and the result can be observed in Figure 9. This figure
represents the box and whiskers plots for the four types of sample (fixed and fresh skull and skin samples). As it
can be seen, the coefficients for fresh and fixed skull samples are obviously significantly different. As this is less
obvious for the skin samples, a Mann-Whitney test was carried out, indicating that the data are significantly
9. different with a probability of equality inferior to 10−6 and 10−11 (see Figure 9). According to these data, no
conclusion can be made concerning the value of the scattering coefficient µs and the anisotropy g, because this
requires Monte Carlo simulations that are still in process. However, it is clear that the attenuation coefficients µ
and reflectivity on surface ρ are different for fixed and fresh samples. These results will be very useful as we are
going to perform simulations of the diffusion in our situation and try to optimize our measurements for in vivo
studies.
Coefficients for skull samples Coefficients for skin samples
Mann−Whitney: P = 1e−06
fixed fixed
fresh fresh
0.0000 0.0005 0.0010 0.0015 0.000 0.005 0.010 0.015 0.020
ρ ρ
Mann−Whitney: P = 6e−12
fixed fixed
fresh fresh
0 50 100 150 200 250 0 50 100 150 200
µ − mm−1 µ − mm−1
Figure 9. Box and whiskers plots of the coefficients ρ and µ measured on skin and skull samples. The fresh samples were
kept few minutes to several hours after the death of the bird in PBS(Phosphate Buffer Saline) solution, while the other
ones were paraformaldehyde-fixed samples.
4. CONCLUSION PERSPECTIVES
Monitoring the brain activation on small songbirds is crucial to understand the neuro-mechanisms that underlie
vocal recognition, emission and learning. The understanding of these complex mechanisms requires a multilevel
approach as well as the use of various complementary monitoring methods. Two-photon imaging has been recently
used for various in vivo studies, demonstrating its potential for brain activation monitoring, unless craniotomy
or thinning of the skull is required. In the particular case of fragile songbirds, craniotomy should be avoided.
Fortunately, we have seen that the skull structure of birds is mostly composed of air and of two thin cortical
bone layers that are quite transparent. The skin itself is quite thin (around 100µm-thick). All these conditions
can also be enhanced by the use of younger birds. In this work, we have showed that the fluorescence of a thin
fiber filled with Rhodamine B can be clearly seen through the skull with a conventional multiphoton microscopy
system. Unfortunately the enlargement of the incident beam is quite important, reaching 200µm in lateral
direction and roughly 1mm in the axial direction. Moreover, the skin and other intracranial tissues were not
present in our simple configuration. Solutions could be found with the use of younger birds, because in this case,
we have seen that the resolution can be kept to a reasonable value of 50µm in the lateral direction and 400µm in
the axial direction. New fluorophores optimized for two-photon absorption17 could be used, as their two-photon
absorption cross section can reach 2000GM, which is ten times the cross section of Rhodamine B.18 As it has
been previoulsy demonstrated,7, 19 the use of a regenerative amplifier is an interesting way to increase the imaging
depth in biological samples. It remains obvious that a compromise has to be found between the resolution and the
imaging depth we require. In our case, simulations of the scattering as well as further experiments are necessary
to investigate if the quite peculiar structure of the bird’s skull will not affect too much our resolution and to find
experimental conditions and approaches that would be likely to enhance this resolution, especially the axial one.
That was the reason why complementary works were presented here as well, concerning the measurement of the
scattering coefficients of the skull and skin, which will be useful for future simulations. Particularly, it has been
showed that the exponential decay of backreflected light is different for fresh and for fixed sample, and this fact
must be taken into account before attempting in vivo experiments.
10. ACKNOWLEDGMENTS
The authors would like to thank the French national agency for research (ANR) for financial support and
Sabine Palle, technical director of the confocal microscopy platform of University Jean-Monnet (PPF microscopie
confocale multiphotonique).
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