Simultaneous two-photon imaging and two-photon optogenetics of cortical circuits in three dimensions

The simultaneous imaging and manipulating of neural activity could enable the functional dissection of neural circuits. Here we have combined two-photon optogenetics with simultaneous volumetric two-photon calcium imaging to measure and manipulate neural activity in mouse neocortex in vivo in three-dimensions (3D) with cellular resolution. Using a hybrid holographic approach, we simultaneously photostimulate more than 80 neurons over 150 μm in depth in layer 2/3 of the mouse visual cortex, while simultaneously imaging the activity of the surrounding neurons. We validate the usefulness of the method by photoactivating in 3D selected groups of interneurons, suppressing the response of nearby pyramidal neurons to visual stimuli in awake animals. Our all-optical approach could be used as a general platform to read and write neuronal activity

A CMOS-based Hartmann-Shack Sensor for Real-Time Adaptive Optical Applications

Adaptive optical systems have a growing field of applications in opthalmology. In every adaptive system there is the need for a sensor and an actuator. The Hartmann-Shack wavefront sensor uses the displacement of spots in the focal plane of a lenslet array for subsequent calculation of the wavefront. The bandwidth of current sensors is mostly limited by software processing of the focal plane image to some tens of Hz, which makes it unsuitable for real-time adaptive optical systems. To overcome the current bandwidth limitations a fast Hartmann-Shack sensor based on an application specific integrated circuit has been developed and tested, that reaches a bandwidth of up to 6 kHz. The sensor includes photodetectors with 40% quantum efficiency at 680 nm wavelength and an image processing, that is especially suitable to reduce the effects of the common mismatching of process parameters in CMOS-based sensors (Complementary Metal Oxide Semiconductor). A special problem in ophthalmic applications is the low available spot power below 1 nW.Adaptive optical systems have a growing field of applications in opthalmology. In every adaptive system there is the need for a sensor and an actuator. The Hartmann-Shack wavefront sensor uses the displacement of spots in the focal plane of a lenslet array for subsequent calculation of the wavefront. The bandwidth of current sensors is mostly limited by software processing of the focal plane image to some tens of Hz, which makes it unsuitable for real-time adaptive optical systems. To overcome the current bandwidth limitations a fast Hartmann-Shack sensor based on an application specific integrated circuit has been developed and tested, that reaches a bandwidth of up to 6 kHz. The sensor includes photodetectors with 40% quantum efficiency at 680 nm wavelength and an image processing, that is especially suitable to reduce the effects of the common mismatching of process parameters in CMOS-based sensors (Complementary Metal Oxide Semiconductor). A special problem in ophthalmic applications is the low available spot power below 1 nW. The developed Hartmann-Shack sensor allowed wavefront measurements with an accuracy of 0.16 dpt defocus at 160 pW spot power. It has been possible for the first time, to measure wavefront aberrations at the living humane eye with 300 Hz repetition rate and to calculate the power spectral density of these aberrations

The effect of aberrations and light scatter on visual performance at photopic and mesopic light levels

The Contrast Acuity Assessment (CAA) test was developed to assess the minimum spatial vision requirements for commercial pilots. The goal of the CAA test was for it to be sensitive to retinal image degradation in subjects who had undergone excimer laser refractive surgery. Increased aberrations and scattered light or abnormal processing of visual information in the retina and/or the visual pathway are the main causes of retinal image degradation. The purpose of this study was to further investigate the effect of aberrations, scatter and other parameters on the CAA test under photopic and mesopic conditions. This could help to provide explanations for previous CAA test results. The effect of contrast, stimulus onset time, pupil size and crowding on the CAA test was examined. This was in order to try to provide explanations for the decline in Landolt ring gap acuity over the central 5 degrees, as observed in previous CAA test results, which had shown a foveal dip to occur under photopic and mesopic conditions. Contrast Sensitivity was measured on eight subjects using 6 and 3 mm artificial pupils using the City University Contrast Sensitivity Test. A significant trend of decreasing contrast sensitivity with increased pupil size occurred for the middle and lower spatial frequencies (1.2 and 6.1 cpd), but not for the highest 19.1 cpd spatial frequency. The effects of using high contrast (125%) rather than low contrast (24%) CAA test targets were investigated, in combination with the use of artificial pupils of 6 mm and 3 mm. We concluded that low contrast could play a role in producing a foveal dip under photopic and mesopic conditions. The effect of crowding and stimulus onset time on the CAA Test was examined on 3 subjects, by reducing the contrast of the fixation guides under mesopic and photopic conditions and increasing stimulus onset time. This gave inconclusive results. No significant conclusions were drawn concerning the effect of crowding or stimulus onset time on the CAA foveal dip. The effect of aberrations on normal subjects on the photopic and mesopic CAA test was examined, to determine whether they may have influenced the foveal dip. 14 subjects were tested with natural pupils, under photopic conditions and 10 subjects, were tested under mesopic conditions. A Shack Hartmann aberrometer was used to take wavefront aberration measurements. No significant regressions were found between aberrations and foveal dip. We concluded that aberrations were probably not the cause of the foveal dip. Q value lenses consisting of Q = -2, Q = -1, Q = 0, Q = +1 and Q = +1.5 contact lenses were tested on subjects with natural pupils, to determine whether the CAA test could pick up larger non-physiological changes in aberrations. Large changes in visual performance were observed. Z (4,0) spherical aberration versus central CAA gap acuity was found to produce a significant quadratic regression under mesopic conditions. Seidel coma and Seidel astigmatism were also found to produce significant linear regressions. under photopic conditions. Scatter was measured in 4 subjects, using 6 mm and 3 mm artificial pupils, to determine whether scatter would increase with the larger pupil size. Linear regressions of scatter k’ versus foveal dip gave results which were not statistically significant. Scatter was measured for 3 subjects using the 5 different Q value contact lenses, to see if the Q values affected the scatter. The results were not statistically significant. The differences in scatter produced were found to be far less than the increase of scatter found in two subjects with pathological conditions. We concluded that scatter played an insignificant role in producing the foveal dip or changing visual performance with the use of Q value contact lenses. The project produced a systematic investigation of the parameters affecting the CAA test. A statistically significant association, described by a quadratic regression curve, exists between CAA mesopic gap acuity and Z (4,0) spherical aberration

Improved methods for functional neuronal imaging with genetically encoded voltage indicators

Voltage imaging has the potential to revolutionise neuronal physiology, enabling high temporal and spatial resolution monitoring of sub- and supra-threshold activity in genetically defined cell classes. Before this goal is reached a number of challenges must be overcome: novel optical, genetic, and experimental techniques must be combined to deal with voltage imaging’s unique difficulties. In this thesis three techniques are applied to genetically encoded voltage indicator (GEVI) imaging. First, I describe a multifocal two-photon microscope and present a novel source localisation control and reconstruction algorithm to increase scattering resistance in functional imaging. I apply this microscope to image population and single-cell voltage signals from voltage sensitive fluorescent proteins in the first demonstration of multifocal GEVI imaging. Second, I show that a recently described genetic technique that sparsely labels cortical pyramidal cells enables single-cell resolution imaging in a one-photon widefield imaging configuration. This genetic technique allows simple, high signal-to-noise optical access to the primary excitatory cells in the cerebral cortex. Third, I present the first application of lightfield microscopy to single cell resolution neuronal voltage imaging. This technique enables single-shot capture of dendritic arbours and resolves 3D localised somatic and dendritic voltage signals. These approaches are finally evaluated for their contribution to the improvement of voltage imaging for physiology.Open Acces

Neuron

Recording the activity of large populations of neurons is an important step toward understanding the\ua0emergent function of neural circuits. Here we present a simple holographic method to simultaneously perform two-photon calcium imaging of neuronal populations across multiple areas and layers of mouse cortex in\ua0vivo. We use prior knowledge of neuronal locations, activity sparsity, and a constrained nonnegative matrix factorization algorithm to extract signals from neurons imaged simultaneously and located in different focal planes or fields of view. Our laser multiplexing approach is simple and fast, and could be used as a general method to image the activity of neural circuits in three dimensions across multiple areas in the brain.R01MH100561/MH/NIMH NIH HHS/United StatesR01MH101218/MH/NIMH NIH HHS/United StatesR01 MH100561/MH/NIMH NIH HHS/United StatesR01 MH101218/MH/NIMH NIH HHS/United StatesDP1 EY024503/EY/NEI NIH HHS/United StatesR01 EY011787/EY/NEI NIH HHS/United StatesR41MH100895/MH/NIMH NIH HHS/United StatesR41 MH100895/MH/NIMH NIH HHS/United StatesDP1EY024503/DP/NCCDPHP CDC HHS/United StatesR01EY011787/EY/NEI NIH HHS/United States2017-01-20T00:00:00Z26774159PMC47242247184vault:2051

Extended field-of-view ultrathin microendoscopes with built-in aberration correction for high-resolution functional imaging with minimal invasiveness

TB

Enlightening axonal activity: Optical approaches to identify ion channels and their function

In this thesis we asked the questions how ion signaling and the periodic organization at the AIS shape the generation of action potentials. In order to answer these questions, we need to bridge functional investigations with high resolution structural reconstructions. In Chapter 2, we used optical calcium recordings and identified three calcium entry pathways in the AIS. As expected, calcium is released from internal stores and enters through calcium channels, however, surprisingly, we also observed calcium entering through sodium channels. We estimated that the conductivity ratio of sodium channels for calcium is small, but because they are present at a high density at the AIS, they do form a major and rapid source of calcium. In Chapter 3, we investigated whether the calcium-dependent BK channel was a downstream target for calcium in the AIS. We implemented a novel technique to use light patterning of a fluorescent voltage reporter to obtain highly accurate measures of the action potential shape in the axon. BK channels were indeed activated during the action potential at the AIS, forming a link between calcium entry and action potential repolarization. Together, the complex of calcium and BK channels mediated high-frequency burst firing, an important feature of the cell type that we studied. In Chapter 4, we developed a novel optical method to perform high resolution microscopy deep inside tissue, where the neurons are in an intact three-dimensional context. Because biological tissue is not transparent, light traveling through tissue suffers from distortions, which makes microscopy at depth problematic. To overcome this obstacle, we used a deformable mirror to counteract the light distortions and enable high resolution microscopy inside biological tissue. We used this method to perform both live experiments and high-resolution microscopy from the same neuron, demonstrating that this method can bridge the structure-function relationship in neurons. Together, the experiments in this thesis shed light on the biophysical properties of axonal ion fluxes and how they are tuned to regulate proper neuronal excitability. The work presented in this thesis shows that optical approaches provide valuable tools in neuroscientific research and open novel avenues for future investigation of the biophysical properties of the neuronal membrane

Microscopy Conference 2017 (MC 2017) - Proceedings

Das Dokument enthält die Kurzfassungen der Beiträge aller Teilnehmer an der Mikroskopiekonferenz "MC 2017", die vom 21. bis 25.08.2017, in Lausanne stattfand

Microscopy Conference 2017 (MC 2017) - Proceedings

Das Dokument enthält die Kurzfassungen der Beiträge aller Teilnehmer an der Mikroskopiekonferenz "MC 2017", die vom 21. bis 25.08.2017, in Lausanne stattfand