PAAQ: Paired Alternating AcQuisitions for virtual high frame rate multichannel cardiac fluorescence microscopy.

In vivo fluorescence microscopy is a powerful tool to image the beating heart in its early development stages. A high acquisition frame rate is necessary to study its fast contractions, but the limited fluorescence intensity requires sensitive cameras that are often too slow. Moreover, the problem is even more complex when imaging distinct tissues in the same sample using different fluorophores.

Efficient compressed sensing reconstruction for 3D fluorescence microscopy using OptoMechanical Modulation Tomography (OMMT) with a 1+2D regularization.

OptoMechanical Modulation Tomography (OMMT) exploits compressed sensing to reconstruct high resolution microscopy volumes from fewer measurement images compared to exhaustive section sampling in conventional light sheet microscopy. Nevertheless, the volumetric reconstruction process is computationally expensive, making it impractically slow to use on large-size images, and prone to generating visual artefacts. Here, we propose a reconstruction approach that uses a 1+2D Total Variation (TV) regularization that does not generate such artefacts and is amenable to efficient implementation using parallel computing.

Aliasing mitigation in optical microscopy of dynamic biological samples by use of temporally modulated color illumination and a standard RGB camera.

Significance: Despite recent developments in microscopy, temporal aliasing can arise when imaging dynamic samples. Modern sampling frameworks, such as generalized sampling, mitigate aliasing but require measurement of temporally overlapping and potentially negative-valued inner products. Conventional cameras cannot collect these directly as they operate sequentially and are only sensitive to light intensity.

Spatially-Variant CNN-based Point Spread Function Estimation for Blind Deconvolution and Depth Estimation in Optical Microscopy.

Optical microscopy is an essential tool in biology and medicine. Imaging thin, yet non-flat objects in a single shot (without relying on more sophisticated sectioning setups) remains challenging as the shallow depth of field that comes with highresolution microscopes leads to unsharp image regions and makes depth localization and quantitative image interpretation difficult. Here, we present a method that improves the resolution of light microscopy images of such objects by locally estimating image distortion while jointly estimating object distance to the focal plane.

Temporal super-resolution microscopy using a hue-encoded shutter.

Limited time-resolution in microscopy is an obstacle to many biological studies. Despite recent advances in hardware, digital cameras have limited operation modes that constrain frame-rate, integration time, and color sensing patterns. In this paper, we propose an approach to extend the temporal resolution of a conventional digital color camera by leveraging a multi-color illumination source.

Direct inversion algorithm for focal plane scanning optical projection tomography.

To achieve approximately parallel projection geometry, traditional optical projection tomography (OPT) requires the use of low numerical aperture (NA) objectives, which have a long depth-of-field at the expense of poor lateral resolution. Particularly promising methods to improve spatial resolution include ad-hoc post-processing filters that limit the effect of the system's MTF and focal-plane-scanning OPT (FPS-OPT), an alternative acquisition procedure that allows the use of higher NA objectives by limiting the effect of their shallow depth of field yet still assumes parallel projection rays during reconstruction. Here, we provide a detailed derivation that establishes the existence of a direct inversion formula for FPS-OPT.

Development of the cardiac conduction system in zebrafish.

The cardiac conduction system (CCS) propagates and coordinates the electrical excitation that originates from the pacemaker cells, throughout the heart, resulting in rhythmic heartbeat. Its defects result in life-threatening arrhythmias and sudden cardiac death. Understanding of the factors involved in the formation and function of the CCS remains incomplete.

Dynamic structure and protein expression of the live embryonic heart captured by 2-photon light sheet microscopy and retrospective registration.

We present an imaging and image reconstruction pipeline that captures the dynamic three-dimensional beating motion of the live embryonic zebrafish heart at subcellular resolution. Live, intact zebrafish embryos were imaged using 2-photon light sheet microscopy, which offers deep and fast imaging at 70 frames per second, and the individual optical sections were assembled into a full 4D reconstruction of the beating heart using an optimized retrospective image registration algorithm. This imaging and reconstruction platform permitted us to visualize protein expression patterns at endogenous concentrations in zebrafish gene trap lines.

Developmental Alterations in Heart Biomechanics and Skeletal Muscle Function in Desmin Mutants Suggest an Early Pathological Root for Desminopathies.

Desminopathies belong to a family of muscle disorders called myofibrillar myopathies that are caused by Desmin mutations and lead to protein aggregates in muscle fibers. To date, the initial pathological steps of desminopathies and the impact of desmin aggregates in the genesis of the disease are unclear. Using live, high-resolution microscopy, we show that Desmin loss of function and Desmin aggregates promote skeletal muscle defects and alter heart biomechanics.

Endothelial cilia mediate low flow sensing during zebrafish vascular development.

The pattern of blood flow has long been thought to play a significant role in vascular morphogenesis, yet the flow-sensing mechanism that is involved at early embryonic stages, when flow forces are low, remains unclear. It has been proposed that endothelial cells use primary cilia to sense flow, but this has never been tested in vivo. Here we show, by noninvasive, high-resolution imaging of live zebrafish embryos, that endothelial cilia progressively deflect at the onset of blood flow and that the deflection angle correlates with calcium levels in endothelial cells.

High-resolution imaging of cardiomyocyte behavior reveals two distinct steps in ventricular trabeculation.

Over the course of development, the vertebrate heart undergoes a series of complex morphogenetic processes that transforms it from a simple myocardial epithelium to the complex 3D structure required for its function. One of these processes leads to the formation of trabeculae to optimize the internal structure of the ventricle for efficient conduction and contraction. Despite the important role of trabeculae in the development and physiology of the heart, little is known about their mechanism of formation.

Discretization of continuous convolution operators for accurate modeling of wave propagation in digital holography.

Discretization of continuous (analog) convolution operators by direct sampling of the convolution kernel and use of fast Fourier transforms is highly efficient. However, it assumes the input and output signals are band-limited, a condition rarely met in practice, where signals have finite support or abrupt edges and sampling is nonideal. Here, we propose to approximate signals in analog, shift-invariant function spaces, which do not need to be band-limited, resulting in discrete coefficients for which we derive discrete convolution kernels that accurately model the analog convolution operator while taking into account nonideal sampling devices (such as finite fill-factor cameras).

Pulse propagation by a capacitive mechanism drives embryonic blood flow.

Pulsatile flow is a universal feature of the blood circulatory system in vertebrates and can lead to diseases when abnormal. In the embryo, blood flow forces stimulate vessel remodeling and stem cell proliferation. At these early stages, when vessels lack muscle cells, the heart is valveless and the Reynolds number (Re) is low, few details are available regarding the mechanisms controlling pulses propagation in the developing vascular network.

4D reconstruction of the beating embryonic heart from two orthogonal sets of parallel optical coherence tomography slice-sequences.

Current methods to build dynamic optical coherence tomography (OCT) volumes of the beating embryonic heart involve synchronization of 2D+time slice-sequences acquired over separate heartbeats. Temporal registration of these sequences is performed either through gating or postprocessing. While synchronization algorithms that exclusively rely on image- intrinsic signals allow forgoing external gating hardware, they are prone to error accumulation, require operator-supervised correction, or lead to nonisotropic resolution.

Motion-based structure separation for label-free, high-speed, 3D cardiac microscopy.

Capturing the dynamics of individual structures in the embryonic heart is an essential step for studying its function and development. Label-free brightfield (BF) microscopy allows for higher acquisition frame-rates than techniques requiring molecular labeling, without interfering with embryo viability or needing complex equipment. However, since different structures contribute similarly to image contrast, label-free microscopy lacks specificity.

Sequential Turning Acquisition and Reconstruction (STAR) method for four-dimensional imaging of cyclically moving structures.

Optical coherence tomography allows for dynamic, three-dimensional (3D+T) imaging of the heart within animal embryos. However, direct 3D+T imaging frame rates remain insufficient for cardiodynamic analysis. Previously, this limitation has been addressed by reconstructing 3D+T representations of the beating heart based on sets of two-dimensional image sequences (2D+T) acquired sequentially at high frame rate and in fixed (and parallel) planes throughout the heart.

Increasing the field-of-view of dynamic cardiac OCT via post-acquisition mosaicing without affecting frame-rate or spatial resolution.

Optical coherence tomography (OCT) allows imaging dynamic structures and fluid flow within scattering tissue, such as the beating heart and blood flow in murine embryos. For any given system, the frame rate, spatial resolution, field-of-view (FOV), and signal-to-noise ratio (SNR) are interconnected: favoring one aspect limits at least one of the others due to optical, instrumentation, and software constraints. Here we describe a spatio-temporal mosaicing technique to reconstruct high-speed, high spatial-resolution, and large-field-of-view OCT sequences.

Characterization of HCN and cardiac function in a colonial ascidian.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate the rhythmic beating of mammalian hearts. We identified an HCN homolog in the colonial ascidian Botryllus schlosseri, a nonvertebrate chordate which possesses a tubular heart that beats bidirectionally. Contractions initiate at one end of the heart and travel across the length of the organ, and these periodically reverse, suggesting the presence of two pacemakers, one on each side.

High-speed multicolor microscopy of repeating dynamic processes.

Images of multiply labeled fluorescent samples provide unique insights into the localization of molecules, cells, and tissues. The ability to image multiple channels simultaneously at high speed without cross talk is limited to a few colors and requires dedicated multichannel or multispectral detection procedures. Simpler microscopes, in which each color is imaged sequentially, produce a much lower frame rate.

Stereotypical cell division orientation controls neural rod midline formation in zebrafish.

The development of multicellular organisms is dependent on the tight coordination between tissue growth and morphogenesis. The stereotypical orientation of cell divisions has been proposed to be a fundamental mechanism by which proliferating and growing tissues take shape. However, the actual contribution of stereotypical division orientation (SDO) to tissue morphogenesis is unclear.