High‐Speed Large‐Field Multifocal Illumination Fluorescence Microscopy

Scanning optical microscopy techniques are commonly restricted to a sub‐millimeter field‐of‐view (FOV) or otherwise employ slow mechanical translation, limiting their applicability for imaging fast biological dynamics occurring over large areas. A rapid scanning large‐field multifocal illumination (LMI) fluorescence microscopy technique is devised based on a beam‐splitting grating and an acousto‐optic deflector synchronized with a high‐speed camera to attain real‐time fluorescence microscopy over a centimeter‐scale FOV. Owing to its large depth of focus, the approach allows noninvasive visualization of perfusion across the entire mouse cerebral cortex, not achievable with conventional wide‐field fluorescence microscopy methods. The new concept can readily be incorporated into conventional wide‐field microscopes to mitigate image blur due to tissue scattering and attain optimal trade‐off between spatial resolution and FOV. It further establishes a bridge between conventional wide‐field macroscopy and laser scanning confocal microscopy, thus it is anticipated to find broad applicability in functional neuroimaging, in vivo cell tracking, and other applications looking at large‐scale fluorescent‐based biodynamics

High-throughput screening of caterpillars as a platform to study host-microbe interactions and enteric immunity.

Mammalian models of human disease are expensive and subject to ethical restrictions. Here, we present an independent platform for high-throughput screening, using larvae of the tobacco hornworm Manduca sexta, combining diagnostic imaging modalities for a comprehensive characterization of aberrant phenotypes. For validation, we use bacterial/chemical-induced gut inflammation to generate a colitis-like phenotype and identify significant alterations in morphology, tissue properties, and intermediary metabolism, which aggravate with disease progression and can be rescued by antimicrobial treatment. In independent experiments, activation of the highly conserved NADPH oxidase DUOX, a key mediator of gut inflammation, leads to similar, dose-dependent alterations, which can be attenuated by pharmacological interventions. Furthermore, the developed platform could differentiate pathogens from mutualistic gastrointestinal bacteria broadening the scope of applications also to microbiomics and host-pathogen interactions. Overall, larvae-based screening can complement mammals in preclinical studies to explore innate immunity and host-pathogen interactions, thus representing a substantial contribution to improve mammalian welfare

Optoacoustic calcium imaging of deep brain activity in an intracardially perfused mouse brain model

One main limitation of established neuroimaging methods is the inability to directly visualize large-scale neural dynamics in whole mammalian brains at subsecond speeds. Optoacoustic imaging has advanced in recent years to provide unique advantages for real-time deep-tissue observations, which have been exploited for three-dimensional imaging of both cerebral hemodynamic parameters and direct calcium activity in rodents. Due to a lack of suitable calcium indicators excitable in the near-infrared window, optoacoustic imaging of neuronal activity at deep-seated areas of the mammalian brain has been impeded by the strong absorption of blood in the visible range of the light spectrum. To overcome this, we have developed and validated an intracardially perfused mouse brain preparation labelled with genetically encoded calcium indicator GCaMP6f that closely resembles in vivo conditions. By overcoming the limitations of hemoglobin-based light absorption, this new technique was used to observe stimulus-evoked calcium dynamics in the brain at penetration depths and spatio-temporal resolution scales not attainable with existing neuroimaging techniques

Monitoring of Stimulus Evoked Murine Somatosensory Cortex Hemodynamic Activity With Volumetric Multi-Spectral Optoacoustic Tomography

Sensory stimulation is an attractive paradigm for studying brain activity using various optical-, ultrasound- and MRI-based functional neuroimaging methods. Optoacoustics has been recently suggested as a powerful new tool for scalable mapping of multiple hemodynamic parameters with rich contrast and previously unachievable spatio-temporal resolution. Yet, its utility for studying the processing of peripheral inputs at the whole brain level has so far not been quantified. We employed volumetric multi-spectral optoacoustic tomography (vMSOT) to non-invasively monitor the HbO, HbR, and HbT dynamics across the mouse somatosensory cortex evoked by electrical paw stimuli. We show that elevated contralateral activation is preserved in the HbO map (invisible to MRI) under isoflurane anesthesia. Brain activation is shown to be predominantly confined to the somatosensory cortex, with strongest activation in the hindpaw region of the contralateral sensorimotor cortex. Furthermore, vMSOT detected the presence of an initial dip in the contralateral hindpaw region in the delta HbO channel. Sensorimotor cortical activity was identified over all other regions in HbT and HbO but not in HbR. Pearson’s correlation mapping enabled localizing the response to the sensorimotor cortex further highlighting the ability of vMSOT to bridge over imaging performance deficiencies of other functional neuroimaging modalities.ISSN:1662-453XISSN:1662-454

High-Speed Large-Field Multifocal Illumination Fluorescence Microscopy

Scanning optical microscopy techniques are commonly restricted to a sub‐millimeter field‐of‐view (FOV) or otherwise employ slow mechanical translation, limiting their applicability for imaging fast biological dynamics occurring over large areas. A rapid scanning large‐field multifocal illumination (LMI) fluorescence microscopy technique is devised based on a beam‐splitting grating and an acousto‐optic deflector synchronized with a high‐speed camera to attain real‐time fluorescence microscopy over a centimeter‐scale FOV. Owing to its large depth of focus, the approach allows noninvasive visualization of perfusion across the entire mouse cerebral cortex, not achievable with conventional wide‐field fluorescence microscopy methods. The new concept can readily be incorporated into conventional wide‐field microscopes to mitigate image blur due to tissue scattering and attain optimal trade‐off between spatial resolution and FOV. It further establishes a bridge between conventional wide‐field macroscopy and laser scanning confocal microscopy, thus it is anticipated to find broad applicability in functional neuroimaging, in vivo cell tracking, and other applications looking at large‐scale fluorescent‐based biodynamics.ISSN:1863-8880ISSN:1863-889

Isolated Murine Brain Model for Large-Scale Optoacoustic Calcium Imaging

Real-time visualization of large-scale neural dynamics in whole mammalian brains is hindered with existing neuroimaging methods having limited capacity when it comes to imaging large tissue volumes at high speeds. Optoacoustic imaging has been shown to be capable of real-time three-dimensional imaging of multiple cerebral hemodynamic parameters in rodents. However, optoacoustic imaging of calcium activity deep within the mammalian brain is hampered by strong blood absorption in the visible light spectrum as well as a lack of activity labels excitable in the near-infrared window. We have developed and validated an isolated whole mouse brain preparation labeled with genetically encoded calcium indicator GCaMP6f, which can closely resemble conditions. An optoacoustic imaging system coupled to a superfusion system was further designed and used for rapid volumetric monitoring of stimulus-evoked calcium dynamics in the brain. These new imaging setup and isolated preparation's protocols and characteristics are described here in detail. Our new technique captures calcium fluxes as true three-dimensional information across the entire brain with temporal resolution of 10 ms and spatial resolution of 150 μm, thus enabling large-scale neural recording at penetration depths and spatio-temporal resolution scales not covered with any existing neuroimaging techniques

Rapid volumetric optoacoustic imaging of neural dynamics across the mouse brain

Efforts to scale neuroimaging towards the direct visualization of mammalian brain-wide neuronal activity have faced major challenges. Although high-resolution optical imaging of the whole brain in small animals has been achieved ex vivo, the real-time and direct monitoring of large-scale neuronal activity remains difficult, owing to the performance gap between localized, largely invasive, optical microscopy of rapid, cellular-resolved neuronal activity and whole-brain macroscopy of slow haemodynamics and metabolism. Here, we demonstrate both ex vivo and non-invasive in vivo functional optoacoustic (OA) neuroimaging of mice expressing the genetically encoded calcium indicator GCaMP6f. The approach offers rapid, high-resolution three-dimensional snapshots of whole-brain neuronal activity maps using single OA excitations, and of stimulus-evoked slow haemodynamics and fast calcium activity in the presence of strong haemoglobin background absorption. By providing direct neuroimaging at depths and spatiotemporal resolutions superior to optical fluorescence imaging, functional OA neuroimaging bridges the gap between functional microscopy and whole-brain macroscopy

A genetically encoded near-infrared fluorescent calcium ion indicator

© 2019, The Author(s), under exclusive licence to Springer Nature America, Inc. We report an intensiometric, near-infrared fluorescent, genetically encoded calcium ion (Ca2+) indicator (GECI) with excitation and emission maxima at 678 and 704 nm, respectively. This GECI, designated NIR-GECO1, enables imaging of Ca2+ transients in cultured mammalian cells and brain tissue with sensitivity comparable to that of currently available visible-wavelength GECIs. We demonstrate that NIR-GECO1 opens up new vistas for multicolor Ca2+ imaging in combination with other optogenetic indicators and actuators

A genetically encoded near-infrared fluorescent calcium ion indicator

We report an intensiometric, near-infrared fluorescent, genetically encoded calcium ion (Ca) indicator (GECI) with excitation and emission maxima at 678 and 704 nm, respectively. This GECI, designated NIR-GECO1, enables imaging of Ca transients in cultured mammalian cells and brain tissue with sensitivity comparable to that of currently available visible-wavelength GECIs. We demonstrate that NIR-GECO1 opens up new vistas for multicolor Ca imaging in combination with other optogenetic indicators and actuators