Precompensation of 3D field distortions in remote focus two-photon microscopy

Remote focusing is widely used in 3D two-photon microscopy and 3D photostimulation because it enables fast axial scanning without moving the objective lens or specimen. However, due to the design constraints of microscope optics, remote focus units are often located in non-telecentric positions in the optical path, leading to significant depth-dependent 3D field distortions in the imaging volume. To address this limitation, we characterized 3D field distortions arising from non-telecentric remote focusing and present a method for distortion precompensation. We demonstrate its applicability for a 3D two-photon microscope that uses an acousto-optic lens (AOL) for remote focusing and scanning. We show that the distortion precompensation method improves the pointing precision of the AOL microscope to < 0.5 µm throughout the 400 × 400 × 400 µm imaging volume

Dynamic wavefront shaping with an acousto-optic lens for laser scanning microscopy

Acousto-optic deflectors (AODs) arranged in series and driven with linearly chirped frequencies can rapidly focus and tilt optical wavefronts, enabling high-speed 3D random access microscopy. Non-linearly chirped acoustic drive frequencies can also be used to shape the optical wavefront allowing a range of higher-order aberrations to be generated. However, to date, wavefront shaping with AODs has been achieved by using single laser pulses for strobed illumination to 'freeze' the moving acoustic wavefront, limiting voxel acquisition rates. Here we show that dynamic wavefront shaping can be achieved by applying non-linear drive frequencies to a pair of AODs with counter-propagating acoustic waves, which comprise a cylindrical acousto-optic lens (AOL). Using a cylindrical AOL we demonstrate high-speed continuous axial line scanning and the first experimental AOL-based correction of a cylindrical lens aberration at 30 kHz, accurate to 1/35th of a wave at 800 nm. Furthermore, we develop a model to show how spherical aberration, which is the major aberration in AOL-based remote-focusing systems, can be partially or fully corrected with AOLs consisting of four or six AODs, respectively

Random-access scanning microscopy for 3D imaging in awake behaving animals

Understanding how neural circuits process information requires rapid measurements of activity from identified neurons distributed in 3D space. Here we describe an acousto-optic lens two-photon microscope that performs high-speed focusing and line scanning within a volume spanning hundreds of micrometers. We demonstrate its random-access functionality by selectively imaging cerebellar interneurons sparsely distributed in 3D space and by simultaneously recording from the soma, proximal and distal dendrites of neocortical pyramidal cells in awake behaving mice

Monitoring synaptic and neuronal activity in 3D with synthetic and genetic indicators using a compact acousto-optic lens two-photon microscope.

Two-photon microscopy is widely used to study brain function, but conventional microscopes are too slow to capture the timing of neuronal signalling and imaging is restricted to one plane. Recent development of acousto-optic-deflector-based random access functional imaging has improved the temporal resolution, but the utility of these technologies for mapping 3D synaptic activity patterns and their performance at the excitation wavelengths required to image genetically encoded indicators have not been investigated

Machine and human observable differences in groups’ collaborative problem-solving behaviours

This paper contributes to our understanding of how to design learning analytics to capture and analyse collaborative problem-solving (CPS) in practice-based learning activities. Most research in learning analytics focuses on student interaction in digital learning environments, yet still most learning and teaching in schools occurs in physical environments. Investigation of student interaction in physical environments can be used to generate observable differences among students, which can then be used in the design and implementation of Learning Analytics. Here, we present several original methods for identifying such differences in groups CPS behaviours. Our data set is based on human observation, hand position (fiducial marker) and heads direction (face recognition) data from eighteen students working in six groups of three. The results show that the high competent CPS groups spend an equal distribution of time on their problem-solving and collaboration stages. Whereas, the low competent CPS groups spend most of their time in identifying knowledge and skill deficiencies only. Moreover, as machine observable data shows, high competent CPS groups present symmetrical contributions to the physical tasks and present high synchrony and individual accountability values. The findings have significant implications on the design and implementation of future learning analytics systems

Beyond carbon and nitrogen: how the microbial energy economy couples elemental cycles in diverse ecosystems

Microbial metabolism couples elemental reactions, driving biogeochemical cycles. Assimilatory coupling of elemental cycles, such as the carbon (C), nitrogen (N), and phosphorus cycles, occurs when these elements are incorporated into biomass or released through its decomposition. In addition, many microbes are capable of dissimilatory coupling, catalyzing energy-releasing reactions linked to transformations in the oxidation state of elements, and releasing the transformed elements to the environment. Different inorganic elements provide varying amounts of energy yield, and the interaction of these processes creates a microbial energy economy. Dissimilatory reactions involving C, N, iron, and sulfur provide particularly important examples where microbially mediated oxidation–reduction (redox) transformations affect nutrient availability for net primary production, greenhouse-gas emissions, levels of contaminants and natural toxic factors, and other ecosystem dynamics. Recent discoveries of previously unrecognized microbial dissimilatory processes are leading to reevaluation of traditional perceptions of biogeochemical cycles

Bacterial variability in the mammalian gut captured by a single-cell synthetic oscillator

Funder: Harvard Medical School; doi: https://doi.org/10.13039/100006691Funder: Wyss Institute for Biologically Inspired EngineeringAbstract: Synthetic gene oscillators have the potential to control timed functions and periodic gene expression in engineered cells. Such oscillators have been refined in bacteria in vitro, however, these systems have lacked the robustness and precision necessary for applications in complex in vivo environments, such as the mammalian gut. Here, we demonstrate the implementation of a synthetic oscillator capable of keeping robust time in the mouse gut over periods of days. The oscillations provide a marker of bacterial growth at a single-cell level enabling quantification of bacterial dynamics in response to inflammation and underlying variations in the gut microbiota. Our work directly detects increased bacterial growth heterogeneity during disease and differences between spatial niches in the gut, demonstrating the deployment of a precise engineered genetic oscillator in real-life settings

A Large Web-Based Observer Reliability Study of Early Ischaemic Signs on Computed Tomography. The Acute Cerebral CT Evaluation of Stroke Study (ACCESS)

BACKGROUND: Early signs of ischaemic stroke on computerised tomography (CT) scanning are subtle but CT is the most widely available diagnostic test for stroke. Scoring methods that code for the extent of brain ischaemia may improve stroke diagnosis and quantification of the impact of ischaemia. METHODOLOGY AND PRINCIPAL FINDINGS: We showed CT scans from patients with acute ischaemic stroke (n = 32, with different patient characteristics and ischaemia signs) to doctors in stroke-related specialties world-wide over the web. CT scans were shown twice, randomly and blindly. Observers entered their scan readings, including early ischaemic signs by three scoring methods, into the web database. We compared observers' scorings to a reference standard neuroradiologist using area under receiver operator characteristic curve (AUC) analysis, Cronbach's alpha and logistic regression to determine the effect of scales, patient, scan and observer variables on detection of early ischaemic changes. Amongst 258 readers representing 33 nationalities and six specialties, the AUCs comparing readers with the reference standard detection of ischaemic signs were similar for all scales and both occasions. Being a neuroradiologist, slower scan reading, more pronounced ischaemic signs and later time to CT all improved detection of early ischaemic signs and agreement on the rating scales. Scan quality, stroke severity and number of years of training did not affect agreement. CONCLUSIONS: Large-scale observer reliability studies are possible using web-based tools and inform routine practice. Slower scan reading and use of CT infarct rating scales improve detection of acute ischaemic signs and should be encouraged to improve stroke diagnosis