Quantitative 3D Imaging of Living Organs-on-Chips with a High-Speed Point-Scanning Confocal System
Organs-on-chips more faithfully recapitulate the 3D architectural and functional complexity of native tissues compared to standard 2D tissue culture systems. Yet these advanced cell culture platforms present technical challenges for imaging-based applications. This Application Note demonstrates how the Nikon A1R HD25 confocal point-scanning system, CFI S Plan Fluor LWD 20XC objective and NIS-Elements software can enable rapid, deep, quantitative imaging of living cells in the Emulate Organ-Chip platform.
Live Imaging of Paneth Cell Secretory Responses in Innate Immunity by Using Three-Dimensional Culture of Small Intestinal Epithelial Cells
Paneth cells, a lineage of small intestinal epithelial cells, secrete granules rich in antimicrobial peptides, α-defensins, in response to cholinergic agents and bacteria, and regulate the intestinal microbiota by killing enteric pathogens, while less killing commensal bacteria. In this Application Note, we introduce examples that clarify the mechanisms of α-defensin secretion by visualization and quanti cation of Paneth cell granule secretory responses ex vivo using enteroid, a three-dimensional culture system of small intestinal epithelial cells.
Super-resolution imaging of double-stained neurons using DNA-Paint
Dr. Noriko Koganezawa (Life Science), Department of Neurobiology and Behavior, Graduate School of Medicine, Gunma University, focuses her research on drebrin, an actin-binding protein that is thought to play an important role in cognitive function. Drebrin accumulates in the posterior synaptic region (dendritic spine) of mature neurons, but when synaptic dysfunction occurs, this accumulation disappears. Also, when the NMDA receptor is activated by glutamate or the like, the accumulation of drebrin is transiently eliminated (Figs.1 and 2). Focusing on the dynamics of drebrin, Dr. Koganezawa is conducting research to verify its effects on cognitive function.
In this Application Note, we introduce examples of image acquisition of changes in the localization of drebrin by glutamate stimulation using the N-STORM super-resolution microscope and quantitative evaluation by cluster analysis.
3D Super-resolution imaging of localization of H3K27me3, Lamin AC and DNA
In this Application Note, we introduce examples of imaging (using the N-SIM super-resolution microscope) performed by pharmaceutical scientist Dr. Tsukasa Oikawa of the Department of Molecular Biology, Hokkaido University Graduate School of Medicine. In the absence of p53, the product of a tumor suppressor gene TP53, K27-trimethylation of histone H3 (H3K27me3) was observed to occur at the perinuclear regions during DNA replication. The researcher then wanted to know the positional relationship between H3K27me3, the nuclear membrane represented by Lamin A/C, and DNA.
The Secret Inside Flowers - Imaging Inside Plants Using ClearSee Clearing Reagent
In this Application Note, we introduce examples of clearing the flowers of Arabidopsis thaliana with ClearSee and observing them with a multiphoton excitation confocal microscope suited for observation of deep parts of organisms. This enables us to observe in detail how each of the pollen tubes elongate in the pistil for fertilization, using the color-coding of the pollen tubes.
Highly accurate segmentation of cell areas based on DIC images using deep learning
Quantification of cell migration and cell confluency is important in biological and medical research on cellular functions. A scratch assay is used to quantitatively measure the speed of cells migrating to a cell-free area (gap) that is physically produced. This is a common technique for evaluating cell migration in cell development and differentiation, as well as in the invasion and metastasis of cancer cells. However, manually processing unstained sample images for quantification takes an immense amount of time. In addition, the Wound Healing function, a special application for scratch assays in the NIS-Elements imaging software, shows roughly correct results, but it has limited accuracy in terms of detailed detection.
In this Application Note, we introduce examples of quantification of a scratch assay using the NIS.ai module of NIS-Elements. These examples proved that NIS.ai can make more accurate inferences compared to the existing Wound Healing function based on a small number of training images.
Visualization of microglia-neuron junctions with super-resolution and confocal microscopy
Microglia are the main immune cells in the brain, and play roles in brain homeostasis and neurological diseases. However, the fundamental mechanisms underlying microglia-neuron communication remain unclear. Dr. Csaba Cserép, Dr. Balázs Pósfai and colleagues, (Laboratory of Neuroimmunology led by Dr. Ádám Dénes, Institute of Experimental Medicine) identified an interaction site between neuronal cell bodies and microglial processes in the mouse brain and studied the function of microglia (C. Cserép and B. Pósfai et al., Science 10.1126/science.aax6752 (2020)). In this Application Note, we introduce how the structure of neuron-microglia junctions was revealed on a nano scale resolution using the confocal and super-resolution microscopes.
Imaging sodium transients in neurons and astrocytes
This application note presents data from Dr Gerkau, Heinrich Heine University, Düsseldorf, Germany on how increases in intracellular sodium in astrocytes and neurons drive the reversal of the sodium/calcium exchange (NCX) during peri-infarct depolarisations (PIDs) in stroke. Nikon’s stable and reliable FN1 microscope combined with its NIS-Elements software were essential in providing high-quality imaging and analysis of intracellular sodium ions in astrocytes and neurons in living brain tissue.
Mapping brain cell types with CARTANA in situ sequencing on the Nikon Ti2-E microscope
In situ sequencing (ISS) kits offered by CARTANA allow scientists to detect transcripts from hundreds of genes directly inside tissue sections with unprecedented throughput and single-cell resolution. The Nikon Ti2-E inverted microscope enables this application by combining its Perfect Focus System 4 focus locking technology and an automated stage with large image scan capabilities. This note demonstrates ISS for mapping transcriptional cell types within the spatial context of mouse brain sections.
Nikon NIS-Elements Denoise.ai Software: utilizing deep learning to denoise confocal data
Noise is a fundamental component of confocal images, a result of discreet digital sampling of continuously emitting photons from samples. The contribution of noise to image quality (signal-to-noise ratio) increases as the signal decreases as a square-root function. Using a trained neural network, we use artificial intelligence to remove the shot noise component from confocal image data, allowing an increase in image quality and the ability to acquire dimmer samples at faster rates. NIS-Elements software’s Denoise.ai deploys this trained network for live or post-acquisition processing.
A1R HD25: the latest in resonant scanning technology allows new live-cell imaging approaches
Capturing the dynamics of living systems requires high acquisition rates. Large samples, such as whole model organisms, additionally require a large field of view. The Nikon A1R HD25 confocal system provides both, combining Nikon’s improved HD high speed resonant scanner with an unprecedented 25 mm field of view. The performance of this system is evaluated in zebrafish embryos.
Increasing Data Collection and Fidelity by Maximizing Confocal Field of View
For years, the field of view (FOV) of confocal systems has been limited by the FOV of the microscope they are attached to. With the release of the Nikon Ti2 inverted microscope, the world’s first 25-mm FOV became available. Now, Nikon has taken advantage of this improvement by building the largest FOV point scanner in the world, the A1 HD25. This Application Note focuses on the impact of this technology on simple, everyday experiments.
Structured Illumination Microscopy (SIM) Imaging Comparison with Confocal
The super-resolution microscopy technique structured illumination microscopy (SIM) imaging of dendritic spines along the dendrite has not been previously performed in fixed tissues, mainly due to deterioration of the stripe pattern of the excitation laser induced by light scattering and optical aberrations.
Reflectance Imaging for Visualization of Unlabeled Structures Using Nikon A1 and N-SIM
Reflectance imaging allows the user to form an intensity image from light backscattered by the sample. Highly reflective markers, including a variety of nanoparticles, allows for imaging with very high signal-to-noise and virtually free of photobleaching, ideal for both confocal and structured illumination microscopies.
Hardware Triggering: Maximizing Speed and Efficiency for Live Cell Imaging
Live cell imaging experiments now require higher speeds and more data throughput than ever before. Nikon Instruments has robust tools that enable hardware triggering of imaging devices in microscopy via direct signaling between hardware. This minimizes delays, synchronizes devices, and reduces the exposure of specimens to light. This Application Note explains how Nikon’s NIS-Elements hardware triggering workflow operates, and details its benefits for common time-lapse acquisition routines.
Application of Patterned Illumination Using a DMD for Optogenetic Control of Signaling
Digital micromirror devices (DMDs) are powerful tools for photostimulation applications, including photoconversion and optogenetic manipulation, owing to their robust ability to produce novel illumination patterns with high spatiotemporal resolution. In this Application Note we showcase recent work describing how DMD technology integrated into a Nikon system can be applied toward light-gated optogenetic control of intracellular signaling.
Robotic Microscopy with the Nikon Ti2 for High-Content Analysis Applications
Robotic Microscopy—a combination of high-content screening methods—enables multivariate experimental approaches with large cell populations and member-level sensitivity. Here we explore how the new Nikon Ti2 line of inverted research microscopes is uniquely suited to Robotic Microscopy applications, focusing on work utilizing induced pluripotent stem cells (iPSCs) as disease models in drug screening.
N-STORM with DNA-PAINT for Reliable Multicolor & 3D Single Molecule Localization Imaging
DNA-PAINT is a technique for single molecule localization microscopy. The sample is immunolabeled with specialized secondary antibodies conjugated to short DNA oligomers known as docking strands. Complimentary oligomers conjugated to fluorophore, known as imaging strands, are introduced in the imaging buffer and will transiently bind to docking strands – allowing single molecules to be localized. Herein multicolor and 3D DNA-PAINT is demonstrated on the Nikon N-STORM 4.0 system.
N-SIM for Quantitative Ultra-Structural Analyses of the Nuclear Lamina
Super-resolution Structured Illumination Microscopy (SIM), available from Nikon via the N-SIM S and N-SIM E systems, allows for the observation of details inaccessible to traditional microscopes, such as confocal and widefield. In this application note we see how the N-SIM system enables quantitative multi-color evaluation of the distribution of different nuclear lamin proteins and the structures they form.
Quantitative Cluster Analysis Applications for N-STORM
Nikon’s N-STORM super-resolution microscope is applied to mapping the distribution of nucleosomes on chromatin fibers, shining a light on the mechanisms of chromatin folding, gene expression, and pluripotency. Multicolor STORM is used to explore how nucleosome distribution differs between differentiated and pluripotent stem cells and correlates with the distribution of other important factors in replication, such as RNA Polymerase II.
Nikon’s Large-Format Multiphoton System for Intravital Imaging
Nikon’s A1R MP multiphoton confocal microscope system provides a choice platform for deep tissue imaging. Researchers at the Allen Institute for Brain Science have applied the A1R MP towards fast dual-color intravital imaging of the mouse brain. The open architecture instrument design allows for the addition of custom stages for large samples, external photostimulation devices, and more – perfect for constantly evolving needs.
Quantitative Analysis Tools and Correlative Imaging Applications for N-STORM
Stochastic Optical Reconstruction Microscopy (STORM) has had a significant impact on our understanding of nano-scale biological processes. In this application note, we see how Nikon’s N-STORM system, combined with quantitative analysis tools, enables single molecule level studies of cannabinoid signaling in the brain. Correlative 3D-STORM, confocal microscopy, and patch-clamp electrophysiology is also explored.
Development of an Integrated Bioprocess for Production of NK-92 Cells for Immunotherapy
Anti-cancer NK-92 lymphocytes are promising for application in immunotherapies. This application note explores a comprehensive workflow for producing clinical-grade NK-92 cells, using Nikon’s BioStation CT to monitor NK-92 phenotype and potency.
Antitumor Lymphocyte Kinetic Cytotoxicity Assay (Collaboration with CCRM)
In this application note we explore the use of the fully robotic Nikon BioStation CT high content screening microscope platform towards quantifying the death rate of a model human leukemia cell line upon co-incubation with anticancer NK-92 lymphocytes. This assay integrates automated imaging of Calcein-AM fluorescence to perform live/dead cell analysis.
A Non-Invasive Method for Counting Human Pluripotent Stem Cell Numbers by Live Cell Imaging
Presented here is a method for counting live pluripotent stem cells using the fluorescent nuclear stain SYTO24 and phase contrast imaging on the Nikon BioStation CT high content screening microscope. The results show that colony coverage area deduced from phase contrast images can be used as an accurate estimator of stem cell count alone, correlating well with haemocytometer-based measurements.
Outline of Stem Cell Applications
The Nikon BioStation CT cell culture observation system and CL-Quant software provide a powerful platform for stem cell research applications, including automated induced pluripotent stem (iPS) cell colony identification, proliferation analysis, and neural differentiation analysis. Real experimental data is provided for each of these use cases.
Screening Assay for PSC Responses to Substrates
An automated imaging-based screening assay for assessing stem cell pluripotency is described. Researchers from Nikon and the Centre for Commercialization of Regenerative Medicine use a Nikon BioStation CT with CL-Quant software to perform stem cell pluripotency maintenance assays in experiments testing feeder cell-free culture vessel substrates. Such commercially available substrates provide a number of process standardization benefits.
Automated Haematopoietic Colony Forming Cell (CFC) Assay
Nikon’s BioStation CT high content live cell screening system is applied in an assay for automated enumeration and identification of haematopoietic colony-forming cells. This assay allows for the identification of multiple colony types, helping distinguish between lineages.
Generation of a Growth Curve for iPS Cells in a Feeder-Free Culture by Non-Invasive Image Analysis
Long-term time-lapse observation of feeder layer-free stem cell cultures, along with automated detection of stem cell colonies, is demonstrated here using a Nikon BioStation CT system. Growth curves for both differentiated and un-differentiated cells were generated for a time period of about 1 week.
Establishment of an Algorithm for Automated Detection of iPS/non-iPS Cells Under a Culture Condition by Non-Invasive Image Analysis
In this application note we detail an automated high throughput method for observing and classifying stem cell colonies with a standardized algorithm and using the Nikon BioStation CT. Automated image analysis discriminates stem cell colonies from non-stem cell colonies using phase contrast imaging and subsequent analysis of distinguishing morphological features.
Automated, Non-Invasive Culture, and Evaluation System for iPS Cells under Neural Differentiation Process
This application note details an integrated workflow for the culture, observation, evaluation, and differentiation of induced pluripotent stem (iPS) cells into neurons, introducing image processing methods for detecting neural rosettes and neurite outgrowth – two important morphological checkpoints in neural differentiation. Automated imaging for this assay is performed using a Nikon BioStation CT.
Embryonic Stem Cell Responses to Commercially Available Substrates for Stem Cell Maintenance
In collaboration with the Centre for Commercialization of Regenerative Medicine (CCRM) we compare the performance of 8 common substrates for stem cell growth and maintenance by developing an assay utilizing the Nikon BioStation CT high content screening microscope and incubation system. Fluorescence imaging of Oct4 is used as a marker for pluripotency.
Survival Analysis of Human In Vitro-derived Neurons Using New Live Cell Extended Time-lapse Imaging Technology
In this application note we demonstrate the Nikon BioStation CT for long-term live-cell imaging of stem cell-derived motor neurons – tracking key attributes such as cell body size and neurite length before, during, and after treatment with different stressors. This approach provides a standardized method for drug discovery studies in neurodegenerative disease models.