We present kilohertz-scale video capture rates in a transmission electron microscope, using a camera normally limited to hertz-scale acquisition. An electrostatic deflector rasters a discrete array of images over a large camera, decoupling the acquisition time per subframe from the camera readout time. Total-variation regularization allows features in overlapping subframes to be correctly placed in each frame. Moreover, the system can be operated in a compressive-sensing video mode, whereby the deflections are performed in a known pseudorandom sequence. Compressive sensing in effect performs data compression before the readout, such that the video resulting from the reconstruction can have substantially more total pixels than that were read from the camera. This allows, for example, 100 frames of video to be encoded and reconstructed using only 15 captured subframes in a single camera exposure. We demonstrate experimental tests including laser-driven melting/dewetting, sintering, and grain coarsening of nanostructured gold, with reconstructed video rates up to 10 kHz. The results exemplify the power of the technique by showing that it can be used to study the fundamentally different temporal behavior for the three different physical processes. Both sintering and coarsening exhibited self-limiting behavior, whereby the process essentially stopped even while the heating laser continued to strike the material. We attribute this to changes in laser absorption and to processes inherent to thin-film coarsening. In contrast, the dewetting proceeded at a relatively uniform rate after an initial incubation time consistent with the establishment of a steady-state temperature profile.

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.

Total holographic characterization (THC) is presented here as an efficient, automated, label-free method of accurately identifying cell viability. THC is a single-particle characterization technology that determines the size and index of refraction of individual particles using the Lorenz-Mie theory of light scattering. Although assessment of cell viability is a challenge in many applications, including biologics manufacturing, traditional approaches often include unreliable labeling with dyes and/or time consuming methods of manually counting cells. In this work we measured the viability of Saccharomyces cerevisiae yeast in the presence of various concentrations of isopropanol as a function of time. All THC measurements were performed in the native environment of the sample with no dilution or addition of labels. Holographic measurements were made with an in-line holographic microscope using a 40[Formula: see text] objective lens with plane wave illumination. We compared our results with THC to manual counting of living and dead cells as distinguished with trypan blue dye. Our findings demonstrate that THC can effectively distinguish living and dead yeast cells by the index of refraction of individual cells.

The heart of the fruit fly, Drosophila melanogaster, is a particularly suitable model for cardiac studies. Optical coherence microscopy (OCM) captures in vivo cross-sectional videos of the beating Drosophila heart for cardiac function quantification. To analyze those large-size multi-frame OCM recordings, human labelling has been employed, leading to low efficiency and poor reproducibility. Here, we introduce a robust and accurate automated Drosophila heart segmentation algorithm, called FlyNet 2.0+, which utilizes a long short-term memory (LSTM) convolutional neural network to leverage time series information in the videos, ensuring consistent, high-quality segmentation. We present a dataset of 213 Drosophila heart videos, equivalent to 604,000 cross-sectional images, containing all developmental stages and a wide range of beating patterns, including faster and slower than normal beating, arrhythmic beating, and periods of heart stop to capture these heart dynamics. Each video contains a corresponding ground truth mask. We expect this unique large dataset of the beating Drosophila heart in vivo will enable new deep learning approaches to efficiently characterize heart function to advance cardiac research.

Visualisation of neurons labeled with fluorescent proteins or compounds generally require exposure to intense light for a relatively long period of time, often leading to bleaching of the fluorescent probe and photodamage of the tissue. Here we created a technique to drastically shorten light exposure and improve the targeting of fluorescent labeled cells that is specially useful for patch-clamp recordings. We applied image tracking and mask overlay to reduce the time of fluorescence exposure and minimise mistakes when identifying neurons.

Inhalation of mineral fibres is associated with the onset of an inflammatory activity in the lungs and the pleura responsible for the development of fatal malignancies. It is known that cell damage is a necessary step for triggering the inflammatory response. However, the mechanisms by which mineral fibres exert cytotoxic activity are not fully understood. In this work, the kinetics of the early cytotoxicity mechanisms of three mineral fibres (i.e., chrysotile, crocidolite and fibrous erionite) classified as carcinogenic by the International Agency for Research on Cancer, was determined for the first time in a comparative manner using time-lapse video microscopy coupled with in vitro assays. All tests were performed using the THP-1 cell line, differentiated into M0 macrophages (M0-THP-1) and exposed for short times (8 h) to 25 μg/mL aliquots of chrysotile, crocidolite and fibrous erionite. The toxic action of fibrous erionite on M0-THP-1 cells is manifested since the early steps (2 h) of the experiment while the cytotoxicity of crocidolite and chrysotile gradually increases during the time span of the experiment. Chrysotile and crocidolite prompt cell death mainly via apoptosis, while erionite exposure is also probably associated to a necrotic-like effect. The potential mechanisms underlying these different toxicity behaviours are discussed in the light of the different morphological, and chemical-physical properties of the three fibres.

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.

A simple method is described to extend image exposure times in video-rate CCD cameras and thereby, increase their sensitivity and reduce noise level of low-light images. Most commercial video cameras lack the capability of extending image exposures since they operate regular television timing formats. The technique described here implements the control of the exposure times by selectively gating the image readout from the CCD sensor. This prevents the cyclic clearing of photo-charges occurring at regular video-rates, allowing image integration beyond the duration of single video field periods. Image readout is controlled by the duration of external gating pulses, giving the camera an efficient operational versatility under different light conditions. This technique is applicable to standard monochrome and color CCD cameras. The evaluations described here using this technique show that the light sensitivity of an standard video-rate CCD camera can be significantly improved, generating high quality images at low-light levels. These were comparable to those obtained with image intensifiers or intensified video cameras. Cameras are still compatible with regular video equipment, since this technique preserves the normal TV synchronization signals. Results in simulated and real experimental situations confirmed that this technique enables the use of affordable video-rate CCD cameras for a variety of fluorescence microscopy and optical recording applications.

Intravital microscopy has proven to be a powerful tool for studying microvascular physiology. In this study, we propose a gas exchange system compatible with intravital microscopy that can be used to impose gas perturbations to small localized regions in skeletal muscles or other tissues that can be imaged using conventional inverted microscopes. We demonstrated the effectiveness of this system by locally manipulating oxygen concentrations in rat extensor digitorum longus muscle and measuring the resulting vascular responses. A computational model of oxygen transport was used to partially validate the localization of oxygen changes in the tissue, and oxygen saturation of red blood cells flowing through capillaries were measured as a surrogate for local tissue oxygenation. Overall, we have demonstrated that this approach can be used to study dynamic and spatial responses to local oxygen challenges to the microenvironment of skeletal muscle.

Quantification of basic cell functions is a preliminary step to understand complex cellular mechanisms, for e.g., to test compatibility of biomaterials, to assess the effectiveness of drugs and siRNAs, and to control cell behavior. However, commonly used quantification methods are label-dependent, and end-point assays. As an alternative, using our lensfree video microscopy platform to perform high-throughput real-time monitoring of cell culture, we introduce specifically devised metrics that are capable of non-invasive quantification of cell functions such as cell-substrate adhesion, cell spreading, cell division, cell division orientation and cell death. Unlike existing methods, our platform and associated metrics embrace entire population of thousands of cells whilst monitoring the fate of every single cell within the population. This results in a high content description of cell functions that typically contains 25,000 - 900,000 measurements per experiment depending on cell density and period of observation. As proof of concept, we monitored cell-substrate adhesion and spreading kinetics of human Mesenchymal Stem Cells (hMSCs) and primary human fibroblasts, we determined the cell division orientation of hMSCs, and we observed the effect of transfection of siCellDeath (siRNA known to induce cell death) on hMSCs and human Osteo Sarcoma (U2OS) Cells.

Interrogation of subcellular biological dynamics occurring in a living cell often requires noninvasive imaging of the fragile cell with high spatiotemporal resolution across all three dimensions. It thereby poses big challenges to modern fluorescence microscopy implementations because the limited photon budget in a live-cell imaging task makes the achievable performance of conventional microscopy approaches compromise between their spatial resolution, volumetric imaging speed, and phototoxicity. Here, we incorporate a two-stage view-channel-depth (VCD) deep-learning reconstruction strategy with a Fourier light-field microscope based on diffractive optical element to realize fast 3D super-resolution reconstructions of intracellular dynamics from single diffraction-limited 2D light-filed measurements. This VCD-enabled Fourier light-filed imaging approach (F-VCD), achieves video-rate (50 volumes per second) 3D imaging of intracellular dynamics at a high spatiotemporal resolution of ~180 nm × 180 nm × 400 nm and strong noise-resistant capability, with which light field images with a signal-to-noise ratio (SNR) down to -1.62 dB could be well reconstructed. With this approach, we successfully demonstrate the 4D imaging of intracellular organelle dynamics, e.g., mitochondria fission and fusion, with ~5000 times of observation.

Based on confocal fluorescence and bright field video microscopy, we present detailed observations on the processes of invasion and egress of erythrocytes by the apicomplexan parasite Babesia divergens. Time-lapse images reveal numerous unexpected findings associated with the dynamics of B. divergens and its ability to manipulate the erythrocyte during both processes in its asexual cycle under in vitro conditions. Despite the speed at which these processes occur and the small size of the parasite, we capture infective merozoites moving vigorously and causing striking deformations in the erythrocyte's plasma membrane during an active invasion. We also observed intraerythrocytic dynamic stages as paired pyriforms, double paired pyriforms, tetrads, unattached pyriform sister cells and multiple parasite stages resulting in the release of large numbers of merozoites over a short period. Of considerable interest is that time-lapse images reveal a novel mechanism of egress used by B. divergens to exit the human erythrocyte. The release occurs when B. divergens parasites establish contacts with the plasma membrane of the erythrocyte from within, before exiting the cell. Visualization and analysis of the images enabled us to obtain useful information and broaden our knowledge of complex and crucial events involved with parasitisation of human erythrocytes by B. divergens.

Microvascular blood flow is crucial for tissue and organ function and is often severely affected by diseases. Therefore, investigating the microvasculature under different pathological circumstances is essential to understand the role of the microcirculation in health and sickness. Microvascular blood flow is generally investigated with Intravital Video Microscopy (IVM), and the captured images are stored on a computer for later off-line analysis. The analysis of these images is a manual and challenging process, evaluating experiments very time consuming and susceptible to human error. Since more advanced digital cameras are used in IVM, the experimental data volume will also increase significantly. This study presents a new two-step image processing algorithm that uses a trained Convolutional Neural Network (CNN) to functionally analyze IVM microscopic images without the need for manual analysis. While the first step uses a modified vessel segmentation algorithm to extract the location of vessel-like structures, the second step uses a 3D-CNN to assess whether the vessel-like structures have blood flowing in it or not. We demonstrate that our two-step algorithm can efficiently analyze IVM image data with high accuracy (83%). To our knowledge, this is the first application of machine learning for the functional analysis of microvascular blood flow in vivo.

Early embryonic development involves complex events such as the regulation of cell division and the establishment of embryonic polarity. To identify genes involved in these events, we collected four-dimensional time-lapse video recordings of the first three cell divisions and analysed terminal phenotypes after RNA interference of 147 embryonic lethal genes previously identified in a systematic screen of Caenorhabditis elegans chromosome I. Over half gave defects in early processes such as meiosis, the assembly or position of the first mitotic spindle, cytokinesis, and proper nuclear positioning. For some phenotypic classes, the majority of genes are involved in a shared biochemical process. In addition, we identified loss-of-function phenotypes for genes of unknown function, but for which homologues exist in other organisms, shedding light on the function of these uncharacterized genes. When applied to the whole genome, this approach should identify the vast majority of genes required for early cell processes, paving the way for a greatly improved understanding of these processes and their regulation at the molecular level.

The aim of this study was to definitively assess the validity of noninvasive high-frequency ultrasound (US) measurements of aortic luminal diameter (ALD) in a murine model of elastase-induced abdominal aortic aneurysm in comparison with in situ video microscopy (VM).

We developed an automated, non-invasive method to detect real-time cardiac contraction in post-larval (1.1-1.7 mm length), juvenile oysters (i.e., oyster spat) via a fiber-optic trans-illumination system. The system is housed within a temperature-controlled chamber and video microscopy imaging of the heart was coupled with video edge-detection to measure cardiac contraction, inter-beat interval, and heart rate (HR). We used the method to address the hypothesis that cool acclimation (10°C vs. 22°C-Ta10 or Ta22, respectively; each n = 8) would preserve cardiac phenotype (assessed via HR variability, HRV analysis and maintained cardiac activity) during acute temperature changes. The temperature ramp (TR) protocol comprised 2°C steps (10 min/experimental temperature, Texp) from 22°C to 10°C to 22°C. HR was related to Texp in both acclimation groups. Spat became asystolic at low temperatures, particularly Ta22 spat (Ta22: 8/8 vs. Ta10: 3/8 asystolic at Texp = 10°C). The rate of HR decrease during cooling was less in Ta10 vs. Ta22 spat when asystole was included in analysis (P = 0.026). Time-domain HRV was inversely related to temperature and elevated in Ta10 vs. Ta22 spat (P < 0.001), whereas a lack of defined peaks in spectral density precluded frequency-domain analysis. Application of the method during an acute cooling challenge revealed that cool temperature acclimation preserved active cardiac contraction in oyster spat and increased time-domain HRV responses, whereas warm acclimation enhanced asystole. These physiologic changes highlight the need for studies of mechanisms, and have translational potential for oyster aquaculture practices.

Motile cilia are found in numerous locations throughout our body and play a critical role in various physiological processes. The most commonly used method to assess cilia motility is to quantify cilia beat frequency (CBF) via video microscopy. However, a large heterogeneity exists within published literature regarding the framerate used to image cilia motility for calculating CBF. The aim of this study was to determine the optimal frame rate required to image cilia motility for CBF assessment, and if the Nyquist theorem may be used to set this rate. One-second movies of cilia were collected at >600 fps from mouse airways and ependyma at room-temperature or 37°C. Movies were then down-sampled to 30-300 fps. CBF was quantified for identical cilia at different framerates by either manual counting or automated MATLAB script. Airway CBF was significantly impaired in 30 fps movies, while ependymal CBF was significantly impaired in both 60 and 30 fps movies. Pairwise comparison showed that video framerate should be at least 150 fps to accurately measure CBF, with minimal improvement in CBF accuracy in movies >150 fps. The automated script was also found to be less accurate for measuring CBF in lower fps movies than manual counting, however, this difference disappeared in higher framerate movies (>150 fps). In conclusion, our data suggest the Nyquist theorem is unreliable for setting sampling rate for CBF measurement. Instead, sampling rate should be 3-4 times faster than CBF for accurate CBF assessment. Especially if CBF calculation is to be automated.

Assessment of the microcirculation is a promising target for the hemodynamic management of critically ill patients. However, just as the sole reliance on macrocirculatory parameters, single static parameters of the microcirculation may not represent a sufficient guide. Our hypothesis was that by serial topical application of acetylcholine (ACH) and nitroglycerin (NG), the sublingual microcirculation can be challenged to determine its endothelial cell-dependent and smooth muscle-dependent physiological reserve capacity.

Cell motility is a critical parameter in many physiological as well as pathophysiological processes. In time-lapse video microscopy, manual cell tracking remains the most common method of analyzing migratory behavior of cell populations. In addition to being labor-intensive, this method is susceptible to user-dependent errors regarding the selection of "representative" subsets of cells and manual determination of precise cell positions.

We evaluate a novel set-up for scanning functional connectivity in brain slices from the somatosensory cortex of the rat. Upright infrared video microscopy for targeted placement of electrodes is combined with rapid photolysis of bath-applied caged neurotransmitter induced by a xenon flash lamp. Flash photolysis of caged glutamate and electrical stimulation produce comparable field potential responses and demonstrate that the viability of the submerged slices exceeds several hours. Glutamate release leads to field potential responses whose two phases are differentially affected by selective blockade of N-methyl-D-aspartate- and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-type glutamate receptors with DL-2-amino-5-phosphonovaleric acid and 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulphonamide, respectively. Rapid computer-controlled scanning of hundreds of distinct stimulation sites with simultaneous recordings at a fixed reference site allows construction of functional input maps from peak amplitudes and delays to peak of field potential responses. Selective laminar expansion of the functional input maps after bicuculline application demonstrates that the combination of this conveniently assembled set-up with pharmacological and physical manipulations can provide insights into the determinants of functional connectivity in brain slices.