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

Detection of cerebral tauopathy in P301L mice using high-resolution large-field multifocal illumination fluorescence microscopy

Current intravital microscopy techniques visualize tauopathy with high-resolution, but have a small field-of-view and depth-of-focus. Herein, we report a transcranial detection of tauopathy over the entire cortex of P301L tauopathy mice using large-field multifocal illumination (LMI) fluorescence microscopy technique and luminescent conjugated oligothiophenes. In vitro assays revealed that fluorescent ligand h-FTAA is optimal for in vivo tau imaging, which was confirmed by observing elevated probe retention in the cortex of P301L mice compared to non-transgenic littermates. Immunohistochemical staining further verified the specificity of h-FTAA to detect tauopathy in P301L mice. The new imaging platform can be leveraged in pre-clinical mechanistic studies of tau spreading and clearance as well as longitudinal monitoring of tau targeting therapeutics

Deep optoacoustic localization microangiography of ischemic stroke in mice

Super-resolution optoacoustic imaging of microvascular structures deep in mammalian tissues has so far been impeded by strong absorption from densely-packed red blood cells. Here we devised 5 µm biocompatible dichloromethane-based microdroplets exhibiting several orders of magnitude higher optical absorption than red blood cells at near-infrared wavelengths, thus enabling single-particle detection in vivo. We demonstrate non-invasive three-dimensional microangiography of the mouse brain beyond the acoustic diffraction limit (<20 µm resolution). Blood flow velocity quantification in microvascular networks and light fluence mapping was also accomplished. In mice affected by acute ischemic stroke, the multi-parametric multi-scale observations enabled by super-resolution and spectroscopic optoacoustic imaging revealed significant differences in microvascular density, flow and oxygen saturation in ipsi- and contra-lateral brain hemispheres. Given the sensitivity of optoacoustics to functional, metabolic and molecular events in living tissues, the new approach paves the way for non-invasive microscopic observations with unrivaled resolution, contrast and speed

Diffuse optical localization imaging for noninvasive deep brain microangiography in the NIR-II window

Fluorescence microscopy is a powerful enabling tool for biological discovery, albeit its effective penetration depth and resolving capacity are limited due to intense light scattering in living tissues. The recently introduced short-wave infrared cameras and contrast agents featuring fluorescence emission in the second near-infrared (NIR-II) window have extended the achievable penetration to about 2 mm. However, the effective spatial resolution progressively deteriorates with depth due to photon diffusion. Here we introduce diffuse optical localization imaging (DOLI) to enable super-resolution deep-tissue fluorescence microscopy beyond the limits imposed by light diffusion. The method is based on localization of flowing microdroplets encapsulating lead sulfide (PbS)-based quantum dots in a sequence of epi-fluorescence images acquired in the NIR-II spectral window. Experiments performed in tissue mimicking phantoms indicate that high-resolution detection of fluorescent particles can be preserved over 4 mm depth range, while in vivo microangiography of murine cerebral vasculature can be accomplished through intact scalp and skull. The method further enables retrieving depth information from planar fluorescence image recordings by exploiting the localized spot size. DOLI operates in a resolution-depth regime previously inaccessible with optical methods, thus massively enhancing the applicability of fluorescence-based imaging techniques

Widefield fluorescence localization microscopy for transcranial imaging of cortical perfusion with capillary resolution

Imaging of cerebral vasculature is impeded with the existing fluorescence microscopy methods due to intense light scattering in living tissues and the need for highly invasive craniotomy procedures to resolve structures on a capillary scale. We propose a widefield fluorescence localization microscopy technique for high-resolution transcranial imaging and quantitative assessment of cortical perfusion in mice. The method is based on tracking single fluorescent microparticles sparsely distributed in the blood stream using a simple CMOS camera and a continuous-wave laser source. We demonstrate quantitative transcranial in vivo mapping of the blood flow velocity and direction at capillary level resolution (5 µm) across the entire cortex. The new technique opens a new high-resolution transcranial window into the brain function in health and disease

Cortex‐wide microcirculation mapping with ultrafast large‐field multifocal illumination microscopy

The recently introduced large‐field multifocal illumination (LMI) fluorescence microscopy technique opened new possibilities for transcranial observations of mouse brain dynamics with a unique combination of capillary level resolution and centimeter‐scale field‐of‐view (FOV). Here we report on a new acceleration scheme for LMI based on raster scan of a lattice pattern combined with a parallel camera exposure scheme, which attains 200 Hz frame rate over 12 × 12 mm2 FOV with 7.5 μm spatial resolution. We demonstrate real‐time transcranial in vivo tracking of particles and imaging of microcirculation across the entire mouse cortex, thus corroborating the superb spatiotemporal resolution performance of LMI unattainable with other techniques. Potential applications include investigations into cerebrovascular function, cell tracking, as well as large‐scale functional neuroimaging

Cortex‐wide microcirculation mapping with ultrafast large‐field multifocal illumination microscopy

The recently introduced large‐field multifocal illumination (LMI) fluorescence microscopy technique opened new possibilities for transcranial observations of mouse brain dynamics with a unique combination of capillary level resolution and centimeter‐scale field‐of‐view (FOV). Here we report on a new acceleration scheme for LMI based on raster scan of a lattice pattern combined with a parallel camera exposure scheme, which attains 200 Hz frame rate over 12 × 12 mm2 FOV with 7.5 μm spatial resolution. We demonstrate real‐time transcranial in vivo tracking of particles and imaging of microcirculation across the entire mouse cortex, thus corroborating the superb spatiotemporal resolution performance of LMI unattainable with other techniques. Potential applications include investigations into cerebrovascular function, cell tracking, as well as large‐scale functional neuroimaging

A four-stage DEA-based efficiency evaluation of public hospitals in China after the implementation of new medical reforms.

This study applied the non-parametric four-stage data envelopment analysis method (Four-Stage DEA) to measure the relative efficiencies of Chinese public hospitals from 2010 to 2016, and to determine how efficiencies were affected by eight factors. A sample of public hospitals (n = 84) was selected from Chongqing, China, including general hospitals and traditional Chinese medicine hospitals graded level 2 or above. The Four-Stage-DEA method was chosen since it enables the control of the impact of environment factors on efficiency evaluation results. Data on the number of staff, government financial subsidies, the number of beds and fixed assets were used as input whereas the number of out-patients and emergency department patients and visits, the number of discharged patients, medical and health service income and hospital bed utilization rate were chosen as study outputs. As relevant environmental variables, we selected GDP per capita, permanent population, population density, number of hospitals and number of available sickbeds in local medical institutions. The relative efficiencies (i.e. technical, pure technical, scale) of sample hospitals were also calculated to analyze the change between the first stage and fourth stage every year. The study found that Four-Stage-DEA can effectively filter the impact of environmental factors on evaluation results, which sets it apart from other models commonly used in existing studies

Coupled responses of the flow-induced vibration and flow-induced rotation of a rigid cylinder-plate body

In this study, coupled responses of flow-induced vibration and rotation for an elastically mounted cylinder-plate body are numerically investigated at a low Reynolds number of 120. A wide vibrational reduced velocity range of Uy = 3–18 under four rotational reduced velocities Uθ = 5, 8, 12, and 18 are considered. The non-bifurcation responses, bifurcation only in rotation responses, and bifurcation in both vibration and rotation responses are identified. Typical vortex-induced vibration (VIV) responses are recognized when considering the passive rotations, different from the full interactions between VIV and galloping for the vibration-only case. As Uθ increases, the peak vibration amplitudes increase, the onset Uy of the lock-in region becomes larger, and the lock-in region is wider. The phase angles of displacements versus lift coefficients experience a jump from 0° to 180° in the lock-in region, and the larger the Uθ, the wider the Uy range of phase jump. Whether the instantaneous posture of the cylinder-plate body is streamlined or not is determined by oscillation amplitudes and phase differences between displacements versus rotation angles. Streamlined profiles can be achieved under small oscillation amplitudes or when the phase angles are nearly 90°. The 2S (two isolated vortices) vortex shedding mode dominates the initial and desynchronization branch, while the 2P (two pairs of vortices), 2S* (two isolated vortices with tendency to split), and 2T (two triplets of vortices) modes appear in the lock-in region. After the symmetry-breaking bifurcation, the reattachment behavior becomes simpler and the length of the recirculation region is significantly increased, as compared with those in non-bifurcation region. With the above study, a new method of improving energy harvesting from flow-induced vibration, by incorporating passive rotations simultaneously, is first introduced. It is found that passive rotations can enhance the vibration responses and thus lead to the increased output power and energy transfer ratio, although they make less contributions to the total power. Generally, this mechanical system presents a promising opportunity for energy harvesting through flow-induced vibration

Multifocal structured illumination optoacoustic microscopy

Optoacoustic (OA) imaging has the capacity to effectively bridge the gap between macroscopic and microscopic realms in biological imaging. High-resolution OA microscopy has so far been performed via point-by-point scanning with a focused laser beam, thus greatly restricting the achievable imaging speed and/or field of view. Herein we introduce multifocal structured illumination OA microscopy (MSIOAM) that attains real-time 3D imaging speeds. For this purpose, the excitation laser beam is shaped to a grid of focused spots at the tissue surface by means of a beamsplitting diffraction grating and a condenser and is then scanned with an acousto-optic deflector operating at kHz rates. In both phantom and in vivo mouse experiments, a 10 mm wide volumetric field of view was imaged with 15 Hz frame rate at 28 μm spatial resolution. The proposed method is expected to greatly aid in biological investigations of dynamic functional, kinetic, and metabolic processes across multiple scales.ISSN:2047-753