Fast Neuronal Imaging using Objective Coupled Planar Illumination Microscopy

Complex computations performed by the brain are produced by activities of neuronal populations. There is a large diversity in the functions of each individual neuron, and neuronal activities occur in the time scale of milliseconds. In order to gain a fundamental understanding of the neuronal populations, one has to measure activity of each neuron at high temporal resolution, while investigating enough neurons to encapsulate the neuronal diversity. Traditional neurotechniques such as electrophysiology and optical imaging are constrained by the number of neurons whose activities can be simultaneously measured or the speed of measuring such activities. We have developed a novel light-sheet based technique called Objective Coupled Planar Illumination: OCPI) microscopy which is capable of measuring simultaneous activities of thousands of neurons at high speeds. In this thesis I pursue the following two aims: * Improve OCPI microscopy by enhancing the spatial resolution deeper in tissue. Tissue inhomogeneity and refractive index mismatch at the surface of the tissue lead to optical aberrations. We have compensated for such aberrations by: 1) miniaturizing the OCPI illumination optics, so as to enable more vertical imaging of the tissue,: 2) correcting for the angular defocus caused by the refraction at the immersion fluid/tissue interface, and: 3) applying adaptive optics to correct for higher order optical aberrations. The improvement in the depth at which one can image tissue will enable the measurement of activities of neuronal populations in cortical areas. * Measure the diversity in the expression pattern of VSNs responsive to sulfated steroids. Nodari et al. have identified sulfated steroids as a novel family of ligands which activate vomeronasal sensory neurons: VSNs). Due to the experimental constraints, it has not been possible to obtain a comprehensive understanding of the number, location and functional characteristics of the sulfated steroid responsive VSNs. Applying OCPI microscopy and calcium imaging to simultaneously image thousands of VSNs, we show that the sulfated steroid responsive neurons: 1) have unique ligand preferences,: 2) are predominantly present in the apical regions of the VNO, and: 3) that the choice of expression of a receptor type is not purely stochastic

Aberrations and their correction in light-sheet microscopy: A low-dimensional parametrization

Light sheet microscopy allows rapid imaging of three-dimensional fluorescent samples, using illumination and detection axes that are orthogonal. For imaging large samples, this often forces the objective to be tilted relative to the sample’s surface; for samples that are not precisely matched to the immersion medium index, this tilt introduces aberrations. Here we calculate the nature of these aberrations for a simple tissue model, and show that a low-dimensional parametrization of these aberrations facilitates online correction via a deformable mirror without introduction of beads or other fiducial markers. We use this approach to demonstrate improved image quality in living tissue

Image-based calibration of a deformable mirror in wide-field microscopy

Optical aberrations limit resolution in biological tissues, and their influence is particularly large for promising techniques like light-sheet microscopy. In principle, image quality might be improved by adaptive optics (AO), in which aberrations are corrected using a deformable mirror (DM). To implement AO in microscopy, one requires a method to measure wavefront aberrations, but the most commonly used methods have limitations for samples lacking point-source emitters. Here we implement an image-based wavefront-sensing technique, a variant of generalized phase-diverse imaging called multi-frame blind deconvolution, and exploit it to calibrate a DM in a light-sheet microscope. We describe two methods of parameterizing the influence of the DM on aberrations: a traditional Zernike expansion requiring 1,040 parameters, and a direct physical model of the DM requiring just 8 or 110 parameters. By randomizing voltages on all actuators, we show that the Zernike expansion successfully predicts wavefronts to an accuracy of approximately 30 nm (rms) even for large aberrations. We thus show that image-based wavefront sensing, which requires no additional optical equipment, allows for a simple but powerful method to calibrate a deformable optical element in a microscope setting

Organization of Vomeronasal Sensory Coding Revealed by Fast Volumetric Calcium Imaging

A long-standing goal in neuroscience is to perform exhaustive recording of each neuron in a functional local circuit. To achieve this goal, one promising approach is optical imaging of fluorescent calcium indicators, but typically the tens or hundreds of cells imaged simultaneously comprise only a tiny percentage of the neurons in an intact circuit. Here, we show that a recent innovation, objective-coupled planar illumination (OCPI) microscopy, permits simultaneous recording from three-dimensional volumes containing many thousand neurons. We used OCPI microscopy to record chemosensory responses in the mouse vomeronasal epithelium, for which expression of hundreds of receptor types implies high functional diversity. The implications of this diversity for sensory coding were examined using several classes of previously reported vomeronasal ligands, including sulfated steroids. A collection of just 12 sulfated steroids activated more than a quarter of the neurons in the apical vomeronasal epithelium; unexpectedly, responses were functionally organized into a modest number of classes with characteristic spatial distribution. Recording from a whole sensory system thus revealed new organizational principles