Three-Dimensional Fluorescent Imaging to Identify Multi-Paths in Polymer Aging

2021 ◽  
Author(s):  
Zekun Zhang ◽  
Rui Tian ◽  
Daolei Lin ◽  
Dezhen Wu ◽  
Chao Lu ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Fluorescent Imaging ◽  
Polymer Aging

scholarly journals Factors to consider when interrogating 3D culture models with plate readers or automated microscopes

2021 ◽  
Author(s):  
Terry Riss ◽  
O. Joseph Trask
Keyword(s):  
Cell Culture ◽  
Three Dimensional ◽  
3D Culture ◽  
Fluorescent Imaging ◽  
Culture Models ◽  
Assay Methods ◽  
Diffusion Rates ◽  
Cell Culture Models ◽  
Dimensional Cell ◽  
Cells Cultured

AbstractAlong with the increased use of more physiologically relevant three-dimensional cell culture models comes the responsibility of researchers to validate new assay methods that measure events in structures that are physically larger and more complex compared to monolayers of cells. It should not be assumed that assays designed using monolayers of cells will work for cells cultured as larger three-dimensional masses. The size and barriers for penetration of molecules through the layers of cells result in a different microenvironment for the cells in the outer layer compared to the center of three-dimensional structures. Diffusion rates for nutrients and oxygen may limit metabolic activity which is often measured as a marker for cell viability. For assays that lyse cells, the penetration of reagents to achieve uniform cell lysis must be considered. For live cell fluorescent imaging assays, the diffusion of fluorescent probes and penetration of photons of light for probe excitation and fluorescent emission must be considered. This review will provide an overview of factors to consider when implementing assays to interrogate three dimensional cell culture models.

scholarly journals The Flexiscope: a low cost, flexible, convertible and modular microscope with automated scanning and micromanipulation

2020 ◽  
Vol 7 (3) ◽  
pp. 191949 ◽  
Author(s):  
Amy Courtney ◽  
Luke M. Alvey ◽  
George O. T. Merces ◽  
Niamh Burke ◽  
Mark Pickering
Keyword(s):  
Experimental Design ◽  
High Performance ◽  
Low Cost ◽  
Three Dimensional ◽  
Cost Savings ◽  
Fluorescent Imaging ◽  
Multiple Roles ◽  
Flexible Work ◽  
Optical Components ◽  
Automated Scanning

With technologies rapidly evolving, many research institutions are now opting to invest in costly, high-quality, specialized microscopes which are shared by many researchers. As a consequence, the user may not have the ability to adapt a microscope to their specific needs and limitations in experimental design are introduced. A flexible work-horse microscopy system is a valuable tool in any laboratory to meet the diverse needs of a research team and promote innovation in experimental design. We have developed the Flexiscope; a multi-functional, adaptable, efficient and high-performance microscopy/electrophysiology system for everyday applications in a neurobiology laboratory. The core optical components are relatively constant in the three configurations described here: an upright configuration, an inverted configuration and an upright/electrophysiology configuration. We have provided a comprehensive description of the Flexiscope. We show that this method is capable of oblique infrared illumination imaging, multi-channel fluorescent imaging and automated three-dimensional scanning of larger specimens. Image quality is conserved across the three configurations of the microscope, and conversion between configurations is possible quickly and easily, while the motion control system can be repurposed to allow sub-micrometre computer-controlled micromanipulation. The Flexiscope provides similar performance and usability to commercially available systems. However, as it can be easily reconfigured for multiple roles, it can remove the need to purchase multiple microscopes, giving significant cost savings. The modular reconfigurable nature allows the user to customize the system to their specific needs and adapt/upgrade the system as challenges arise, without requiring specialized technical skills.

scholarly journals Three-Dimensional Visualization for Early-Stage Evolution of Polymer Aging

2020 ◽  
Vol 6 (5) ◽  
pp. 771-778 ◽  
Author(s):  
Zekun Zhang ◽  
Rui Tian ◽  
Pudun Zhang ◽  
Chao Lu ◽  
Xue Duan
Keyword(s):  
Early Stage ◽  
Three Dimensional ◽  
Polymer Aging

Abstract 196: Self-Organized Formation of Cardiac Pacemaker Tissues in Embryoid Bodies

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Sherin I Hashem ◽  
William C Claycomb
Keyword(s):  
Cell Line ◽  
Es Cells ◽  
Three Dimensional ◽  
Cardiac Pacemaker ◽  
Model System ◽  
Embryoid Bodies ◽  
Fluorescent Imaging ◽  
Es Cell ◽  
Altered Expression ◽  
Self Organized

The pacemaker tissues of the heart are a complex set of specialized cells that initiate the rhythmic heart beat. The sinoatrial node (SAN) serves as the primary pacemaker, whereas the atrioventricular node (AVN) serves as a subsidiary pacemaker under conditions of SAN failure. The elucidation of genetic networks regulating the development of these tissues is crucial for understanding the mechanisms underlying arrhythmias and for the design of biological pacemakers. At present, there is no in vitro model system in which these specialized cells are defined or targeted. Here we report efficient self-organized formation of the two pacemaker nodes in three-dimensional aggregate cultures of mouse embryonic stem (ES) cells known as embryoid bodies (EBs), as well as the isolation of ES cell-derived AVN cells. A Shox2-lacZ knockin ES cell line carrying a Cx30.2 enhancer-RFP construct was used to generate EBs. The Shox2 gene, a determinant of the SAN genetic cascade, was used to delineate the SAN in EBs. The Cx30.2 enhancer was used to direct RFP expression to the AVN in EBs. Using live fluorescent imaging and immunohistochemistry, we demonstrate that these genetic markers reproducibly delineate cell clusters which express nodal proteins. These clusters are functionally connected with, and consistently located adjacent to the contracting region of the EB in an organized manner. We demonstrate that EBs generated using Shox2 knockout ES cells exhibit a hypoplastic SAN phenotype that includes reduction in spontaneous contraction rates and altered expression of Shox2 downstream targets. We isolated an ES cell-derived AVN cell line using a genetic selection technique and demonstrated that these cells display nodal characteristics such as calcium dependent spontaneous depolarizations and an AVN gene expression profile. When these cells were grown as three-dimensional aggregates they induced synchronous contraction of surrounding cardiac myocytes in co-culture experiments. Using molecular markers, we have generated a reproducible model system and have isolated an AVN cell line that will be invaluable tools for studying the molecular pathways regulating the development of the cardiac pacemaker tissues and molecular composition of these specialized cells.

Generation and Control of Wide-Field Three-Dimensional Structured Illumination for Advanced Microscopic Imaging

2012 ◽  
Vol 516 ◽  
pp. 640-644
Author(s):  
Shin Usuki ◽  
Hiroyoshi Kanaka ◽  
Kenjiro Takai Miura
Keyword(s):  
Three Dimensional ◽  
Fluorescent Imaging ◽  
Wide Field ◽  
Structured Illumination ◽  
Axial Resolution ◽  
Structured Illumination Microscopy ◽  
Spatial Control ◽  
Microscopic Imaging ◽  
Point Spread ◽  
Resolution Improvement

In a variety of practical microscopic imaging applications, many industries require not only lateral resolution improvement but also axial resolution improvement. The resolution in optical microscopy is limited by diffraction and determined by the wavelength of the incident light and the numerical aperture (NA) of the objective lens. The diffraction limit is mathematically described by a point spread function in the imaging system, and three-dimensional (3D) point spread functions describe both the lateral and axial resolutions. Thus, it is useful to focus on exceeding this limit and improving the resolution of optical imaging by the spatial control of structured illumination. Structured illumination microscopy is a familiar technique to improve resolution in fluorescent imaging, and it is expected to be applied to industrial applications. Microscopic imaging is convenient, non-destructive, and has a high-throughput performance and compatibility with a number of applications. However, the spatial resolution of conventional light microscopy is limited to wavelength scale and the depth of field is shallow; hence, it is difficult to obtain detailed 3D spatial data of the object to be measured. Here, we propose a new technique for generating and controlling wide-field 3D structured illumination. The technique, based on the 3D interference of multiple laser beams, provides lateral and axial resolution improvement, and a wide 3D field of view. The spatial configuration of the beams was theoretically examined and the optimal incident angle of the multiple beams was confirmed. Numerical simulations using the finite difference time domain (FDTD) method were carried out and confirmed the generation of 3D structured illumination and spatial control of the illumination by using the phase shift of incident beams.

scholarly journals Data of a fluorescent imaging-based analysis of anti-cancer drug effects on three-dimensional cultures of breast cancer cells

Data in Brief ◽  
2015 ◽  
Vol 5 ◽  
pp. 429-433 ◽  
Author(s):  
Junji Itou ◽  
Sunao Tanaka ◽  
Wenzhao Li ◽  
Yoshiaki Matsumoto ◽  
Fumiaki Sato ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Three Dimensional ◽  
Drug Effects ◽  
Fluorescent Imaging ◽  
Cancer Drug ◽  
Anti Cancer

Three-dimensional DNA nanostructures for dual-color microRNA imaging in living cells via hybridization chain reaction

2020 ◽  
Vol 56 (49) ◽  
pp. 6668-6671
Author(s):  
Meng-Mei Lv ◽  
Zhan Wu ◽  
Ru-Qin Yu ◽  
Jian-Hui Jiang
Keyword(s):  
Three Dimensional ◽  
Living Cells ◽  
Fluorescent Imaging ◽  
Hybridization Chain Reaction ◽  
Dna Nanostructures ◽  
Chain Reaction ◽  
Dna Nanostructure ◽  
Dual Color ◽  
Dna Tetrahedron

A well-defined 3D DNA nanostructure was developed by combination of DNA tetrahedron and Y-shaped DNA, which allowed multiplexed, signal amplified fluorescent imaging of miRNAs in living cells via hybridization chain reaction.

scholarly journals A H2O2-activatable nanoprobe for diagnosing interstitial cystitis and liver ischemia-reperfusion injury via multispectral optoacoustic tomography and NIR-II fluorescent imaging

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Junjie Chen ◽  
Longqi Chen ◽  
Yinglong Wu ◽  
Yichang Fang ◽  
Fang Zeng ◽  
...  
Keyword(s):  
Ischemia Reperfusion Injury ◽  
Inflammatory Diseases ◽  
Ischemia Reperfusion ◽  
Three Dimensional ◽  
Fluorescent Imaging ◽  
Pluronic F127 ◽  
Molecular Probe ◽  
Optoacoustic Tomography ◽  
Ultrasound Signal

AbstractDeveloping high-quality NIR-II fluorophores (emission in 1000–1700 nm) for in vivo imaging is of great significance. Benzothiadiazole-core fluorophores are an important class of NIR-II dyes, yet ongoing limitations such as aggregation-caused quenching in aqueous milieu and non-activatable response are still major obstacles for their biological applications. Here, we devise an activatable nanoprobe to address these limitations. A molecular probe named BTPE-NO2 is synthesized by linking a benzothiadiazole core with two tetraphenylene groups serving as hydrophobic molecular rotors, followed by incorporating two nitrophenyloxoacetamide units at both ends of the core as recognition moieties and fluorescence quenchers. An FDA-approved amphiphilic polymer Pluronic F127 is then employed to encapsulate the molecular BTPE-NO2 to render the nanoprobe BTPE-NO2@F127. The pathological levels of H2O2 in the disease sites cleave the nitrophenyloxoacetamide groups and activate the probe, thereby generating strong fluorescent emission (950~1200 nm) and ultrasound signal for multi-mode imaging of inflammatory diseases. The nanoprobe can therefore function as a robust tool for detecting and imaging the disease sites with NIR-II fluorescent and multispectral optoacoustic tomography (MSOT) imaging. Moreover, the three-dimensional MSOT images can be obtained for visualizing and locating the disease foci.

scholarly journals Development of a Multi-Compartment Micro-Cell Culture Device as a Future On-Chip Human: Fabrication of a Three-Compartment Device and Immobilization of Mature Rat Adipocytes for the Evaluation of Chemical Distributions

2007 ◽  
Vol 19 (5) ◽  
pp. 544-549 ◽  
Author(s):  
Hidenari Nakayama ◽  
◽  
Hiroshi Kimura ◽  
Kikuo Komori ◽  
Teruo Fujii ◽  
...  
Keyword(s):  
Cell Culture ◽  
Animal Experiments ◽  
Three Dimensional ◽  
Nonwoven Fabric ◽  
Fluorescent Imaging ◽  
Culture Solution ◽  
Kinetic Evaluation ◽  
Rat Adipocytes ◽  
Hydrophobic Chemical

Absorption, distribution, metabolism and excretion (ADME) are important in estimating the influence of chemicals on health. Distribution among internal organs is difficult to estimate without animal experiments. In vitro development targets the perfusion culturing of multiple cells derived from organs such as the liver, stomach, small intestines, and kidney. Using cheep, easily molded polydimethylsiloxane (PDMS), we produced a cell-culture microdevice having three compartments to determine the kinetic distribution of hydrophobic chemicals imaged by fluorescent imaging based on the presence of mature rat adipocytes. The disposable device uses liquid feeding using a magnetic stirrer. Separate and complete perfusion modes are easily changed by a valve after nurturing organ-derived cells in the device under separate conditions, enabling kinetic evaluation. To stabilize, disperse, and fix mature adipocytes whose specific gravity is lower than the culture solution, nonwoven fabric is used as a three-dimensional scaffold. When fluoranthene, a fluorescent hydrophobic chemical, is added during perfusion culturing, fluoranthene selectively accumulates in a fat compartment after six hours in a device to which adipocytes are added, enabling in vitro determination of hydrophobic chemical accumulation determining the distribution of chemicals in adipocytes. By introducing cells of target organs and metabolic organs in to other compartments, the device is extremely effective in experimentally determining the ADME of chemicals and the development of toxicity in vitro.

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