Despite its importance for understanding human infertility and congenital diseases, early mammalian development has remained inaccessible to in toto imaging. We developed an inverted light-sheet microscope that enabled us to image mouse embryos from zygote to blastocyst, computationally track all cells and reconstruct a complete lineage tree of mouse pre-implantation development. We used this unique data set to show that the first cell fate specification occurs at the 16-cell stage.
Selective-plane illumination microscopy has proven to be a powerful imaging technique due to its unsurpassed acquisition speed and gentle optical sectioning. However, even in the case of multiview imaging techniques that illuminate and image the sample from multiple directions, light scattering inside tissues often severely impairs image contrast. Here we combine multiview light-sheet imaging with electronic confocal slit detection implemented on modern camera sensors. In addition to improved imaging quality, the electronic confocal slit detection doubles the acquisition speed in multiview setups with two opposing illumination directions allowing simultaneous dual-sided illumination. Confocal multiview light-sheet microscopy eliminates the need for specimen-specific data fusion algorithms, streamlines image post-processing, easing data handling and storage.
Light-field microscopy (LFM) has emerged as a powerful tool for fast volumetric image acquisition in biology, but its effective throughput and widespread use has been hampered by a computationally demanding and artefact-prone image reconstruction process. Here, we present a novel framework consisting of a hybrid light-field light-sheet microscope and deep learning-based volume reconstruction, where single light-sheet acquisitions continuously serve as training data and validation for the convolutional neural network reconstructing the LFM volume. Our network delivers high-quality reconstructions at video-rate throughput and we demonstrate the capabilities .
Morphology of aggregation intermediates, polymorphism of amyloid fibrils and aggregation kinetics of the “Arctic” mutant of the Alzheimer’s amyloid β-peptide, Aβ(1-40)(E22G), in a physiologically relevant TRIS buffer (pH 7.4) were thoroughly explored in comparison with the human wild type Alzheimer’s amyloid peptide, wt-Aβ(1-40), using both in situ atomic force and electron microscopy, circular dichroism and thioflavin T fluorescence assays. For arc-Aβ(1-40) at the end of the ‘lag’-period of fibrillization an abrupt appearance of ~3 nm size ‘spherical aggregates’ with a homogeneous morphology, was identified. Then, the aggregation proceeds with a rapid growth of amyloid fibrils with a variety of morphologies, while the spherical aggregates eventually disappeared during in situ measurements. Arc-Aβ(1-40) was also shown to form fibrils at much lower concentrations than wt-Aβ(1-40): ≤2.5 μM and 12.5 μM, respectively. Moreover, at the same concentration, 50 μM, the aggregation process proceeds more rapidly for arc-Aβ(1-40): The first amyloid fibrils were observed after ca 72 hours from the onset of incubation as compared to approximately 7 days for wt-Aβ(1-40). Amyloid fibrils of arc-Aβ(1-40) exhibit a large variety of polymorphs, at least five, both coiled and non-coiled distinct fibril structures were recognized by AFM, while at least four types of arc-Aβ(1-40) fibrils were identified by TEM and STEM and their mass-per-length statistics were collected suggesting supramolecular structures with two, four and six β-sheet laminae. Our results suggest a pathway of fibrillogenesis for full-length Alzheimer’s peptides with small and structurally ordered transient spherical aggregates as on-pathway immediate precursors of amyloid fibrils.
By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen's (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR) 2 ; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction 2 and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations 3. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.
We present a plane-scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light-sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent molecular state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS-RESOLFT nanoscopy offers wide-field 3D imaging of living biological specimens with low light dose and axial resolution far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5-12-fold compared with their conventional diffraction-limited LS analogs.ar-field nanoscopy (1, 2) methods discern features within subdiffraction distances by briefly forcing their molecules to two distinguishable states for the time period of detection. Typically, fluorophores are switched between a signaling "on" and a nonsignaling (i.e., dark) "off" state. Depending on the switching and fluorescence registration strategy used, these superresolution techniques can be categorized into coordinate-stochastic and coordinatetargeted approaches (2). The latter group of methods, comprising the so-called RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] (1, 3-7) approaches, have been realized using patterns of switch-off light with one or more zero-intensity points or lines, to single out target point (zero-dimensional) or line (1D) coordinates in space where the fluorophores are allowed to assume the on state. The RESOLFT idea can also be implemented in the inverse mode, by using switch-on light and confining the off state. In any case, probing the presence of molecules in new sets of points or lines at every scanning step produces images.Owing to the nature of the on and off states involved--first excited electronic and ground state--stimulated emission depletion (STED) (3) and saturated structured illumination microscopy (SSIM) (8), which both qualify as variants of the RESOLFT principle, typically apply light intensities in the range of MW/cm 2 and above. Especially when imaging sensitive samples where photoinduced changes must be avoided, RESOLFT is preferably realized with fluorophores which lead to the same factor of resolution improvement at much lower intensities of state-switching light. Reversibly switchable fluorescent proteins (RSFPs) are highly suitable for this purpose (4-7, 9), as transitions between their metastable on and off states require 5 orders of magnitude lower threshold intensities than STED/SSIM to guarantee switch-off. Suitable spectral properties, relatively fast millisecond switching kinetics, and high photostability of recently developed yellow-green-emitting RSFPs like rsEGFP (5), rsEGFP2 (7), and rsEGFP(N205S) (10) compared with early RSFPs have indeed enabled RESOLFT nanoscopy in living cells and tissues. To date, RSFP-based RESOLFT has achieved resolution improvements by facto...
Graphical Abstract Highlights d Neuronal processing by microglia depends on Slc37a2mediated phagosomal shrinkage d Phagosomes fuse with the gastrosome, a unique compartment in the phagocytic pathway d Loss of Slc37a2 blocks phagosomal shrinkage, resulting in gastrosomal expansion d Gastrosomal expansion affects microglia phagocytosis and migration toward injuries SUMMARYPhagocytic immune cells such as microglia can engulf and process pathogens and dying cells with high efficiency while still maintaining their dynamic behavior and morphology. Effective intracellular processing of ingested cells is likely to be crucial for microglial function, but the underlying cellular mechanisms are poorly understood. Using both living fish embryos and mammalian macrophages, we show that processing depends on the shrinkage and packaging of phagosomes into a unique cellular compartment, the gastrosome, with distinct molecular and ultra-structural characteristics. Loss of the transporter Slc37a2 blocks phagosomal shrinkage, resulting in the expansion of the gastrosome and the dramatic bloating of the cell. This, in turn, affects the ability of microglia to phagocytose and migrate toward brain injuries. Thus, this work identifies a conserved crucial step in the phagocytic pathway of immune cells and provides a potential entry point for manipulating their behavior in development and disease.
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