A microscopy-based service for cellular signaling pathway analysis
Accurately capturing changes in cellular signaling pathways due to drug addition is essential for target discovery and mechanistic characterization of new drugs. Many signaling pathways are regulated through the localization of proteins into intracellular compartments such as cell membrane lipid rafts, scaffold proteins, and nuclei. Recently, it has been discovered that protein droplets formed by liquid-liquid phase separation are involved in signal enhancement and branching. Observing protein localization patterns is a key component of elucidating the modes of action of new drugs and compounds, but conventional biochemical analysis does not provide adequate spatial information. In contrast, microscopy enables detailed quantification of the spatiotemporal behavior of signaling proteins. Nikon leverages these capabilities to provide a new service that estimates signaling pathways activated in cells based only on microscopy images. We call this method PLOM-CON*1. In this paper, we present an example of applying this method to analyze insulin signaling pathways.
Keywords: drug discovery, signaling pathway, localization, liquid-liquid phase separation, droplet, protein condensates, organelle, microscopeimages
Confocal imaging of 3D biological tissues using the HUMIMIC Chip
In this application note, we present an intestinal model, a bone marrow model, and a vascular tissue model cultured in TissUse’s HUMIMIC Chips, which provide a structure that imitates the human body. By visualizing the tissue-specific marker with fluorescence and observing it with a confocal microscope, we confirmed that the tissue created in the chip forms the same structure as the corresponding living tissue. These models have the capability to close the gap between systemic animal models, which are physiological different to human, and standard in vitro assays performed in human derived cells, which cannot reproduce the human physiologic conditions.
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.
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.
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.
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.
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.
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.