Universal adaptive optics for microscopy through embedded neural network control.

The resolution and contrast of microscope imaging is often affected by aberrations introduced by imperfect optical systems and inhomogeneous refractive structures in specimens. Adaptive optics (AO) compensates these aberrations and restores diffraction limited performance. A wide range of AO solutions have been introduced, often tailored to a specific microscope type or application.

Systematic analysis of YFP traps reveals common mRNA/protein discordance in neural tissues.

While post-transcriptional control is thought to be required at the periphery of neurons and glia, its extent is unclear. Here, we investigate systematically the spatial distribution and expression of mRNA at single molecule sensitivity and their corresponding proteins of 200 YFP trap lines across the intact Drosophila nervous system. 97.

Democratising "Microscopi": a 3D printed automated XYZT fluorescence imaging system for teaching, outreach and fieldwork.

Commercial fluorescence microscope stands and fully automated XYZt fluorescence imaging systems are generally beyond the limited budgets available for teaching and outreach. We have addressed this problem by developing "Microscopi", an accessible, affordable, DIY automated imaging system that is built from 3D printed and commodity off-the-shelf hardware, including electro-mechanical, computer and optical components. Our design features automated sample navigation and image capture with a simple web-based graphical user interface, accessible with a tablet or other mobile device.

Absolute quantitation of individual SARS-CoV-2 RNA molecules provides a new paradigm for infection dynamics and variant differences.

Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single-molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, subgenomic RNAs, and viral proteins.

CryoSIM: super-resolution 3D structured illumination cryogenic fluorescence microscopy for correlated ultrastructural imaging.

Rapid cryopreservation of biological specimens is the gold standard for visualizing cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo-electron microscopy or cryo-soft X-ray tomography.

Microscope-Cockpit: Python-based bespoke microscopy for bio-medical science.

We have developed "Microscope-Cockpit" (Cockpit), a highly adaptable open source user-friendly Python-based Graphical User Interface (GUI) environment for precision control of both simple and elaborate bespoke microscope systems. The user environment allows next-generation near instantaneous navigation of the entire slide landscape for efficient selection of specimens of interest and automated acquisition without the use of eyepieces. Cockpit uses "Python-Microscope" (Microscope) for high-performance coordinated control of a wide range of hardware devices using open source software.

CytoCensus, mapping cell identity and division in tissues and organs using machine learning.

A major challenge in cell and developmental biology is the automated identification and quantitation of cells in complex multilayered tissues. We developed CytoCensus: an easily deployed implementation of supervised machine learning that extends convenient 2D 'point-and-click' user training to 3D detection of cells in challenging datasets with ill-defined cell boundaries. In tests on such datasets, CytoCensus outperforms other freely available image analysis software in accuracy and speed of cell detection.

Testing Models of mRNA Localization Reveals Robustness Regulated by Reducing Transport between Cells.

Robust control of gene expression in both space and time is of central importance in the regulation of cellular processes and for multicellular development. However, the mechanisms by which robustness is achieved are generally not identified or well understood. For example, messenger RNA (mRNA) localization by molecular motor-driven transport is crucial for cell polarization in numerous contexts, but the regulatory mechanisms that enable this process to take place in the face of noise or significant perturbations are not fully understood.

IsoSense: frequency enhanced sensorless adaptive optics through structured illumination.

We present IsoSense, a wavefront sensing method that mitigates sample dependency in image-based sensorless adaptive optics applications in microscopy. Our method employs structured illumination to create additional high spatial frequencies in the image through custom illumination patterns. This improves the reliability of image quality metric calculations and enables sensorless wavefront measurement even in samples with sparse spatial frequency content.

Accessibility to Peptidoglycan Is Important for the Recognition of Gram-Positive Bacteria in Drosophila.

In Drosophila, it is thought that peptidoglycan recognition proteins (PGRPs) SA and LC structurally discriminate between bacterial peptidoglycans with lysine (Lys) or diaminopimelic (DAP) acid, respectively, thus inducing differential antimicrobial transcription response. Here, we find that accessibility to PG at the cell wall plays a central role in immunity to infection. When wall teichoic acids (WTAs) are genetically removed from S.

Super-Resolution Single Molecule FISH at the Drosophila Neuromuscular Junction.

The lack of an effective, simple, and highly sensitive protocol for fluorescent in situ hybridization (FISH) at the Drosophila larval neuromuscular junction (NMJ) has hampered the study of mRNA biology. Here, we describe our modified single molecule FISH (smFISH) methods that work well in whole mount Drosophila NMJ preparations to quantify primary transcription and count individual cytoplasmic mRNA molecules in specimens while maintaining ultrastructural preservation. The smFISH method is suitable for high-throughput sample processing and 3D image acquisition using any conventional microscopy imaging modality and is compatible with the use of antibody colabeling and transgenic fluorescent protein tags in axons, glia, synapses, and muscle cells.

Localized Translation of gurken/TGF-α mRNA during Axis Specification Is Controlled by Access to Orb/CPEB on Processing Bodies.

In Drosophila oocytes, gurken/TGF-α mRNA is essential for establishing the future embryonic axes. gurken remains translationally silent during transport from its point of synthesis in nurse cells to its final destination in the oocyte, where it associates with the edge of processing bodies. Here we show that, in nurse cells, gurken is kept translationally silent by the lack of sufficient Orb/CPEB, its translational activator.

A single and rapid calcium wave at egg activation in Drosophila.

Activation is an essential process that accompanies fertilisation in all animals and heralds major cellular changes, most notably, resumption of the cell cycle. While activation involves wave-like oscillations in intracellular Ca(2+) concentration in mammals, ascidians and polychaete worms and a single Ca(2+) peak in fish and frogs, in insects, such as Drosophila, to date, it has not been shown what changes in intracellular Ca(2+) levels occur. Here, we utilise ratiometric imaging of Ca(2+) indicator dyes and genetically encoded Ca(2+) indicator proteins to identify and characterise a single, rapid, transient wave of Ca(2+) in the Drosophila egg at activation.

Subcellular mRNA localisation at a glance.

mRNA localisation coupled to translational regulation provides an important means of dictating when and where proteins function in a variety of model systems. This mechanism is particularly relevant in polarised or migrating cells. Although many of the models for how this is achieved were first proposed over 20 years ago, some of the molecular details are still poorly understood.

Drosophila patterning is established by differential association of mRNAs with P bodies.

The primary embryonic axes in flies, frogs and fish are formed through translational regulation of localized transcripts before fertilization. In Drosophila melanogaster, the axes are established through the transport and translational regulation of gurken (grk) and bicoid (bcd) messenger RNA in the oocyte and embryo. Both transcripts are translationally silent while being localized within the oocyte along microtubules by cytoplasmic dynein.

Preparing individual Drosophila egg chambers for live imaging.

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis (1). Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions.

A cell biologist's guide to high resolution imaging.

Fluorescence microscopy is particularly well suited to the study of cell biology, due to its noninvasive nature, high sensitivity detection of specific molecules, and high spatial and temporal resolution. In recent years, there has been an important transition from imaging the static distributions of molecules as a snapshot in time in fixed material to live-cell imaging of the dynamics of molecules in cells: in essence visualizing biochemical processes in living cells. Furthermore, in the last 5 years, there have been important advances in so-called "super-resolution" imaging methods that have overcome the resolution limits imposed by the diffraction of light in optical systems.

Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy.

Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion.

OMX: a new platform for multimodal, multichannel wide-field imaging.

Bioimaging is currently undergoing an exciting revolution. This includes all aspects of imaging from probe development, specimen preparation, and instrumentation to image analysis and quantitation. Perhaps the most exciting developments are new platforms for imaging that radically advance the capabilities for collecting high spatial and temporal resolution data.

A PAR-1-dependent orientation gradient of dynamic microtubules directs posterior cargo transport in the Drosophila oocyte.

Cytoskeletal organization is central to establishing cell polarity in various cellular contexts, including during messenger ribonucleic acid sorting in Drosophila melanogaster oocytes by microtubule (MT)-dependent molecular motors. However, MT organization and dynamics remain controversial in the oocyte. In this paper, we use rapid multichannel live-cell imaging with novel image analysis, tracking, and visualization tools to characterize MT polarity and dynamics while imaging posterior cargo transport.

Nanometric depth resolution from multi-focal images in microscopy.

We describe a method for tracking the position of small features in three dimensions from images recorded on a standard microscope with an inexpensive attachment between the microscope and the camera. The depth-measurement accuracy of this method is tested experimentally on a wide-field, inverted microscope and is shown to give approximately 8 nm depth resolution, over a specimen depth of approximately 6 µm, when using a 12-bit charge-coupled device (CCD) camera and very bright but unresolved particles. To assess low-flux limitations a theoretical model is used to derive an analytical expression for the minimum variance bound.

How the sea squirt nucleus tells mesoderm not to be endoderm.

Sea squirts are simple invertebrate chordates. In this issue of Developmental Cell, Takatori et al. show nuclear migration within ascidian mesendodermal cells enables polarized localization of Not mRNA, which encodes a homeobox protein that distinguishes mesoderm from endoderm fates.

ParticleStats: open source software for the analysis of particle motility and cytoskeletal polarity.

The study of dynamic cellular processes in living cells is central to biology and is particularly powerful when the motility characteristics of individual objects within cells can be determined and analysed statistically. However, commercial programs only offer a limited range of inflexible analysis modules and there are currently no open source programs for extensive analysis of particle motility. Here, we describe ParticleStats (http://www.

Making the message clear: visualizing mRNA localization.

Localized mRNA provides spatial and temporal protein expression essential to cell development and physiology. To explore the mechanisms involved, considerable effort has been spent in establishing new and improved methods for visualizing mRNA. Here, we discuss how these techniques have extended our understanding of intracellular mRNA localization in a variety of organisms.