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Atomic and Molecular Physics, and Optics and Electronic, Optical and Magnetic Materials

Multifocus microscopy enables recording of entire volumes in a single camera exposure. In dense samples, multifocus microscopy is severely hampered by background haze. Here, we introduce a scalable multifocus method that incorporates optical sectioning and offers improved axial resolution capabilities. In our method, a dithered oblique light-sheet scans the sample volume during a single exposure, while fluorescence from each illuminated plane in the sample is mapped onto a line on the camera with a multifocus optical element. A synchronized rolling shutter readout realizes optical sectioning. We describe the technique theoretically and verify its optical sectioning and resolution improvement capabilities. We demonstrate a prototype system with a multifocus beam splitter cascade and record monolayers of endothelial cells at 35 volumes per second. We furthermore image uncleared engineered human heart tissue and visualize the distribution of mitochondria at high axial resolution. Our method manages to capture sub-diffraction sized mitochondria-derived vesicles up to 30 µm deep into the tissue.

Image quality, Animal Scales, 02 engineering and technology, 01 natural sciences, Fluorescence, 010309 optics, Optics, Salmon, 0103 physical sciences, Microscopy, Fluorescence microscope, Animals, Lighting, Common emitter, Physics, Total internal reflection, Photons, Total internal reflection fluorescence microscope, Super-resolution microscopy, business.industry, Optical Imaging, 021001 nanoscience & nanotechnology, Atomic and Molecular Physics, and Optics, VDP::Mathematics and natural science: 400::Physics: 430::Electromagnetism, acoustics, optics: 434, Microscopy, Fluorescence, VDP::Matematikk og Naturvitenskap: 400::Fysikk: 430::Elektromagnetisme, akustikk, optikk: 434, Epidermis, 0210 nano-technology, business, and Refractive index

Photonic chip-based total internal reflection fluorescence microscopy (c-TIRFM) is an emerging technology enabling a large TIRF excitation area decoupled from the detection objective. Additionally, due to the inherent multimodal nature of wide waveguides, it is a convenient platform for introducing temporal fluctuations in the illumination pattern. The fluorescence fluctuation-based nanoscopy technique multiple signal classification algorithm (MUSICAL) does not assume stochastic independence of the emitter emission and can therefore exploit fluctuations arising from other sources, as such multimodal illumination patterns. In this work, we demonstrate and verify the utilization of fluctuations in the illumination for super-resolution imaging using MUSICAL on actin in salmon keratocytes. The resolution improvement was measured to be 2.2–3.6-fold compared to the corresponding conventional images.

Electrical and Electronic Engineering, Atomic and Molecular Physics, and Optics, Electronic, Optical and Magnetic Materials, and Biotechnology

symbols.namesake, Fourier transform, business.industry, Deep learning, Microscopy, Independent parameter, symbols, Frequency space, Artificial intelligence, business, Algorithm, Sample (graphics), and Mathematics

Parameter estimation is crucial for phase retrieval in Fourier ptychographic microscopy. We cast this task as object detection problem in frequency space, thus permitting the use of a neural network that performs with unprecedented accuracy.

Materials science, Microscope, Cell, General Physics and Astronomy, 02 engineering and technology, 01 natural sciences, Retinal ganglion, General Biochemistry, Genetics and Molecular Biology, law.invention, 010309 optics, 03 medical and health sciences, Optics, law, 0103 physical sciences, Microscopy, medicine, Animals, General Materials Science, 030304 developmental biology, VDP::Mathematics and natural science: 400, Neurons, 0303 health sciences, High contrast, Photons, Total internal reflection fluorescence microscope, business.industry, 010401 analytical chemistry, General Engineering, General Chemistry, VDP::Matematikk og Naturvitenskap: 400, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Rats, medicine.anatomical_structure, Microscopy, Fluorescence, Photonics, 0210 nano-technology, business, and Waveguide

Large fields of view (FOVs) in total internal reflection fluorescence microscopy (TIRFM) via waveguides have been shown to be highly beneficial for single molecule localisation microscopy on fixed cells [1, 2] and have also been demonstrated for short-term live-imaging of robust cell types [3–5], but not yet for delicate primary neurons nor over extended periods of time. Here, we present a waveguide-based TIRFM set-up for live-cell imaging of demanding samples. Using the developed microscope, referred to as the ChipScope, we demonstrate successful culturing and imaging of fibroblasts, primary rat hippocampal neurons and axons of Xenopus retinal ganglion cells (RGC). The high contrast and gentle illumination mode provided by TIRFM coupled with the exceptionally large excitation areas and superior illumination homogeneity offered by photonic waveguides have potential for a wide application span in neuroscience applications.

Point spread function, 0303 health sciences, Image quality, Computer science, Structured illumination microscopy, ComputingMethodologies_IMAGEPROCESSINGANDCOMPUTERVISION, Imagej plugin, Image processing, computer.software_genre, 01 natural sciences, Atomic and Molecular Physics, and Optics, Article, 010309 optics, 03 medical and health sciences, VDP::Teknologi: 500::Medisinsk teknologi: 620, Computer graphics (images), 0103 physical sciences, Plug-in, MATLAB, computer, VDP::Technology: 500::Medical technology: 620, 030304 developmental biology, Biotechnology, and computer.programming_language

We present an open-source implementation of the fluctuation-based nanoscopy method MUSICAL for ImageJ. This implementation improves the algorithm’s computational efficiency and takes advantage of multi-threading to provide orders of magnitude faster reconstructions than the original MATLAB implementation. In addition, the plugin is capable of generating super-resolution videos from large stacks of time-lapse images via an interleaved reconstruction, thus enabling easy-to-use multi-color super-resolution imaging of dynamic systems.

Microscope, Materials science, Optical sectioning, Confocal, Bioengineering, 01 natural sciences, Article, law.invention, 010309 optics, 03 medical and health sciences, Optics, law, Confocal microscopy, 0103 physical sciences, Microscopy, Fluorescence microscope, 1 Underpinning research, 4018 Nanotechnology, 40 Engineering, 030304 developmental biology, 0303 health sciences, business.industry, Resolution (electron density), 1.5 Resources and infrastructure (underpinning), Atomic and Molecular Physics, and Optics, 3. Good health, Interferometry, business, 51 Physical Sciences, and Biotechnology

Wide-field fluorescence microscopy, while much faster than confocal microscopy, suffers from a lack of optical sectioning and poor axial resolution. 3D structured illumination microscopy (SIM) has been demonstrated to provide optical sectioning and to double the resolution limit both laterally and axially, but even with this the axial resolution is still worse than the lateral resolution of unmodified wide-field microscopy. Interferometric schemes using two high numerical aperture objectives, such as 4Pi confocal and I5M microscopy, have improved the axial resolution beyond that of the lateral, but at the cost of a significantly more complex optical setup. Here, we investigate a simpler dual-objective scheme which we propose can be easily added to an existing 3D-SIM microscope, providing lateral and axial resolutions in excess of 125 nm with conventional fluorophores and without the need for interferometric detection.

0301 basic medicine, Materials science, Placenta, Structured illumination microscopy, law.invention, 03 medical and health sciences, 0302 clinical medicine, Syncytiotrophoblast, law, Pregnancy, Microscopy, Fluorescence microscope, medicine, Humans, VDP::Medisinske Fag: 700, 030304 developmental biology, 0303 health sciences, 030219 obstetrics & reproductive medicine, Super-resolution microscopy, VDP::Technology: 500, 030302 biochemistry & molecular biology, Resolution (electron density), Placental tissue, Obstetrics and Gynecology, Fluorescence, 3. Good health, VDP::Medical disciplines: 700, VDP::Teknologi: 500, 030104 developmental biology, medicine.anatomical_structure, Reproductive Medicine, Microscopy, Fluorescence, embryonic structures, Ultrastructure, Female, Deconvolution, Electron microscope, Chorionic Villi, Developmental Biology, and Biomedical engineering

Super-resolution fluorescence microscopy is a widely employed technique in cell biology research, yet remains relatively unexplored in the field of histo-pathology. Here, we describe the sample preparation steps and acquisition parameters necessary to obtain fluorescent multicolor super-resolution structured illumination microscopy (SIM) images of both formalin-fixed paraffin-embedded and cryo-preserved placental tissue sections. We compare super-resolved images of chorionic villi against diffraction-limited deconvolution microscopy and demonstrate the significant contrast and resolution enhancement attainable with SIM. We show that SIM resolves ultrastructural details such as the syncytiotrophoblast’s microvilli brush border, which up until now has been only resolvable by electron microscopy.

biology, Chemistry, Xenopus, Translation (biology), biology.organism_classification, Retinal ganglion, and Cell biology

Materials science, business.industry, Resolution (electron density), Hyperspectral imaging, Iterative reconstruction, law.invention, symbols.namesake, VDP::Mathematics and natural science: 400::Physics: 430::Electromagnetism, acoustics, optics: 434, Optics, Fourier transform, law, VDP::Matematikk og Naturvitenskap: 400::Fysikk: 430::Elektromagnetisme, akustikk, optikk: 434, Microscopy, symbols, Photonics, business, Phase retrieval, and Waveguide

SPIE Article-Sharing Policies https://www.spiedigitallibrary.org/article-sharing-policies In this project it was found that Fourier ptychographic microscopy can be improved far beyond its conventional limits via waveguide-based optical systems. Extensive in silico studies showed that images obtained on highrefractive index material waveguide chips in conjunction with hyperspectral illumination light and finely designed waveguide geometries can be combined via a modified phase-retrieval algorithm to yield a resolution below 150 nm.

0301 basic medicine, Computer science, ComputingMethodologies_IMAGEPROCESSINGANDCOMPUTERVISION, lcsh:Medicine, Bioengineering, Imaging techniques, 01 natural sciences, Optical projection tomography, 5105 Medical and Biological Physics, Article, 010309 optics, Set (abstract data type), 03 medical and health sciences, 0302 clinical medicine, 46 Information and Computing Sciences, Computer graphics (images), 0103 physical sciences, Microscopy, lcsh:Science, Lung, 030304 developmental biology, 0303 health sciences, Multidisciplinary, 639/766/930/2735, lcsh:R, Sample (graphics), 030104 developmental biology, Networking and Information Technology R&D (NITRD), 14/63, Biomedical Imaging, lcsh:Q, 51 Physical Sciences, Biological fluorescence, 030217 neurology & neurosurgery, and 631/57/2267

The three-dimensional imaging of mesoscopic samples with Optical Projection Tomography (OPT) has become a powerful tool for biomedical phenotyping studies. OPT uses visible light to visualize the 3D morphology of large transparent samples. To enable a wider application of OPT, we present OptiJ, a low-cost, fully open-source OPT system capable of imaging large transparent specimens up to 13 mm tall and 8 mm deep with 50 µm resolution. OptiJ is based on off-the-shelf, easy-to-assemble optical components and an ImageJ plugin library for OPT data reconstruction. The software includes novel correction routines for uneven illumination and sample jitter in addition to CPU/GPU accelerated reconstruction for large datasets. We demonstrate the use of OptiJ to image and reconstruct cleared lung lobes from adult mice. We provide a detailed set of instructions to set up and use the OptiJ framework. Our hardware and software design are modular and easy to implement, allowing for further open microscopy developments for imaging large organ samples.

Ribosomal Proteins, 0301 basic medicine, mRNA, Neurogenesis, neural wiring, VDP::Mathematics and natural science: 400::Basic biosciences: 470::Cell biology: 471, Library science, Regulatory Sequences, Ribonucleic Acid, Article, General Biochemistry, Genetics and Molecular Biology, ribosome remodeling, Xenopus laevis, 03 medical and health sciences, local translation, 0302 clinical medicine, Political science, Rps4x, Animals, RNA, Messenger, lcsh:QH301-705.5, health care economics and organizations, Cells, Cultured, axon, European research, Brain, Axons, Engineering and Physical Sciences, axon branching, 030104 developmental biology, ribosome, Axonal branching, lcsh:Biology (General), nervous system, Research council, Ribosomes, 030217 neurology & neurosurgery, axonal protein synthesis, and VDP::Matematikk og Naturvitenskap: 400::Basale biofag: 470::Cellebiologi: 471

Summary Ribosome assembly occurs mainly in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding many ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Here, we report a physical and functional interaction between locally synthesized RPs and ribosomes in the axon. We show that axonal RP translation is regulated through a sequence motif, CUIC, that forms an RNA-loop structure in the region immediately upstream of the initiation codon. Using imaging and subcellular proteomics techniques, we show that RPs synthesized in axons join axonal ribosomes in a nucleolus-independent fashion. Inhibition of axonal CUIC-regulated RP translation decreases local translation activity and reduces axon branching in the developing brain, revealing the physiological relevance of axonal RP synthesis in vivo. These results suggest that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus in neurons.
Graphical Abstract
Highlights • Axonal ribosomal protein (RP) synthesis is cue regulated via a 5′ UTR loop-forming motif • Axonal RP synthesis generates a free cytosolic pool of ribosomal proteins in axons • Axonally synthesized RPs join axonal ribosomes in a nucleolus-independent manner • Local RP synthesis is required for ribosome function and branch architecture
Local protein synthesis in axons supplies new ribosomal proteins far from the nucleolus, the known site of ribosome biogenesis. Shigeoka et al. provide evidence that axonally synthesized ribosomal proteins join pre-existing ribosomes and maintain translation activity in axons, which is required for axon terminal branching.

0303 health sciences, Adaptive imaging, Endosome, Computer science, Endoplasmic reticulum, Dynamics (mechanics), VDP::Technology: 500, 020206 networking & telecommunications, 02 engineering and technology, Multiple signal classification algorithm, Superresolution, 03 medical and health sciences, VDP::Teknologi: 500, Microtubule, Live cell imaging, 0202 electrical engineering, electronic engineering, information engineering, Biological system, and 030304 developmental biology

Copyright 2019 Society of Photo‑Optical Instrumentation Engineers (SPIE). One print or electronic copy may be made for personal use only. Systematic reproduction and distribution, duplication of any material in this publication for a fee or for commercial purposes, and modification of the contents of the publication are prohibited. In this work we have explored the live-cell friendly nanoscopy method Multiple Signal Classification Algorithm (MUSICAL) for multi-colour imaging of various organelles and sub-cellular structures in the cardiomyoblast cell line H2c9. We have tested MUSICAL for fast (up to 230Hz), multi-colour time-lapse sequences of various sub-cellular structures (mitochondria, endoplasmic reticulum, microtubules, endosomes and nuclei) in living cells using low excitation-light dose. Challenges and opportunities with applying MUSICAL for studying rapid sub-cellular dynamics are discussed.