Nanolive’s 3D Cell Explorer allows for the creation of very powerful 3D images and 4D time lapses
of living cells with very high spatio-temporal resolution (x,y:180nm; z:400nm; t:1.7sec). Moreover,
imaging with the 3D Cell Explorer does not require the use of any labels since the microscope directly
measures the refractive index of the different substructures of the cell. However, in the context of
molecular or cellular biology investigations, it can be useful to follow fluorescent markers combined
with the refractive index distribution to validate specific structures or to correlate fluorescent signals
and cellular states.
For this purpose, Nanolive developed the 3D Cell Explorer-fluo (https://nanolive.ch/fluo/) – a
complete solution that combines high precision tomographic data with high quality tri- or fourchannel
epifluorescence provided by a CoolLed module (https://www.coolled.com/).
In this application note we will present the time lapse imaging of mouse embryonic stem cells
(mESCs) that have been genetically modified to express the fluorescence ubiquitination cell cycle
indicator (FUCCI), a two-color (red and green) indicator that allows to monitor the cell cycle phases.
We will explain how to use the 3D Cell Explorer-fluo to record movies that require both fluorescence
and 3D refractive index imaging and will propose a solution to analyze the resulting time lapse
experiment.
Investigating cellular metabolism with the 3D Cell ExplorerMathieuFRECHIN
The 3D Cell Explorer microscope allows for unprecedented live imaging of subcellular structures like mitochondria and lipid droplets due to its high spatiotemporal resolution and lack of phototoxicity. This enables long-term observation of organelle dynamics and interactions in an unperturbed state. Experiments demonstrated unique imaging of mitochondrial network perturbations and rescues as well as quantitative tracking of lipid droplet features over time. The system provides new opportunities to study metabolism at the subcellular level.
Microdialysis is an integral part of preclinical research to determine extracellular fluid and blood concentrations of metabolites, hormones, drugs, etc, and is often used in quantifying the biochemistry of brain and peripheral tissues. However, it is a molecular-only technique and other imaging modalities are needed to provide the researcher with functional and anatomical information of the animal in vivo.
This document discusses how nanomedicine uses molecular tools and knowledge of the human body to diagnose, treat, and prevent disease at the molecular level. It provides examples of how nanotechnology can enhance drug solubility and bioavailability, enable imaging for diagnosis via MRI, CT, and PET scans, and allow for targeted drug delivery through passive mechanisms like the EPR effect or active targeting using ligands. Future applications discussed include nanorobots for repairing damage, inducing blood clots, enhancing brain cells, and assisting with dental and surgical procedures. Nanoparticles may also enable more effective vaccine development.
Flow cytometry is a powerful analytical tool that can analyze up to 10,000 individual cells per second. It works by passing single cells in suspension through a flow cell where they are exposed to a laser. Light scattering and fluorescence emissions from stained cells are then measured using detectors. Flow cytometry has many applications including analyzing cell viability, cell cycle, apoptosis, and cell sorting. It provides benefits like rapid analysis of single cells but also has limitations such as not showing intracellular protein localization and requiring cell preparation.
20150924 smb noviocell juliette van den dolderSMBBV
1. Noviocell aims to accelerate stem cell research by developing a 3D polyisocyanopeptide hydrogel cell culture system that mimics the natural extracellular matrix.
2. The hydrogel provides a synthetic, reproducible scaffold that allows stem cells to grow in 3D, unlike traditional 2D cultures, while also enabling easy recovery of intact cells and tissues.
3. The hydrogel has been shown to support the growth and organization of various cell types in a 3D environment, and exhibits biomechanical properties similar to human collagen matrices.
This document summarizes flow cytometry, a technique used to count and examine microscopic particles suspended in fluid. It describes key components of modern flow cytometers including lasers, detectors, and computer systems that can analyze thousands of particles per second. The document outlines the principles of how each cell passes through a laser, scatters and emits light, which is detected and analyzed by software. Common applications like cell sorting, fluorescence detection using labeled antibodies, and measurable parameters are discussed. Terminology related to instrumentation, optical systems, data analysis and compensation are also introduced.
This document describes how laser-induced breakdown spectroscopy (LIBS) was used to generate 3D elemental images of nanoparticle distribution in biological tissue at multiple scales. Sliced kidney tissue sections were mapped using LIBS to reconstruct the global nanoparticle distribution throughout the entire organ. Higher resolution LIBS imaging was also performed on specific regions of interest by repeatedly ablating the same tissue volume. This proof-of-concept study demonstrates that LIBS can quantitatively image both endogenous and exogenous elements in 3D within entire organs.
Investigating cellular metabolism with the 3D Cell ExplorerMathieuFRECHIN
The 3D Cell Explorer microscope allows for unprecedented live imaging of subcellular structures like mitochondria and lipid droplets due to its high spatiotemporal resolution and lack of phototoxicity. This enables long-term observation of organelle dynamics and interactions in an unperturbed state. Experiments demonstrated unique imaging of mitochondrial network perturbations and rescues as well as quantitative tracking of lipid droplet features over time. The system provides new opportunities to study metabolism at the subcellular level.
Microdialysis is an integral part of preclinical research to determine extracellular fluid and blood concentrations of metabolites, hormones, drugs, etc, and is often used in quantifying the biochemistry of brain and peripheral tissues. However, it is a molecular-only technique and other imaging modalities are needed to provide the researcher with functional and anatomical information of the animal in vivo.
This document discusses how nanomedicine uses molecular tools and knowledge of the human body to diagnose, treat, and prevent disease at the molecular level. It provides examples of how nanotechnology can enhance drug solubility and bioavailability, enable imaging for diagnosis via MRI, CT, and PET scans, and allow for targeted drug delivery through passive mechanisms like the EPR effect or active targeting using ligands. Future applications discussed include nanorobots for repairing damage, inducing blood clots, enhancing brain cells, and assisting with dental and surgical procedures. Nanoparticles may also enable more effective vaccine development.
Flow cytometry is a powerful analytical tool that can analyze up to 10,000 individual cells per second. It works by passing single cells in suspension through a flow cell where they are exposed to a laser. Light scattering and fluorescence emissions from stained cells are then measured using detectors. Flow cytometry has many applications including analyzing cell viability, cell cycle, apoptosis, and cell sorting. It provides benefits like rapid analysis of single cells but also has limitations such as not showing intracellular protein localization and requiring cell preparation.
20150924 smb noviocell juliette van den dolderSMBBV
1. Noviocell aims to accelerate stem cell research by developing a 3D polyisocyanopeptide hydrogel cell culture system that mimics the natural extracellular matrix.
2. The hydrogel provides a synthetic, reproducible scaffold that allows stem cells to grow in 3D, unlike traditional 2D cultures, while also enabling easy recovery of intact cells and tissues.
3. The hydrogel has been shown to support the growth and organization of various cell types in a 3D environment, and exhibits biomechanical properties similar to human collagen matrices.
This document summarizes flow cytometry, a technique used to count and examine microscopic particles suspended in fluid. It describes key components of modern flow cytometers including lasers, detectors, and computer systems that can analyze thousands of particles per second. The document outlines the principles of how each cell passes through a laser, scatters and emits light, which is detected and analyzed by software. Common applications like cell sorting, fluorescence detection using labeled antibodies, and measurable parameters are discussed. Terminology related to instrumentation, optical systems, data analysis and compensation are also introduced.
This document describes how laser-induced breakdown spectroscopy (LIBS) was used to generate 3D elemental images of nanoparticle distribution in biological tissue at multiple scales. Sliced kidney tissue sections were mapped using LIBS to reconstruct the global nanoparticle distribution throughout the entire organ. Higher resolution LIBS imaging was also performed on specific regions of interest by repeatedly ablating the same tissue volume. This proof-of-concept study demonstrates that LIBS can quantitatively image both endogenous and exogenous elements in 3D within entire organs.
Flow cytometry is a standard laser-based technology that is used in the detection and measurement of physical and chemical characteristics of cells or particles in a heterogeneous fluid mixture.
This document provides an overview of flow cytometry including:
- An introduction to flow cytometry techniques and applications from multiple speakers
- Descriptions of key components and parameters measured in flow cytometry like scatter, fluorescence, and fluorochromes
- Examples of flow cytometry applications in fields like cell viability, proliferation, and surface marker analysis
- A discussion of antibody conjugation methods and considerations for multi-color flow cytometry experiments
The document provides an overview of the basic principles and components of flow cytometry. It discusses how flow cytometry works by measuring the properties of cells in fluid flow, using a combination of fluidics to introduce cells, optics to generate and collect light signals, and electronics to convert signals to digital data. Key aspects summarized include how cells are hydrodynamically focused and interrogated by light scatter and fluorescence to derive information on their size, granularity, and marker expression that can be analyzed using software.
This document provides an overview of flow cytometry and fluorescence-activated cell sorting (FACS). It describes flow cytometry as a technique for measuring physical and chemical characteristics of cells as they flow in a fluid stream, allowing for single cell analysis. FACS extends this by using fluorescence to identify cell characteristics and sort cells into separate collections based on these characteristics. The key components of a flow cytometer are described as lasers, optics including filters and detectors, fluidics to hydrodynamically focus cells, and electronics to convert optical signals to digital data. Applications including cell phenotyping, apoptosis analysis, and cell cycle analysis are discussed. Cell sorting and quantitative analysis of cell cycle phases are also summarized.
The document provides an overview of flow cytometry, including its history, principles, and applications. It discusses how flow cytometry allows for the measurement of cellular characteristics like fluorescence and light scattering at high speeds. Key developments include the first apparatus for detecting bacteria in a fluid stream in 1947 and the first cell sorter in 1965. The term "fluorescence activated cell sorter" or FACS was coined in 1972. Flow cytometry integrates technologies like lasers, optics, fluidics, and electronics to analyze individual cells and measure parameters such as cell size, granularity, and receptor expression. It has various applications in fields like immunology, genetics, and microbiology.
The Microscopy & Imaging Shared Resource (MISR) provides advanced cellular imaging and image analysis services and instrumentation. MISR offers techniques like TIRF fluorescence microscopy for membrane studies, confocal microscopy for live cell imaging up to 500um deep, and spinning disk confocal for fast 3D imaging. Staff provide training, technical assistance, and access to image analysis software like Metamorph and Volocity. The goal is to support tumor biology research through quantitative imaging of subcellular changes.
This document summarizes a lecture on microfluidics and their applications including lab-on-a-chip devices. Key topics discussed include microfluidic applications in areas like blood analysis, biochemical detection, chemical synthesis, and DNA sequencing. Common microfluidic materials like silicon, glass, and polymers are also summarized. Fabrication techniques for polymers include casting, hot embossing, and injection molding. Lab-on-a-chip offers advantages of low cost, small sample/reagent sizes, and minimized harmful byproducts compared to conventional laboratories.
This document provides an introduction to flow cytometry. It defines flow cytometry as a method for sensing individual cells in a fluid stream as they pass through a laser beam, measuring light scattering and fluorescence. Key aspects of flow cytometry systems and methodology are described, including hydrodynamic focusing of cells, light scattering measurements, use of fluorescent markers, optical and electronic components, data acquisition and analysis techniques like gating and compensation. The history of technological developments in flow cytometry is also summarized.
This document provides an introduction to flow cytometry. It defines flow cytometry as the measurement of physical and chemical characteristics of cells as they flow in a fluid stream through a beam of light. It describes the key components of a flow cytometer including fluidics to deliver cells to the laser, optics to excite and collect light, and electronics to amplify and process signals. It explains the different types of signals detected including light scatter and fluorescence, and how these can be used to characterize cells. The document provides guidance on choosing fluorochromes and considerations for multi-color panels such as spectral overlap. It outlines some common applications of flow cytometry and contact details.
Mapping Inhibitory Neuronal Ircuits By Laser Scanning Photostimula TionTaruna Ikrar
1) The document describes a technique called laser scanning photostimulation (LSPS) combined with whole-cell patch clamp recording to map local inhibitory neuronal circuits.
2) LSPS uses laser pulses to selectively activate neurons via glutamate uncaging, allowing mapping of excitatory and inhibitory synaptic inputs onto recorded neurons from many stimulation sites.
3) An example is provided showing excitatory synaptic input maps for a fast-spiking inhibitory interneuron in mouse somatosensory cortex, revealing strong input from layer 4 and deeper layers.
An introduction to flow cytometry- Ashwini.RAshwini R
The document provides an introduction to flow cytometry. It describes flow cytometry as a technique that allows simultaneous multiparametric analysis of physical and chemical characteristics of single cells suspended in a fluid stream. Key components of a flow cytometer include fluidics, optics, detectors, and electronics. Cells are hydrodynamically focused into a single file stream and pass through a laser beam, where light scattering and fluorescence emissions provide information about cellular properties. Photodetectors convert light signals into electrical pulses that are analyzed. Flow cytometry has various applications including immunophenotyping, cell sorting, DNA content analysis, and cell cycle/proliferation analysis.
Flow cytometry allows the measurement of physical characteristics of single cells as they flow through a laser beam. It measures size, granularity, and fluorescence. Applications include immunophenotyping cancers and leukemias, monitoring transplant rejection and HIV, and determining DNA content and proliferation. Recent advances include improved instruments, new antibodies, and assessment of cytoplasmic/nuclear antigens and T-cell clonality.
Fish (Fish (fluorescence in situ hybridization))HAM ZAD
FISH is a molecular cytogenetic technique that uses fluorescent probes to bind specifically to parts of chromosomes. It is used to detect and localize DNA sequences on chromosomes. The FISH assay involves fixing cells, designing probes, denaturing DNA, hybridizing probes to complementary DNA, and using fluorescence microscopy to view results. FISH has applications like detecting Down syndrome and provides advantages over other methods like being less labor-intensive, though imperfect hybridization or non-specific binding can produce errors.
Sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology cannot read whole genomes in one go, but rather reads small pieces of between 20 and 30,000 bases, depending on the technology used. Typically the short fragments, called reads, result from shotgun sequencing genomic DNA, or gene transcript (ESTs).
The problem of sequence assembly can be compared to taking many copies of a book, passing each of them through a shredder with a different cutter, and piecing the text of the book back together just by looking at the shredded pieces. Besides the obvious difficulty of this task, there are some extra practical issues: the original may have many repeated paragraphs, and some shreds may be modified during shredding to have typos. Excerpts from another book may also be added in, and some shreds may be completely unrecognizable.
Using multiple imaging techniques, the study found:
1) Fluorescence microscopy with transgenic mice expressing fluorescent reporters allowed visualization of axonal damage over time.
2) Confocal microscopy revealed reactive changes in axotomized neurons such as sprouting.
3) Multi-photon microscopy enabled in vivo and in vitro imaging at greater depths with less phototoxicity.
This document provides an introduction to flow cytometry. It defines flow cytometry as the measurement of physical and chemical characteristics of cells as they flow in a fluid stream through a beam of light. The key components of a flow cytometer are described as the fluidics system, optics system, and electronics. The fluidics system controls the hydrodynamic focusing of cells in the flow cell for interrogation by lasers. The optics system allows for excitation of cells by lasers and collection of emitted light, which is then analyzed by the electronics. Common applications and parameters that can be measured by flow cytometry are also outlined.
This document discusses the use of fluorescent proteins in current biological research. It begins with an overview of the development of optical microscopy and fluorescence techniques. It then focuses on the green fluorescent protein (GFP) and how it has been used as a molecular tag to study protein expression and interactions in living cells through techniques like gene delivery, transfection, viral infection, FRET, and optogenetics. The document concludes that fluorescent proteins have revolutionized cell biology by enabling the real-time visualization and control of molecular pathways and signaling processes in living systems.
Flow cytometry is a standard laser-based technology that is used in the detection and measurement of physical and chemical characteristics of cells or particles in a heterogeneous fluid mixture.
This document provides an overview of flow cytometry including:
- An introduction to flow cytometry techniques and applications from multiple speakers
- Descriptions of key components and parameters measured in flow cytometry like scatter, fluorescence, and fluorochromes
- Examples of flow cytometry applications in fields like cell viability, proliferation, and surface marker analysis
- A discussion of antibody conjugation methods and considerations for multi-color flow cytometry experiments
The document provides an overview of the basic principles and components of flow cytometry. It discusses how flow cytometry works by measuring the properties of cells in fluid flow, using a combination of fluidics to introduce cells, optics to generate and collect light signals, and electronics to convert signals to digital data. Key aspects summarized include how cells are hydrodynamically focused and interrogated by light scatter and fluorescence to derive information on their size, granularity, and marker expression that can be analyzed using software.
This document provides an overview of flow cytometry and fluorescence-activated cell sorting (FACS). It describes flow cytometry as a technique for measuring physical and chemical characteristics of cells as they flow in a fluid stream, allowing for single cell analysis. FACS extends this by using fluorescence to identify cell characteristics and sort cells into separate collections based on these characteristics. The key components of a flow cytometer are described as lasers, optics including filters and detectors, fluidics to hydrodynamically focus cells, and electronics to convert optical signals to digital data. Applications including cell phenotyping, apoptosis analysis, and cell cycle analysis are discussed. Cell sorting and quantitative analysis of cell cycle phases are also summarized.
The document provides an overview of flow cytometry, including its history, principles, and applications. It discusses how flow cytometry allows for the measurement of cellular characteristics like fluorescence and light scattering at high speeds. Key developments include the first apparatus for detecting bacteria in a fluid stream in 1947 and the first cell sorter in 1965. The term "fluorescence activated cell sorter" or FACS was coined in 1972. Flow cytometry integrates technologies like lasers, optics, fluidics, and electronics to analyze individual cells and measure parameters such as cell size, granularity, and receptor expression. It has various applications in fields like immunology, genetics, and microbiology.
The Microscopy & Imaging Shared Resource (MISR) provides advanced cellular imaging and image analysis services and instrumentation. MISR offers techniques like TIRF fluorescence microscopy for membrane studies, confocal microscopy for live cell imaging up to 500um deep, and spinning disk confocal for fast 3D imaging. Staff provide training, technical assistance, and access to image analysis software like Metamorph and Volocity. The goal is to support tumor biology research through quantitative imaging of subcellular changes.
This document summarizes a lecture on microfluidics and their applications including lab-on-a-chip devices. Key topics discussed include microfluidic applications in areas like blood analysis, biochemical detection, chemical synthesis, and DNA sequencing. Common microfluidic materials like silicon, glass, and polymers are also summarized. Fabrication techniques for polymers include casting, hot embossing, and injection molding. Lab-on-a-chip offers advantages of low cost, small sample/reagent sizes, and minimized harmful byproducts compared to conventional laboratories.
This document provides an introduction to flow cytometry. It defines flow cytometry as a method for sensing individual cells in a fluid stream as they pass through a laser beam, measuring light scattering and fluorescence. Key aspects of flow cytometry systems and methodology are described, including hydrodynamic focusing of cells, light scattering measurements, use of fluorescent markers, optical and electronic components, data acquisition and analysis techniques like gating and compensation. The history of technological developments in flow cytometry is also summarized.
This document provides an introduction to flow cytometry. It defines flow cytometry as the measurement of physical and chemical characteristics of cells as they flow in a fluid stream through a beam of light. It describes the key components of a flow cytometer including fluidics to deliver cells to the laser, optics to excite and collect light, and electronics to amplify and process signals. It explains the different types of signals detected including light scatter and fluorescence, and how these can be used to characterize cells. The document provides guidance on choosing fluorochromes and considerations for multi-color panels such as spectral overlap. It outlines some common applications of flow cytometry and contact details.
Mapping Inhibitory Neuronal Ircuits By Laser Scanning Photostimula TionTaruna Ikrar
1) The document describes a technique called laser scanning photostimulation (LSPS) combined with whole-cell patch clamp recording to map local inhibitory neuronal circuits.
2) LSPS uses laser pulses to selectively activate neurons via glutamate uncaging, allowing mapping of excitatory and inhibitory synaptic inputs onto recorded neurons from many stimulation sites.
3) An example is provided showing excitatory synaptic input maps for a fast-spiking inhibitory interneuron in mouse somatosensory cortex, revealing strong input from layer 4 and deeper layers.
An introduction to flow cytometry- Ashwini.RAshwini R
The document provides an introduction to flow cytometry. It describes flow cytometry as a technique that allows simultaneous multiparametric analysis of physical and chemical characteristics of single cells suspended in a fluid stream. Key components of a flow cytometer include fluidics, optics, detectors, and electronics. Cells are hydrodynamically focused into a single file stream and pass through a laser beam, where light scattering and fluorescence emissions provide information about cellular properties. Photodetectors convert light signals into electrical pulses that are analyzed. Flow cytometry has various applications including immunophenotyping, cell sorting, DNA content analysis, and cell cycle/proliferation analysis.
Flow cytometry allows the measurement of physical characteristics of single cells as they flow through a laser beam. It measures size, granularity, and fluorescence. Applications include immunophenotyping cancers and leukemias, monitoring transplant rejection and HIV, and determining DNA content and proliferation. Recent advances include improved instruments, new antibodies, and assessment of cytoplasmic/nuclear antigens and T-cell clonality.
Fish (Fish (fluorescence in situ hybridization))HAM ZAD
FISH is a molecular cytogenetic technique that uses fluorescent probes to bind specifically to parts of chromosomes. It is used to detect and localize DNA sequences on chromosomes. The FISH assay involves fixing cells, designing probes, denaturing DNA, hybridizing probes to complementary DNA, and using fluorescence microscopy to view results. FISH has applications like detecting Down syndrome and provides advantages over other methods like being less labor-intensive, though imperfect hybridization or non-specific binding can produce errors.
Sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology cannot read whole genomes in one go, but rather reads small pieces of between 20 and 30,000 bases, depending on the technology used. Typically the short fragments, called reads, result from shotgun sequencing genomic DNA, or gene transcript (ESTs).
The problem of sequence assembly can be compared to taking many copies of a book, passing each of them through a shredder with a different cutter, and piecing the text of the book back together just by looking at the shredded pieces. Besides the obvious difficulty of this task, there are some extra practical issues: the original may have many repeated paragraphs, and some shreds may be modified during shredding to have typos. Excerpts from another book may also be added in, and some shreds may be completely unrecognizable.
Using multiple imaging techniques, the study found:
1) Fluorescence microscopy with transgenic mice expressing fluorescent reporters allowed visualization of axonal damage over time.
2) Confocal microscopy revealed reactive changes in axotomized neurons such as sprouting.
3) Multi-photon microscopy enabled in vivo and in vitro imaging at greater depths with less phototoxicity.
This document provides an introduction to flow cytometry. It defines flow cytometry as the measurement of physical and chemical characteristics of cells as they flow in a fluid stream through a beam of light. The key components of a flow cytometer are described as the fluidics system, optics system, and electronics. The fluidics system controls the hydrodynamic focusing of cells in the flow cell for interrogation by lasers. The optics system allows for excitation of cells by lasers and collection of emitted light, which is then analyzed by the electronics. Common applications and parameters that can be measured by flow cytometry are also outlined.
This document discusses the use of fluorescent proteins in current biological research. It begins with an overview of the development of optical microscopy and fluorescence techniques. It then focuses on the green fluorescent protein (GFP) and how it has been used as a molecular tag to study protein expression and interactions in living cells through techniques like gene delivery, transfection, viral infection, FRET, and optogenetics. The document concludes that fluorescent proteins have revolutionized cell biology by enabling the real-time visualization and control of molecular pathways and signaling processes in living systems.
Optical coherence tomography (OCT) is used to image tissue samples but currently can only image one sample at a time. The document describes a device that allows multiple smaller samples like fruit flies to be imaged concurrently using a rotating sample dish. A prototype was created that places samples in a circle and uses a stepper motor and timing belt to rotate the dish, automatically imaging each new position. Initial testing showed it could successfully image printed numbers and internal structures of fruit fly larvae over time. Further improvements are still needed but this approach shows promise for more efficient multi-sample OCT imaging.
ADAPTIVE SEGMENTATION OF CELLS AND PARTICLES IN FLUORESCENT MICROSCOPE IMAGEJournal For Research
The document presents an adaptive segmentation method for segmenting cells and particles in fluorescent microscope images. It involves applying a coherence-enhancing diffusion filter to reduce noise and enhance structures, followed by using the Chan-Vese model to detect cell boundaries. The method allows simultaneous tracking of multiple cells over time by integrating both fast level set and graph cut frameworks with a topological prior. It is demonstrated on 2D and 3D time-lapse images of stem cells and carcinoma cells.
Monitoring live cell viability Comparative studyWerden Keeler
This document compares three live cell imaging techniques: fluorescence microscopy, oblique incidence reflection microscopy, and phase contrast microscopy. It finds that oblique incidence reflection microscopy is the simplest, least expensive, and least phototoxic method, causing the least damage to live cells during long-term monitoring of cell viability. The document describes the equipment and cell lines used, including normal and cancerous cell lines tagged with fluorescent proteins or unlabeled, to evaluate the stresses induced by different illumination techniques.
Automated platform for multiparameter stimulus response studies of metabolic ...Shashaanka Ashili
This document describes an automated platform for performing multiparameter stimulus-response studies on single cells and small groups of interacting cells. The platform allows for minimally invasive monitoring of cell phenotypes while applying various physiological stimuli through microfluidic systems. It features integrated fluorescent probes to monitor intracellular and extracellular physiological changes with high sensitivity. The platform consists of a confocal microscope, microfluidic cassette for single cell confinement and application of stimuli, and software/hardware for automated control and data collection. Preliminary results characterizing metabolic responses of single esophageal cells are presented.
This document provides protocols for optimizing high-dimensional flow cytometry panels using spectral flow cytometry. It begins with evaluating spectral reference controls to ensure accurate unmixing. Next, it involves evaluating unmixing of a fully stained sample to verify resolution of all markers. Marker resolution is then directly assessed. Finally, data quality is analyzed using gating and dimensionality reduction. A 24-color panel for identifying human T-cell subsets is used as an example. The protocols guide optimization from theoretical design to practical implementation and troubleshooting of spectral flow cytometry panels.
While Phosphorous (31P) MRS (I) has been promising in experimental and clinical settings since the early 70s, it has been beset by prohibitively lower sensitivity, limited spectral-spatial resolution, and prolonged acquisition. This manuscript and proceedings of the annual scientific meeting of ISMRM in 2022 (REF1) and 2023 (REF2) demonstrate that our novel acquisition strategy, the novel Rosette Trajectory for fast and flexible MR(S)I contrast (Shen et al. 2023 (REF3), later we renamed it as PETALUTE after the translation to the preclinical scanners of 7T and 9.4T), enables operator-independent (1) rapid acquisition (~7 minutes), (2) reconstruction, and (3) processing pipeline, resulting in phosphorous metabolite ratio maps (10 x 10 x 10 mm3) of the whole brain.
In response to the “Repeat it with Me” challenge organized by the Reproducible Research study group of ISMRM, we demonstrated the power of this technique in 5 healthy volunteers at three different institutions with different experimental setups (2nd Place: UTE 31P 3D Rosette MRSI Reproducibility Team, REF4). Since the proposed acquisition/reconstruction/processing pipeline was operator/scanner/coil-independent, the Reproducer sub-teams successfully replicated the findings of the original proceeding in 2022 (REF1). As part of this challenge, we provided some MATLAB scripts and k-space data to reproduce some of the results described in this manuscript. The software and data can be downloaded from https://purr.purdue.edu/projects/ismrm31pmrsi.
These results will likely be of broad interest across clinical settings since the proposed acquisition strategy is not specific to any region, nuclei, or magnetic field and is operator-independent. This study's resolution and signal-to-noise ratios permit the metabolite maps in an experimentally and clinically feasible timeframe at 3 Tesla and 7T.
REF1 Bozymski B, Shen X, Ozen AC, Ibey S, Chiew M, Thomas A, Dydak U, Emir UE. Ultra-Short Echo Time 31P 3D MRSI at 3T with Novel Rosette k-space Trajectory. Proceedings 30th Scientific Meeting, International Society for Magnetic Resonance in Medicine, 2022.
REF2 Farley N, Bozymski B, Dydak U, Emir UE*. Fast 3D 31P MRSI Using Novel Rosette Petal Trajectory at 3T with x4 Accelerated Compressed Sensing. Proceedings 31st Scientific Meeting, International Society for Magnetic Resonance in Medicine, 2023.
REF3 Shen X, Özen AC, Sunjar A, Ilbey S, Sawiak S, Shi R, Chiew M, Emir UE. Ultrashort T2 components imaging of the whole brain using 3D dual-echo UTE MRI with rosette k-space pattern. Magnetic Resonance in Medicine. 2023;89(2):508–521.
REF4 https://challenge.ismrm.org/2023-24-reproducibility-challenge/results-22-23/
Review on laser scanning confocal microscopyUdayan Ghosh
This document provides an overview of laser scanning confocal microscopy (LSCM). It discusses the working principle of LSCM, highlighting how it uses pinholes and laser excitation to optically section samples. Advantages include high resolution 3D imaging without physical sectioning. Applications described include biology, materials science, and semiconductor fabrication. LSCM is presented as a valuable non-destructive characterization tool across many fields due to its high resolution and ability to image living samples.
CASSS Forum Poster - Enabling robist cloning method qualification in cell lin...IanTaylor50
This document describes methods for using fluorescence imaging to statistically qualify cell cloning methods. Cells can be labelled with different fluorescent dyes and deposited using cloning methods like limiting dilution or FACS. Imaging the cells with fluorescence on day 0 allows analysis of deposition statistics, such as detecting "ghost cells" not initially seen. Regular fluorescence imaging also enables quality control of the cloning method over time to ensure consistent performance. The high resolution fluorescence imaging capabilities of the Solentim Cell Metric system support validation and qualification of cell cloning methods in cell line development.
White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving miceFUJIFILM VisualSonics Inc.
Fiberoptic fluorescence microscopy (FFM) employs optical fibers as small as 300 micrometers in diameter and offers the ability to image cellular and subcellular processes in deep brain structures including the Ventral Tegmental Area (VTA) and the substantia nigra (Sn).
Lightoptical nanoscopy for the use in biomedical applications and material sciences, detection in attomolar concentrations
* Use of standard fluorophores like GFP, RFP, YFP, Alexa, Fluorescein (no photoswitch necessary)
2CLM Two Color Localisation microscopy in the nanoscale
* Optical resolution 10 nm in 2D, 40 nm in 3D
* Very fast in processing, complete picture (2000 images) with processing in 3 minutes
The document summarizes the development and validation of a hyperspectral imaging microscopy system called Xanoscope for pathology applications. Key points:
1) Xanoscope allows for fast, automated acquisition of hyperspectral images over contiguous wavelength bands with high spatial resolution, reducing scan times significantly compared to previous methods.
2) Validation experiments showed Xanoscope produces quantitative measurements and improves signal-to-noise ratio through optimized camera settings and image accumulation.
3) Xanoscope was used to scan 10 multiplexed fluorophores in cell lines, and linear unmixing was able to measure the individual contribution of each fluorophore at pixel-level for advanced pathological analysis.
MPEF (multiphoton excitation fluorescence) microscopy uses ultrafast lasers to enable deep tissue imaging of living samples with little damage. It is widely used in neuroscience and cancer research. While primarily a research tool, MPEF microscopy has potential for clinical applications through developments like multiphoton endoscopy. Key benefits of MPEF include deeper imaging into tissues, low photodamage, and inherent 3D resolution without the need for a confocal pinhole. The technology continues to advance through increased laser powers, new fluorescent probes, and application-specific devices.
dokumen.tips_immunofluorescence-and-fluoroscence-microscopy.pdfBassem Ahmed
Immunofluorescence is a technique that uses fluorescent-labeled antibodies to detect specific target antigens in cells or tissues. It allows visualization of the target under a fluorescence microscope. There are two main methods - direct immunofluorescence, which uses pre-labeled antibodies, and indirect immunofluorescence, which uses a secondary antibody labeled with a fluorophore. Immunofluorescence is widely used in research and clinical diagnosis to study the distribution of proteins, glycoproteins and other molecules in cells and tissues.
Immunofluorescence and fluoroscence microscopyManjubala Us
This document provides information about immunofluorescence and fluorescence microscopy techniques. It discusses immunofluorescence, which uses fluorescent-labeled antibodies to detect target antigens. It describes direct and indirect immunofluorescence methods. It also discusses fluorescence microscopy, including different types of fluorescent dyes, fluorescence microscopes, applications such as visualizing viral plaques and detecting proteins in cells, and considerations for effective immunofluorescence applications. Flow cytometry is also summarized, which uses fluorescence to examine particles like cells that are passed through a flow chamber.
The Extended Nijboer-Zernike diffraction theory provides an analytic solution to the diffraction integral that describes image formation by optical systems. This thesis presents the ENZ theory as well as two of its applications. First, an ENZ imaging model is constructed that accurately simulates image formation with few approximations. Second, it is shown that the aberrations of an optical system can be determined from intensity measurements of a point object image alone, providing an alternative to interferometric methods. Examples illustrate the potential of the ENZ formalism for simulating and analyzing advanced optical imaging systems with high accuracy.
This document summarizes a new microfluidic platform that allows for complete mammalian cell culture. Key innovations include growing cells on patterned islands on an array of electrodes, rapidly exchanging media using digital microfluidics, and detaching and collecting cells for subculturing to fresh sites. This represents the first microfluidic system that can perform all the steps of standard cell culture, including repeated passaging of cells. The technique was demonstrated by culturing several cell lines in 150 nL droplets for weeks, with cells exhibiting normal growth and morphology. Complete cellular microculture could enable automated cell-based applications requiring repeated passaging.
Flow Cytometry (FC) determines multiple physical and biological characteristics of cells by detecting the intensity of the scattered or emitted light of a single cell in a linear flow state before laser irradiation. This technology can analyze cells rapidly at a rate of tens of thousands of cells per second. The types of samples can be various types of cells (such as peripheral blood, bone marrow, solid tissues, cells in suspension or adherent culture), microorganisms, synthetic microspheres, etc. https://www.creative-proteomics.com/services/flow-cytometry-facs-service.htm
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Using the 3D Cell Explorer-fluo for fluorescence and holotomographic imaging
1. Using the 3D Cell Explorer-fluo for
fluorescence and holotomographic imaging
Nanolive SA
Chemin de la Dent d‘Oche 1a | 1024 Ecublens | Switzerland
Application Note by Nanolive SA
2. Application Note by Nanolive SA
1
Abstract
Nanolive’s 3D Cell Explorer allows for the creation of very powerful 3D images and 4D time lapses
of living cells with very high spatio-temporal resolution (x,y:180nm; z:400nm; t:1.7sec). Moreover,
imaging with the 3D Cell Explorer does not require the use of any labels since the microscope directly
measures the refractive index of the different substructures of the cell. However, in the context of
molecular or cellular biology investigations, it can be useful to follow fluorescent markers combined
with the refractive index distribution to validate specific structures or to correlate fluorescent signals
and cellular states.
For this purpose, Nanolive developed the 3D Cell Explorer-fluo (https://nanolive.ch/fluo/) – a
complete solution that combines high precision tomographic data with high quality tri- or four-
channel epifluorescence provided by a CoolLed module (https://www.coolled.com/).
In this application note we will present the time lapse imaging of mouse embryonic stem cells
(mESCs) that have been genetically modified to express the fluorescence ubiquitination cell cycle
indicator (FUCCI), a two-color (red and green) indicator that allows to monitor the cell cycle phases.
We will explain how to use the 3D Cell Explorer-fluo to record movies that require both fluorescence
and 3D refractive index imaging and will propose a solution to analyze the resulting time lapse
experiment.
1. Introduction
Long-term imaging of fine cellular dynamics is today’s biggest challenge in cell biology (Frechin et
al., 2015; Kruse & Jülicher, 2005; Kueh, Champhekhar, Nutt, Elowitz, & Rothenberg, 2013; Skylaki,
Hilsenbeck, & Schroeder, 2016). The goal is not only to acquire snapshots of dynamic biological
systems, but to actually see such active processes (Muzzey, Gómez-Uribe, Mettetal, & van
Oudenaarden, 2009). We mentioned in the previous application note “Growing and Filming Stem
Cells with the 3D Cell Explorer” (https://nanolive.ch/wp-content/uploads/nanolive-application-
note-stem-cells-movies-web-1.pdf) that fluorescence microscopy, while being the current method
of choice in high-content live imaging approaches, induces phototoxicity when the sample is
stimulated at various wavelengths. This stress induces cellular damages and limits live imaging
possibilities especially with sensitive cell lines such as mammalian embryonic stem cells. Since the
3D Cell Explorer’s laser irradiates the sample with 100 times less energy than the gentlest of the
current fluorescent imaging approaches, it is technically possible to perform endless live imaging of
the 3D refractive index map of cells.
However, advanced molecular or cellular biology investigations rely more than ever on fluorescence
imaging to follow specific cellular structures, to cross-correlate fluorescent reporters or to follow
Figure 1: mESCs-FUCCI time lapse imaging. Follow this link to see the full movie: https://vimeo.com/271257958
3. Application Note by Nanolive SA
2
cellular states or signaling dynamics via specific fluorescent reporters. In this context, offering the
possibility to perform fluorescent imaging combined with 3D refractive index imaging is essential
and is now possible with the 3D Cell Explorer-fluo. The combination of fluorescence imaging and
label-free holotomography allows to further mitigate the phototoxic effect of fluorescence while
still benefiting from its advantages, especially the possibility to follow fluorescent proteins. The
alliance of these two techniques reduces the need of multiple light stimulation on the sample since
holotomography provides structural and dynamic information that can, given proper correlative
investigation and image analysis, replace fluorescence imaging or bridge the gap between two
consecutive fluorescent acquisitions.
In this application note we will show you how to monitor mouse embryonic stem cells over their entire
cell cycle by using holotomography combined with epifluorescence. We will first cover the required
equipment and knowledge, then guide you through the interface of the 3D Cell Explorer-fluo. Finally,
we will propose the analysis of a movie of mESCs that express the fluorescence ubiquitination cell
cycle indicator (FUCCI) construct which allows to monitor the cell cycle by observing the cells in both
the red and green fluorescent channels.
2. Prerequisites
First, you will need mouse Embryonic Stem Cells expressing the FUCCI construct (Sakaue-Sawano
et al., 2008) (mESCs-FUCCI), growing in a glass bottom dish compatible with the 3D Cell Explorer
(http://nanolive.ch/wp-content/uploads/nanolive-ibidi-labware.pdf) inside a typical cell culture
incubator. The sensor FUCCI consists of two fluorescent proteins, red (RFP) and a green (GFP), fused
respectively to cdt1 and geminin which are regulators of the cell cycle. In the G1 phase of the cell
cycle, geminin-GFP is degraded, which leaves only cdt1-RFP expressing red fluorescence within the
nuclei. In the S, G2, and M phases, cdt1-RFP is degraded and only geminin-GFP remains, resulting in
cells with green fluorescent nuclei.
During the G1/S transition, cdt1-RFP decreases and geminin-GFP increases, yet, both proteins are
present, therefore the nucleus appears yellow fluorescent since the green and red images are
overlaid. Thus, during the cell cycle, a cell’s nucleus will display colors from red to yellow to green
before dividing when no color is observed in the nucleus anymore.
Notes:
1) This document is not about mESCs cell culture which is a research field on its own. If you wish
to start a cell culture of this sort in your laboratory, please approach a specialist who will explain
the necessary dish coating and medium composition for maintaining this very specific cell type.
2) The content of this application note can be applied to any fluorescent cell line by adapting it to
the specifics of the used fluorescent marker or reporters.
Secondly, you will need Nanolive’s top stage incubator equipment. This includes the top stage
incubation chamber, a controller pad, and a humidity system. We recommend using our CO2
mixer and air pump that will ensure a proper control of CO2
proportions and will help you save
some money on compressed air. Learn how to set up your Nanolive top stage incubator here:
http://nanolive.ch/supporting-material/. Learn how to set up a perfect environment control for mESCs
here: https://nanolive.ch/wp-content/uploads/nanolive-application-note-stem-cells-movies-web-1.pdf
Finally, you will need the latest version of STEVE (Version 1.6 at least) to benefit from the latest export
functionalities and image post-processing features. You will also need the software Cell Profiler
3 (http://cellprofiler.org/releases/) as well as FIJI (https://fiji.sc/#download). For data plotting and
presentation, which is an entire topic on its own, we recommend a Python IDE, R or Matlab.
4. Application Note by Nanolive SA
3
Application Note by Nanolive SA
3. Setting up a fluorescence acquisition
As mentioned in a previous application note, the 3D Cell Explorer is capable of unmatched time
lapse performances because of the absence of phototoxicity. Given proper data management
and environment control, it is possible to perform endless live imaging of fragile samples at great
temporal resolution (1 image every 1.7 seconds). We will see here that having the possibility to do
holotomography and fluorescence imaging simultaneously has significant advantages, however, this
implies that the phototoxicity induced by the fluorescence imaging regime needs to be managed.
Once your cells of interest are in focus and ready to be imaged in the incubation chamber, click on
the 4D acquisition button in STEVE. You will be asked to define the time lapse base frequency, and
how the refractive index (RI) and the individual fluorescent channels should be programmed over
this base frequency. The goal here is to reduce phototoxicity by reducing fluorescence acquisition
frequency but to maintain a high imaging frequency with the RI acquisition.
In this application note we define 15s between each image, with an RI volume acquired every 15
seconds while the green and red fluorescent channels are acquired every 30 time points i.e. every 7
minutes and 30 seconds. This is a rather challenging fluorescence imaging regime for stem cells, but
since the microscope has a powerful camera and a formidable development has been made on the
FUCCI construct that produces a very bright signal, we are taking advantage of these tools to reduce
the light exposure to its strict minimum. Click OK, set up the green and red fluorescent channels to
100 milliseconds, 1% power and 50% of gain. Finally, define a location for your acquisition file to be
stored (Figure 2).
If these settings do not provide nice results and you consider to further stimulate your fluorescent
dye, it is better to increase the exposure time before increasing light power in order to maintain
acute light exposure low while still increasing the stimulation of the fluorescent proteins, GFP and
RFP in this case.
Figure 2: Set-up of fluorescence time lapse imaging
Figure 3: mESCs-FUCCI time lapse imaging
5. Application Note by Nanolive SA
4
4. Data export
The export tool (covered extensively in the previous application note: “Growing and Filming Stem
Cells with the 3D Cell Explorer” (https://nanolive.ch/wp-content/uploads/nanolive-application-note-
stem-cells-movies-web-1.pdf) allows to export each time point and each channel separately and to
organize your dataset properly with the green and red fluorescent channels in .tif (or .png) format.
5. Creating a set of nuclei signal images
Note: Here we propose one out of many image analysis solutions. What follows should serve
as inspiration for your own fluorescence combined with holotomography experiment which
will most likely contain very different fluorescent signals, cellular structures and dynamics.
Using FIJI you can now open the red and green image sequences (File > Import > Image sequence) in
two independent stacks by adding the proper name based on your image channel.
Then combine the two stacks so that the green and red signals become a single additive signal
that will serve for nuclei signal (Process > Calculator Plus...). Save the frames as single images using
the same format as the green and red channel image sets in the same folder and with a clear file
identifier.
6. Analysis of mESCs cell cycle
We will now briefly introduce you to Cell Profiler 3 and will quickly mention the image pipeline
that you can build for tracking cells and extracting dynamic values from living cells in your movie.
However, Cell Profiler 3 is a wonderful tool that requires some experience and we recommend
you follow their tutorial first, (http://cellprofiler.org/tutorials/) since it is not possible to cover all
necessary Cell Profiler’s details here.
We created above three sets of images: the green channel, the red channel and a sum of these
two channels that will serve as the nuclear signal. Load these three sets of images and use the
nuclear signal to feed the module for primary object detection. It is then easy to obtain a nice nuclei
segmentation and individual nucleus objects. If you wish, each z-slice of the RI signal of your cell can
be used for detecting the cellular boundaries at a given height.
To do so, create the appropriate image sequences in separate folders (repeat paragraph 4 and 5 on
the RI slices of interest), load them and use them for secondary object detection with the segmented
nuclei as the seed primary object. Note that with the 3D Cell Explorer-fluo you could add a third blue
or far red channel to observe a dedicated stain for the nuclei, which would ease the object detection
steps. However, you would need a proper cell line expressing such markers and you would need to
consider the extra amount of phototoxicity that this extra exposure would induce.
The segmented nuclei can now be tracked with the track object module as they are large and well
defined. If you extract their features with the various “measure—“ modules of Cell Profiler, you can
produce this type of data representation for various cell parameters. For advanced ideas on how to
use the FUCCI tag please look at (Sakaue-Sawano et al., 2008).
6. Application Note by Nanolive SA
5
7. General Hardware & Software Requirements
3D Cell Explorer models:
3D Cell Explorer-fluo
Incubation system:
Nanolive Top Stage Incubator
Microscope stage:
Normal 3D Cell Explorer stage
Hi-grade 3D Cell Explorer manual stage
Software:
STEVE – version 1.6 and higher.
FIJI
Cell Profiler 3
Figure 4: tracking FUCCI green and red fluorescent signals recorded with the 3D Cell Explorer-fluo
7. Application Note by Nanolive SA
6
8. References
Frechin, M., Stoeger, T., Daetwyler, S., Gehin, C., Battich, N., Damm, E.-M., ... Pelkmans, L. (2015). Cell-
intrinsic adaptation of lipid composition to local crowding drives social behaviour. Nature, 523(7558),
88–91. https://doi.org/10.1038/nature14429
Kruse, K., & Jülicher, F. (2005). Oscillations in cell biology. Current Opinion in Cell Biology, 17(1), 20–26.
https://doi.org/10.1016/j.ceb.2004.12.007
Kueh, H. Y., Champhekhar, A., Nutt, S. L., Elowitz, M. B., & Rothenberg, E. V. (2013). Positive Feedback
Between PU.1 and the Cell Cycle Controls Myeloid Differentiation. Science (New York, N.Y.), 670. https://
doi.org/10.1126/science.1240831
Muzzey, D., Gómez-Uribe, C. a., Mettetal, J. T., & van Oudenaarden, A. (2009). A Systems-Level Analysis
of Perfect Adaptation in Yeast Osmoregulation. Cell, 138(1), 160–171. https://doi.org/10.1016/j.
cell.2009.04.047
Sakaue-Sawano, A., Kurokawa, H., Morimura, T., Hanyu, A., Hama, H., Osawa, H., ... Miyawaki, A. (2008).
Visualizing Spatiotemporal Dynamics of Multicellular Cell-Cycle Progression. Cell, 132(3), 487–498.
https://doi.org/10.1016/j.cell.2007.12.033
Skylaki, S., Hilsenbeck, O., & Schroeder, T. (2016). Challenges in long-term imaging and quantification of
single-cell dynamics. Nature Biotechnology, 34(11), 1137–1144. https://doi.org/10.1038/nbt.3713