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Probe-based confocal endomicroscopy provides real time videos of autoflourescent elastin structures within the alveoli. With it, multiple changes in the elastin structure due to different diffuse parenchymal lung diseases have previously been described. However, these evaluations have mainly relied on qualitative evaluation by the examiner and manually selected parts post-examination. To develop a fully automatic method for quantifying structural properties of the imaged alveoli elastin and to perform a preliminary assessment of their diagnostic potential. 46 patients underwent probe-based confocal endomicroscopy, of which 38 were divided into 4 groups categorizing different diffuse parenchymal lung diseases. 8 patients were imaged in representative healthy lung areas and used as control group. Alveolar elastin structures were automatically segmented with a trained machine learning algorithm and subsequently evaluated with two methods developed for quantifying the local thickness and structural connectivity. The automatic segmentation algorithm performed generally well and all 4 patient groups showed statistically significant differences with median elastin thickness, standard deviation of thickness and connectivity compared to the control group. Alveoli elastin structures can be quantified based on their structural connectivity and thickness statistics with a fully-automated algorithm and initial results highlight its potential for distinguishing parenchymal lung diseases from normal alveoli.

Super-resolved structured illumination microscopy (SR-SIM) is among the fastest fluorescence microscopy techniques capable of surpassing the optical diffraction limit. Current custom-build instruments are able to deliver two-fold resolution enhancement with high acquisition speed. SR-SIM is usually a two-step process, with raw-data acquisition and subsequent, time-consuming post-processing for image reconstruction. In contrast, wide-field and (multi-spot) confocal techniques produce high-resolution images instantly. Such immediacy is also possible with SR-SIM, by tight integration of a video-rate capable SIM with fast reconstruction software. Here we present instant SR-SIM by VIGOR (Video-rate Immediate GPU-accelerated Open-Source Reconstruction). We demonstrate multi-color SR-SIM at video frame-rates, with less than 250 ms delay between measurement and reconstructed image display. This is achieved by modifying and extending high-speed SR-SIM image acquisition with a new, GPU-enhanced, network-enabled image-reconstruction software. We demonstrate high-speed surveying of biological samples in multiple colors and live imaging of moving mitochondria as an example of intracellular dynamics. Sequential acquisition and image reconstruction in super-resolved structured illumination microscopy (SR-SIM) is time-consuming. Here the authors optimise both acquisition and reconstruction software to achieve multicolour SR-SIM at video frame-rates with reconstructed images displaying with only milliseconds delay during the experiment.

The use of a spatial light modulator for illuminating the sample in structured-illumination microscopy (SIM) increases imaging speed by three orders of magnitude. The resulting 100-nm resolution and 11-Hz frame rate allowed video imaging of tubulin polymerization and depolymerization as well as kinesin movement on microtubules. Structured-illumination microscopy can double the resolution of the widefield fluorescence microscope but has previously been too slow for dynamic live imaging. Here we demonstrate a high-speed structured-illumination microscope that is capable of 100-nm resolution at frame rates up to 11 Hz for several hundred time points. We demonstrate the microscope by video imaging of tubulin and kinesin dynamics in living Drosophila melanogaster S2 cells in the total internal reflection mode.

A series of technical and analytical improvements to light sheet microscopy is described, permitting dynamic imaging of the beating zebrafish heart at cellular resolution. The heart′s continuous motion makes it difficult to capture high-resolution images of this organ in vivo . We developed tools based on high-speed selective plane illumination microscopy (SPIM), offering pristine views into the beating zebrafish heart. We captured three-dimensional cardiac dynamics with postacquisition synchronization of multiview movie stacks, obtained static high-resolution reconstructions by briefly stopping the heart with optogenetics and resolved nonperiodic phenomena by high-speed volume scanning with a liquid lens.

High-resolution, three-dimensional protein structures can be solved using MicroED, an electron diffraction method that uses three-dimensional microcrystals. An improved MicroED data collection approach described here increases data quality and resolution and extends its broad applicability. MicroED uses very small three-dimensional protein crystals and electron diffraction for structure determination. We present an improved data collection protocol for MicroED called 'continuous rotation'. Microcrystals are continuously rotated during data collection, yielding more accurate data. The method enables data processing with the crystallographic software tool MOSFLM, which resulted in improved resolution for the model protein lysozyme. These improvements are paving the way for the broad implementation and application of MicroED in structural biology.

Optical microscopy is one of the most widely used diagnostic methods in scientific, industrial, and biomedical applications. However, while useful for detailed examination of a small number (< 10,000) of microscopic entities, conventional optical microscopy is incapable of statistically relevant screening of large populations (> 100,000,000) with high precision due to its low throughput and limited digital memory size. We present an automated flow-through single-particle optical microscope that overcomes this limitation by performing sensitive blur-free image acquisition and nonstop real-time image-recording and classification of microparticles during high-speed flow. This is made possible by integrating ultrafast optical imaging technology, self-focusing microfluidic technology, optoelectronic communication technology, and information technology. To show the system’s utility, we demonstrate high-throughput image-based screening of budding yeast and rare breast cancer cells in blood with an unprecedented throughput of 100,000 particles/s and a record false positive rate of one in a million.

Since the 1980s, semi-automated sperm motility analysis of phase contrast microscopy videos has been used to measure and categorize sperm motility patterns. Motility categories are determined from various kinematic parameters such as Curvilinear Velocity (VCL) and Beat Cross Frequency (BCF). These measures ultimately rely on the quality of the tracking for each individual sperm in the microscopy video. However, common approaches to sperm tracking require sample dilution and shortening the time window of observation (less than 1 to 2 seconds) to avoid tracking errors that occur when sperm cross paths. The post-ejaculatory lifespan of sperm can exceed several hours to days in some species, and long-term adaptive changes in motility pattern may be an important distinguishing factor for predictive modeling of sperm fertilizing competence. Improving the predictive value of computer assisted semen analysis will require accurate tracking of sperm trajectories over physiologically-relevant time scales and at the high cell densities typically found in semen. In this work, we identify a framework for accurately assessing the quality of sperm trajectory tracking that is independent of standard motility measures. We utilize cell tracking metrics adapted from the more common task of tracking adherent somatic cells and propose modifications based on the unique challenges of sperm video-microscopy. We also provide a small dataset of microscopy videos that includes 340 labeled sperm trajectories to allow for future comparisons and developments. Finally, we demonstrate that variations in configuration can lead to as much as a 30% improvement on metrics, showcasing their effectiveness at analyzing tracking quality.

The ability of microbial active motion, morphology, and optical properties to serve as biosignatures was investigated by in situ video microscopy in a wide range of extreme field sites where such imaging had not been performed previously. These sites allowed for sampling seawater, sea ice brines, cryopeg brines, hypersaline pools and seeps, hyperalkaline springs, and glaciovolcanic cave ice. In all samples except the cryopeg brine, active motion was observed without any sample treatment. Active motion was observed in the cryopeg brines when samples were subjected to a temperature gradient above in situ . In general, levels of motility were low in the field samples collected at temperatures <  4ºC. Non-motile cells could be distinguished from microminerals by differences in passive motion (e.g., density measured by sinking/floating), refractive index and/or absorbance, or morphology in the case of larger eukaryotes. Dramatic increases in the fraction of motile cells were seen with simple stimuli such as warming or the addition of L-serine. Chemotaxis and thermotaxis were also observed in select samples. An open-source, autonomous software package with computational requirements that can be scaled to spaceflight computers was used to classify the data. These results demonstrate the utility of volumetric light microscopy for life detection, but also suggest the importance of developing methods to stimulate cells in situ and process data using the restrictions imposed by mission bandwidth, as well as instruments to capture cell-like objects for detailed chemical analysis.

Various noninvasive microscopic camera technologies have been used to visualize the sublingual microcirculation in patients. We describe a comprehensive approach to bedside in vivo sublingual microcirculation video image capture and analysis techniques in the human clinical setting. We present a user perspective and guide suitable for clinical researchers and developers interested in the capture and analysis of sublingual microcirculatory flow videos. We review basic differences in the cameras, optics, light sources, operation, and digital image capture. We describe common techniques for image acquisition and discuss aspects of video data management, including data transfer, metadata, and database design and utilization to facilitate the image analysis pipeline. We outline image analysis techniques and reporting including video preprocessing and image quality evaluation. Finally, we propose a framework for future directions in the field of microcirculatory flow videomicroscopy acquisition and analysis. Although automated scoring systems have not been sufficiently robust for widespread clinical or research use to date, we discuss promising innovations that are driving new development.

It is well recognized that isolated cardiac muscle cells beat in a periodic manner. Recently, evidence indicates that other, non-muscle cells, also perform periodic motions that are either imperceptible under conventional lab microscope lens or practically not easily amenable for analysis of oscillation amplitude, frequency, phase of movement and its direction. Here, we create a real-time video analysis tool to visually magnify and explore sub-micron rhythmic movements performed by biological cells and the induced movements in their surroundings. Using this tool, we suggest that fibroblast cells perform small fluctuating movements with a dominant frequency that is dependent on their surrounding substrate and its stiffness.