Macro to micro skeletal muscle fiber formation mechanism captured with large FOV confocal microscope
Skeletal muscle formation begins with myoblast differentiation by expression of master transcription factors (Pax7, MyoD, Myogenin) in mesenchymal stem cells. Subsequently, molecules such as Myomaker and Myomixer fuse (multinucleate) myoblasts and mature them into skeletal muscle fibers. When forming skeletal muscle fibers, mononuclear myoblasts reorganize their cytoskeleton, consisting of actin, tubulin, etc., and show an elongated morphology. At higher densities, these elongated cells align with each other to form a locally ordered phase.
When this cell population is observed from a macro viewpoint, it shows a characteristic spiral pattern, which is inherited by the subsequent skeletal muscle fibers. On the other hand, parts where the cell population is not well oriented are called topological defects with respect to the ordered phase and were observed as having an accumulation of mononuclear round cells. The spiral pattern formation of such myoblast populations may be important for subsequent multinucleation and maturation into skeletal muscle fibers.
Mr. Yoshizuki Fumoto, Dr. Tsukasa Oikawa, et al. at the Department of Molecular Biology, Hokkaido University Graduate School of Medicine, aim to elucidate the molecular mechanism that is a necessary condition for myoblast populations to form spiral patterns. To that end, they are challenging the question of how microscopic molecular dynamics such as transcription factors, membrane fusion molecules and cytoskeletal molecules relate to macroscopic phenotypes at the cell population and tissue level.
In this application note, we introduce the behavior of related molecules in cells and the macroscopic pattern of cell populations, as captured by a confocal microscope.
Ecology of a fossilized cockroach in amber was revealed by confocal microscopy and thin sectioning technology
Insect sensory organs play an essential role in detecting information about their surrounding environment. Despite their small size and few sensory neurons, they have excellent abilities to process information, comparable to those of vertebrates. This is considered one of the primary reasons for the great success of insects, which account for 70% of all animal species. It is therefore important to investigate the sensory organs in evolutionary paleontological studies on insects.
Ryo Taniguchi and Associate Professor Yasuhiro Iba of the Graduate School of Science, Hokkaido University, Assistant Professor Hiroshi Nishino of the Research Institute of Electronic Science, Hokkaido University, Dr. Shûhei Yamamoto of the Hokkaido University Museum, and Associate Professor Hidehiro Watanabe of the Department of Earth System Science, Fukuoka University, reported a method for removing the amber substrate from a male fossilized cockroach in amber, Huablattula hui, as much as possible, and creating thin section specimens with the sensory organs still enclosed. The results of confocal microscopy observation of the specimen show that analysis of micro sensory organs is extremely effective in reconstructing detailed lifestyles of fossilized insects. In this application note, we introduce an example of the contribution of the laser scanning confocal microscope to the results of this research.
Confocal Imaging of CAR-T Cell Dynamics Using an Organ-on-a-chip Platform
Evaluation of the immune effect of CAR-T (Chimeric Antigen Receptor T) cell therapy is usually performed using a model organism, which is costly and time consuming. This application note introduces an example of building a simple 3D immune cell-mediated killing assay model using AIM Biotech’s 3D cell culture chips, and measuring the immune effects of T cells by in vitro imaging. The 3D assay model makes it easy to probe different conditions in vitro such as the cancer microenvironment and T cell regulation, and it can be customized in various ways according to the purpose of the research. This assay reproduces the more spatiotemporal dynamics of cells in vitro and enables the analysis of immune cell-mediated killing under more physiological conditions as compared to 2D models.
Automatic morphometric extraction of the rat osteocytic lacunae using Segment.ai
Osteocytes form an intercellular network similar to a neuronal network in bone. Drs. Tadahiro Iimura and Takanori Sato at the Department of Pharmacology, Faculty of Dental Medicine, Hokkaido University, visualize and measure this network structure, the functional significance of which remains to be elucidated. Since osteocytes have a structure that is continuous with osteocytic processes (countless cellular processes protruding from the cell body), extracting the morphologies of individual cells using the conventional fluorescence binarization method is a tall order. In this application note, we introduce an example of the use of Segment.ai, one of the functions of the NIS.ai module for microscopes, to automate segmentation of osteocytic lacunae in order to facilitate measurement of their numbers and morphology.
Time-lapse imaging analysis of angiogenesis induction using a 3D model
It is known that a decrease in the homeostasis function of blood vessels is involved in the onset and progression of various diseases and pathological conditions such as cancer, arteriosclerosis, chronic inflammation, and ischemia. Against this background, it is very important to understand the detailed mechanisms relating to new vascular structure formation by angiogenesis etc. and microvessel damage. This application note introduces an example of detailed observation and analysis of a 3D angiogenesis structure (in vitro perfusion angiogenesis model) with an AX/ AX R confocal microscope, using the Mimetas’ OrganoPlate® 3D tissue culture platform.
Morphogenesis imaging of mCherry-expressing Magnaporthe oryzae transformants with DIC and confocal microscopy
Rice blast disease is the most serious rice disease. The ascomycete fungus Magnaporthe oryzae is known as a hemibiotrophic pathogen that causes rice blast disease. M. oryzae infects rice leaves, stems and panicles, and causes severe reductions in yield. To establish a new control method for this disease and to develop resistant rice varieties, it is important to clarify the details of gene-to-gene and protein-protein interactions between M. oryzae and rice.
In this application note, we will introduce an example of imaging with differential interference contrast (DIC) and confocal laser scanning microscopy using the CFI Apochromat Lambda S 40XC WI objective, in a paper concerning the identification of a novel pathogenic gene using the differential gene expression evaluation method during plant-pathogenic fungus interactions reported by Professor Hiromasa Saitoh at Tokyo University of Agriculture.
High-speed, high-resolution 3D imaging using resonant scanner and Denoise.ai
Although resonant scanners are suitable for live cell imaging as they enable confocal imaging with high temporal resolution, in terms of image resolution, galvano scanners are superior. However, the resonant scanner of the AX R confocal microscope system can capture high-resolution images at high speed because it achieves 2K pixel resolution. Moreover, using Denoise.ai, the AI module of NIS-Elements imaging software, can remove shot noise generated by resonant scanning. As this can shorten exposure times and reduce photobleaching, it is also effective for capturing images of fixed samples. This application note introduces examples of acquisition of high-speed, high definition images using a resonant scanner together with Denoise.ai.
High-Definition Imaging of Mouse Neuromuscular Junction Using a Resonant Scanner
Since a resonant scanner can perform confocal imaging with higher temporal resolution than a galvano scanner, it is used in many cases to acquire life phenomena occurring at high speeds. In contrast, because the resonant scanner of the new generation AX R confocal microscope system supports up to 2K x 2K acquisition, it can be used for a wide range of purposes, from high-speed imaging to high-resolution imaging. This application note introduces examples of the structure of a neuromuscular junction in a mouse captured by high-definition 3D imaging using a resonant scanner.
3D enhanced resolution confocal imaging of cortical excitatory mouse neurons uncovers dendritic spinule subsets differing in dynamics, regulation, and function
In this application note, we used strong fluorescence labeling of neuronal elements and optimized Nikon laser scanning confocal imaging parameters, followed by post-acquisition iterative 3D deconvolution using NIS-Elements software (Zaccard, et al., 2021). This technique enabled the visualization and tracking of individual spinules in relation to presynaptic and postsynaptic markers, revealing spinule subtypes that differ in dynamics, length, lifespan, regulation, and function (Zaccard et al., 2020).
Efficient Confocal Imaging of Dynamics and Changes of Cells utilizing a Large Field of View
The Research Group led by Professor Masaru Ishii (immunology and cell biology) at the Graduate School of Medicine and Frontier Biosciences, Osaka University, is working on visualizing tumor cell movement in vivo using the microscopic imaging technique. This application note introduces an image acquisition example in which the dynamics and state changes of tumor cells were accurately captured using Nikon's AX R confocal microscope, and by making the maximum use of the large field of view that is one of the advantages of this microscope.
In vivo Confocal Imaging of Mouse Organs that Clearly Captures Fast Dynamics
The Research Group led by Professor Masaru Ishii (immunology and cell biology) at the Graduate School of Medicine and Frontier Biosciences, Osaka University, is studying the mechanism of immune cell movement in vivo by visualizing cell motility using the microscopic imaging technique. This application note introduces an image acquisition example in which quick movement of cells rolling in the blood vessel is captured in vivo using Nikon’s AX R confocal microscope, and by utilizing the high speed resonant scanning that is one of the advantages of this microscope.
The Advantages of Resonant Scanning with Ultra Short Laser Exposure Times in Live Imaging
Enteroids are an excellent tool for studying intestinal epithelial functions, such as secretion of antimicrobial peptides, α-defensins by Paneth cells in innate enteric immunity. However, enteroids are very sensitive not only to such environmental factors as temperature and humidity, but also laser exposure by laser scanning microscopes, so they must be handled with care during experiments. In this application note, we will show the advantages of conducting observations with ultra-short laser exposure times using a resonant scanner by evaluating not only photobleaching of fluorescent dyes but also intestinal epithelial cell functions focusing on Paneth cell granule secretion.
Development of Technology to Visualize Interaction of Osteoblasts and Osteoclasts in Living Bone Tissue
A research group led by Professor Masaru Ishii (Immune cell biology), Graduate School of Medicine, Osaka University, has developed a technology to observe the inside of living bones, and to simultaneously visualize osteoblasts, which make new bone, and osteoclasts, which dissolve old bone, using the A1R MP+ multi-photon confocal microscope, which is capable of deep tissue imaging. This application note introduces an example of the world’s first successful imaging of the moment of direct contact and communication between osteoblasts and osteoclasts in living bone tissue.
Wide FOV, High-Resolution Confocal Imaging of Podosomes in Osteoclasts ~ Macro and micro observation ~
Dr. Tadahiro Iimura, Dr. Ji-Won Lee, et al. of the Department of Pharmacology, Faculty and Graduate School of Dental Medicine, Hokkaido University have been studying lifelong changes in bones and joints from the viewpoint of skeletal development, maintenance of homeostasis, aging, and pharmacology. There is a ring structure called the “actin ring” on the adhesive surface between osteoclasts and bone, and electron microscope level resolution was required up until now in order to observe the microstructure of ”podosomes”, which are the components of this actin ring. This application note introduces examples of osteoclast macro-observation and podosome micro-observation using the new generation AX confocal microscope, and quantitative analysis thereof.
Selecting the Right Objectives - Bright, Sharp Imaging of Structures down to Deep Areas
Spherical aberrations caused by a mismatch of refractive indices may lead to a reduction in image resolution and brightness, and are one of the key problems in imaging. In this application note, we will demonstrate the effects of spherical aberration using 3D imaging of an enteroid, which is a 3D culture system for small intestinal epithelial cells, as an example, and show how to select the appropriate immersion liquid and objective.
3D Imaging of Intestinal Organoid
To perform high resolution observation of the deep regions of specimens during confocal imaging, selecting the objective is very important. This application note, prepared with the cooperation of Dr. Hidenori Akutsu and Dr. Tomoyuki Kawasaki of the Department of Reproductive Medicine, Center for Regenerative Medicine, National Center for Child Health and Development, will introduce an example of 3D imaging of a small intestine organoid using two different objectives, comparing the difference in image quality between them.
Capturing the physiological complexity of human tissues in vitro with organ-on-a-chip
3D cell cultures enable the recreation of physiological compositions and spatial arrangements of cells in vitro more accurately than 2D cell cultures. This application note shows that we have developed a 3D tissue culture model using OrganoPlate® (MIMETAS) and Nikon’s A1R HD25 confocal microscope system.
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.
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.
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.