Microscopie par excitation à deux photons

vignette|350px|Microscopie par excitation à 2 photons de l'intestin d'une souris. Rouge: actine. Vert: noyaux des cellules. Bleu: mucus des cellules caliciformes. Obtenu à 780 nm avec un laser Ti-sapph. La microscopie par excitation à deux photons (M2P, TPEF ou 2PEF en anglais, aussi appelée « microscopie 2 photons ») est une technique d'imagerie optique combinant les principes de microscopie à fluorescence et de l'absorption à deux photons, faisant partie de la famille des microscopies multiphotons. Elle a été développée en 1990 par Watt W. Webb, Winfried Denk et Jim Strickler à l'Université Cornell. Elle utilise la fluorescence pour imager des tissus vivants jusqu'à environ un millimètre de profondeur, mais diffère de la microscopie à fluorescence classique puisque la lumière excitatrice a une fréquence lumineuse plus petite que celle émise (c’est l’inverse en fluorescence à un photon). Cette excitation est généralement dans le proche infrarouge, qui peut également exciter les colora

Sparse, reliable, and long-term stable representation of periodic whisker deflections in the mouse barrel cortex

Johannes Maria Mayrhofer

The rodent whisker system is a preferred model for studying plasticity in the somatosensory cortex (barrel cortex). Contrarily, only a small amount of research has been conducted to characterize the stability of neuronal population activity in the barrel cortex. We used the mouse whisker system to address the neuronal basis of stable perception in the somatosensory cortex. Cortical representation of periodic whisker deflections was studied in populations of neurons in supragranular layers over extended time periods (up to 3 months) with long-term two-photon Ca2+ imaging in anesthetized mice. We found that inmost of the neurons (87%), Ca2+ responses increased sublinearly with increasing number of contralateral whisker deflections. The imaged population of neurons was activated in a stereotypic way over days and for different deflection rates (pulse frequencies). Thus, pulse frequencies are coded by response strength rather than by distinct neuronal sub-populations. A small population of highly responsive neurons (similar to 3%) was sufficient to decode the whisker stimulus. This conserved functional map, led by a small set of highly responsive neurons, might form the foundation of stable sensory percepts. (C) 2015 Elsevier Inc. All rights reserved.

Academic Press Inc Elsevier Science2015

Biomedical optical techniques for intracochlear cellular imaging

Marilisa Romito

Imaging the inner ear microanatomy is of great importance for the assessment of the state of the cells responsible for sound detection. Hearing disorders are mainly due to a malfunction of the cochlea, the bone containing the cells inside the inner ear. Under this situation, using suitable imaging techniques would be extremely helpful in detecting disease and diagnosing pathologies of the inner ear. However, cochlear small size and encasement in bone provide challenging obstacles, preventing visualization of intracochlear microanatomy using standard clinical imaging modalities. In this thesis, we explore optical techniques to image inner ear cells and we report an innovative method to observe intracochlear structures through the scattering cochlear bone. Firstly, we report different imaging techniques we used for intracochlear cellular investigation, starting with conventional optical microscopy. Two-photon excitation fluorescence (TPEF) microscopy is the most suitable technique for high quality images of the hair cells within the organ of Corti. Our results from extracted mouse organ of Corti show that we are able to distinguish between healthy and damaged sensory cells and nerve fibers, analyzing TPEF images from different mouse samples: young, old, exposed to noise and not exposed to noise. We then describe different noninvasive methods that have been used to image intact cochleae, such as Optical Coherence Tomography (OCT), X-ray Computed Tomography (X-Ray CT), micro-Computed Tomography (X-Ray µCT) and Optical Diffraction Tomography (ODT). These techniques allow the observation of the internal ear anatomy, despite the bone. We report tomographic volumetric reconstructions of mouse cochlea and we compare them in terms of achievable image resolution. µ-CT is the most appropriate techniques providing both penetration through bone and high-level resolution images down to 2µm. However, the high dose of radiation used in µCT studies prevent translation of these techniques to living humans. Due to the discussed challenges for imaging the hair cells through the highly scattering bone, the aim of my thesis is to develop a new method for intracochlear cellular imaging with minimum trauma. The last chapter represents the core of this PhD project. In this chapter, after considering the advantages and disadvantages of all the possible imaging techniques, we report our innovative technique for visualization of cochlear cells through the overlying scattering bone by combining femtosecond laser bone ablation and TPEF microscopy. The controlled ultrafast laser ablation reduces the optical scattering of the cochlear bone while the TPEF provides a contrast mechanism to resolve individual cells behind the bone. We implemented a simultaneous OCT with the laser ablation to enhance the precision of ablation and prevent inadvertent violation of the delicate cells hidden behind bone. An additional bright-field camera shows real-time images of the sample. We demonstrate that our approach improves the light focusing capability through the cochlear bone, allowing imaging of intracochlear structures in intact cochleae with high resolution. This extremely highly promising approach can be further developed for clinical ablation instruments and endoscopes that can reach the cochlea and produce images of cells within the inner ear to establish precise diagnosis, guide treatment and assess response to therapies, which is not yet possible in the clinic.

EPFL2019

Uncovering the neural substrates of communication between the brain and ventral nerve cord in Drosophila melanogaster (italic)

Florian Aymanns

Despite a long history of research in motor control, the exact mechanism of how the brain communicates with the the invertebrate ventral nerve cord (VNC) and the vertebrate spinal cord on a single neuron basis remains largely elusive.Drosophila melanogaster is an ideal model organism to address the role of individual neurons, thanks to its stereotyped nervous system.Descending neurons (DNs), interneurons in the brain that project to the invertebrate ventral nerve cord or vertebrate spinal cord, have been shown to elicit specific behaviors upon optogenetic activation in Drosophila.These results suggest that behavior is controlled by sparse sets of command neurons, each eliciting a particular behavior.However, it remains to be seen how many DNs are involved in the control of naturalistic behaviors and what other information they might encode.In order to avoid commands leading to physically impossible or destabilizing actions, the brain has to be aware of the current behavior state.Ascending neurons (ANs), interneurons in the invertebrate ventral nerve cord or vertebrate spinal cord projecting to the brain, are likely conveying this information to the brain.To which degree of fidelity and in what way ANs encode behavior state remains unclear.To address these questions, we developed a dissection approach that allows functional imaging of ANs and DNs in the cervical connective.This dissection gives us optical access to the cervical connective and parts of the VNC in behaving adult Drosophila such that a two-photon fluorescence microscope can be used to image neural activity via calcium indicators.We began by imaging a new library of sparse split GAL4 driver lines targeting ANs.Our recordings from 247 genetically identifiable ANs revealed neurons encoding walking, resting, turning, eye grooming, and foreleg movements.Anatomical characterization of these ANs showed that they predominantly project to two brain regions: the anterior ventrolateral protocerebrum (AVLP) and the gnathal ganglion (GNG).Our results suggest that ANs encoding the motion of the animal with respect to the surrounding environment (self-motion) predominantly project to the AVLP, an integrative sensory hub. ANs projecting to the GNG mostly encode discrete actions.We then proceeded to image populations of DNs.The majority of DNs we recorded encoded walking behavior, with smaller fractions encoding resting and head grooming.Some of these neurons are likely to drive walking, but we also identified neurons encoding walking speed and turning.This suggests that a core set of DNs drives a behavior whose features can be modulated by additional DNs.Besides encoding behaviors, some DNs encode sensory information, including the presence of odors and deflection of the antennae.

EPFL2022