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.
Flow cytometry (FCM) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument.
Introduction, the principle of immunofluorescence, Technique, Fluorescent microscope and its components, Application and types of immunofluorescence, Direct and indirect immunofluorescence, FACS (Fluorescence-activated cell sorting), Uses and limitations of Immunofluorescence
Flow cytometry is a technique used to detect and measure physical and chemical characteristics of cells or particles in a fluid sample. A sample is injected into the flow cytometer where cells pass through a laser beam, scattering light in a characteristic way. Cells are often labeled with fluorescent markers that are detected. Data on multiple parameters can be collected simultaneously. The flow cytometer has fluidic, optic, and electronic systems. Applications include cell sorting, apoptosis detection, cell cycle analysis, and clinical uses like diagnosing hematologic malignancies.
Flow cytometry is a technique that uses lasers to detect and measure physical characteristics of cells or particles in a fluid mixture. Cells passing through the laser beam scatter light and may fluoresce if stained with fluorescent antibodies. Forward scatter detects cell size while side scatter detects internal complexity. Fluorescence identifies protein or nucleic acid expression. Data is converted to electrical signals and analyzed by a computer. Flow cytometry is used in clinical applications like detecting malignancy and monitoring treatment response. It provides information about cell phenotypes that helps diagnose hematological conditions.
Flow cytometry definition, principle, parts, steps, types, usesGayathri Devi S
Flow cytometry is a technique that uses lasers to detect and measure physical and chemical characteristics of cells or particles in fluid suspension. Cells pass through a laser beam, which scatters light and causes fluorescence that is detected by sensors. Measurements of scattered and fluorescent light provide information about cell size, granularity, and expression of targeted proteins or nucleic acids. Flow cytometry allows rapid multi-parameter analysis of individual cells in heterogeneous populations and is widely used for clinical, research, and industrial applications.
Flow cytometry is an optical technique used to analyze physical and chemical characteristics of cells and other biological particles as they flow in a fluid stream through a beam of light. It allows for multiparameter analysis of cells based on light scattering, fluorescence, and other optical properties. Key components include a flow cell to hydrodynamically focus cells into a single file, lasers as light sources, optical collection systems, and detectors. Flow cytometry finds applications in research, clinical diagnostics, and agriculture.
This presentation discusses immunofluorescence techniques. Immunofluorescence involves tagging antibodies with fluorescent dyes so that antigen-antibody complexes can be visualized under a fluorescent microscope. It describes the history and principles of direct and indirect immunofluorescence. Direct immunofluorescence detects in vivo antibodies bound to tissue antigens, while indirect immunofluorescence detects antibodies in patient serum. Both techniques have advantages like sensitivity but also disadvantages like potential cross-reactivity.
Flow cytometry is a technique used in immunology that analyzes physical and chemical properties of cells flowing through a laser beam. Cells are hydrodynamically focused into a stream and passed through the laser one by one. Forward and side scattered light and fluorescence emissions are detected and analyzed by software. Flow cytometry uses monoclonal antibodies to identify and quantify immune cell subsets and expression levels. It has numerous applications in clinical and research immunology due to its ability to rapidly analyze multiple parameters on thousands of individual cells.
Flow cytometry (FCM) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument.
Introduction, the principle of immunofluorescence, Technique, Fluorescent microscope and its components, Application and types of immunofluorescence, Direct and indirect immunofluorescence, FACS (Fluorescence-activated cell sorting), Uses and limitations of Immunofluorescence
Flow cytometry is a technique used to detect and measure physical and chemical characteristics of cells or particles in a fluid sample. A sample is injected into the flow cytometer where cells pass through a laser beam, scattering light in a characteristic way. Cells are often labeled with fluorescent markers that are detected. Data on multiple parameters can be collected simultaneously. The flow cytometer has fluidic, optic, and electronic systems. Applications include cell sorting, apoptosis detection, cell cycle analysis, and clinical uses like diagnosing hematologic malignancies.
Flow cytometry is a technique that uses lasers to detect and measure physical characteristics of cells or particles in a fluid mixture. Cells passing through the laser beam scatter light and may fluoresce if stained with fluorescent antibodies. Forward scatter detects cell size while side scatter detects internal complexity. Fluorescence identifies protein or nucleic acid expression. Data is converted to electrical signals and analyzed by a computer. Flow cytometry is used in clinical applications like detecting malignancy and monitoring treatment response. It provides information about cell phenotypes that helps diagnose hematological conditions.
Flow cytometry definition, principle, parts, steps, types, usesGayathri Devi S
Flow cytometry is a technique that uses lasers to detect and measure physical and chemical characteristics of cells or particles in fluid suspension. Cells pass through a laser beam, which scatters light and causes fluorescence that is detected by sensors. Measurements of scattered and fluorescent light provide information about cell size, granularity, and expression of targeted proteins or nucleic acids. Flow cytometry allows rapid multi-parameter analysis of individual cells in heterogeneous populations and is widely used for clinical, research, and industrial applications.
Flow cytometry is an optical technique used to analyze physical and chemical characteristics of cells and other biological particles as they flow in a fluid stream through a beam of light. It allows for multiparameter analysis of cells based on light scattering, fluorescence, and other optical properties. Key components include a flow cell to hydrodynamically focus cells into a single file, lasers as light sources, optical collection systems, and detectors. Flow cytometry finds applications in research, clinical diagnostics, and agriculture.
This presentation discusses immunofluorescence techniques. Immunofluorescence involves tagging antibodies with fluorescent dyes so that antigen-antibody complexes can be visualized under a fluorescent microscope. It describes the history and principles of direct and indirect immunofluorescence. Direct immunofluorescence detects in vivo antibodies bound to tissue antigens, while indirect immunofluorescence detects antibodies in patient serum. Both techniques have advantages like sensitivity but also disadvantages like potential cross-reactivity.
Flow cytometry is a technique used in immunology that analyzes physical and chemical properties of cells flowing through a laser beam. Cells are hydrodynamically focused into a stream and passed through the laser one by one. Forward and side scattered light and fluorescence emissions are detected and analyzed by software. Flow cytometry uses monoclonal antibodies to identify and quantify immune cell subsets and expression levels. It has numerous applications in clinical and research immunology due to its ability to rapidly analyze multiple parameters on thousands of individual cells.
This document provides an overview of flow cytometry, including its history, principles, components, applications, and quality control. Flow cytometry involves measuring physical and chemical properties of cells or particles as they pass through a fluid stream. Key developments included Moldavan's early work in 1934 and the coinage of the term "flow cytometry" in the mid-1970s. The three main systems of a flow cytometer are fluidics to transport particles, optics like lasers and detectors, and electronics to convert light signals to data. Applications include clinical uses like detection of bacteria and characterization of cells and particles across many fields.
This document discusses flow cytometry, which measures properties of cells as they flow through a fluid stream. It describes the principles and components of a flow cytometer, including the flow system that orders cells into a single-file stream, the optical system that illuminates cells and detects light scattering/fluorescence, and the electronic system that converts signals to digital data. The document outlines how flow cytometry is used to analyze physical/antigen characteristics of cells and identifies different cell types. It provides examples of clinical applications like leukemia diagnosis and CD4 counting in HIV/AIDS.
Flow cytometry is a technique that uses lasers and fluorescence to count, examine, and sort cells suspended in fluid based on their optical and physical properties. Cells are stained with fluorescent markers and passed in a stream through a laser beam, which causes them to emit light at different wavelengths. Detectors then measure the light scattered and emitted to identify cell characteristics like size, granularity, and marker expression. Data is analyzed using software to identify cell populations and phenotypes. Flow cytometry has many applications in fields like immunology, hematology, and stem cell research.
Flow cytometry is a technique that uses lasers and fluorescence to count, examine, and sort cells suspended in fluid based on their optical and physical properties. Cells are stained with fluorescent markers and passed in a stream through a laser beam, which causes them to emit light at different wavelengths. Detectors then measure the light scattered and emitted to identify cell characteristics like size, granularity, and marker expression. Data is analyzed using software to identify cell populations and phenotypes. Flow cytometry has many applications in fields like immunology, hematology, and oncology for analyzing blood, tissue, and other cell samples.
Flow cytometry is a technique that uses laser light scattering and fluorescence to detect and measure physical and chemical characteristics of cells or particles in a fluid mixture. A flow cytometer passes cells in a fluid stream through a laser beam, measuring light scattering and fluorescence to identify cell types and count cell populations. Measurements of forward-scattered light, side-scattered light, and fluorescence emissions are used to distinguish cell types and detect the expression of cellular molecules like proteins or nucleic acids.
Flow cytometry is a technique used in biotechnology that uses lasers to measure cells labeled with fluorescent antibodies as they flow in a liquid stream. It allows for cell counting, sorting, biomarker detection, and analysis of cell properties. In preclinical studies, flow cytometry is used to measure immune cells, tissue culture cells, and antibodies binding to immune molecules. It utilizes multiple fluorochrome-conjugated antibodies that emit photons at distinct wavelengths when excited by lasers, enabling detection and analysis of specific cell populations. Working with flow cytometry experts can help propel preclinical research programs forward more quickly through specialized sample analysis.
Immunoelectron microscopy combines electron microscopy with antibody labeling to localize antigens within cells and tissues at the nanoscale level. It works by labeling samples with antibodies attached to electron-dense gold particles, which are then visible under the electron microscope. This allows researchers to determine the precise subcellular localization of proteins and study biological processes and structures at high resolution. Key applications include analyzing host-pathogen interactions, plant virus diagnosis, and identifying protein localization in neurons.
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
Principle and applications of flow cytometryDinesh Gangoda
Flow cytometry is a technique used to analyze physical and chemical characteristics of cells or particles in suspension as they flow in a fluid stream past a laser. It works by fluorescently labeling cells and components, then passing them in single file through a laser which detects scattered and fluorescent light. This allows for quantitative and qualitative analysis of cell populations. Properties like size, granularity, and fluorescence intensity can be measured. Main applications include immunophenotyping, cell sorting, cell cycle analysis, apoptosis analysis, and measuring intracellular calcium flux and cell proliferation in response to stimuli.
Flow cytometry is a laser-based technique used to analyze physical and chemical properties of cells/particles as they flow in a fluid stream through a laser beam. It allows for rapid, multiparameter analysis of individual cells. The key components of a flow cytometer are fluidics, optics, and electronics systems. Lasers illuminate cells and light is scattered and detected to analyze properties like size, granularity, and fluorescence. Applications in food microbiology include quantifying bacteria in foods and beverages, monitoring dairy starters, and studying probiotics under stress conditions.
This presentation shows a basic overview of all aspects of Fluorescence Microscopy including its description, history, mechanism, applications, advantages, limitations, and some examples of studies that used this technique.
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 is a technique that uses laser-based technology to count, sort, and profile cells in a fluid mixture. A flow cytometer passes cells in single file through a laser, and measures light scatter and fluorescence to obtain quantifiable data on physical and chemical characteristics of cells. Key components include a fluidic system to hydrodynamically focus cells through the laser, an optics system using lasers and detectors to measure light signals, and an electronics system to convert these signals into electronic data that can be analyzed. Common applications include immunophenotyping, apoptosis measurement, and cell sorting.
Introduction
Definition
Basic mechanism
Prerequisite of flow cytometer
Components of flow cytometry
Flow system
Optics system
Concept of scattering
Advantage
Limitation
Application
Conclusion
References
Flow cytometry is a technique used in cell biology that allows for the analysis of physical and chemical characteristics of cells as they flow in a fluid stream through a beam of light. It provides rapid multi-parametric analysis of cells based on light scattering, fluorescence, and other optical properties. Flow cytometry gives information about cell size, granularity, and the expression of cell surface markers or intracellular proteins through the use of fluorescent probes. It has many applications in fields like immunology, cancer research, and infectious disease.
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.
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 discusses several common microbiological techniques used to study microbes. It describes microscopy techniques like brightfield and phase contrast microscopy used to view microbes. It also discusses gel electrophoresis to separate DNA, RNA, and proteins by size and charge. Flow cytometry is described as a method to analyze physical and chemical properties of particles using fluorescence. Additional techniques covered include cell counting, spectrophotometry, plating, and polymerase chain reaction (PCR) to amplify DNA segments.
This document discusses various principles and techniques for measuring antigen-antibody interactions, including agglutination tests, precipitation tests, radioimmunoassays, enzyme-linked immunosorbent assays, immunofluorescence, and complement fixation. It defines key terms like affinity, specificity, cross-reactivity and describes how factors like antigen-antibody ratio affect measurements. Specific techniques covered include agglutination, passive agglutination, agglutination inhibition, radial immunodiffusion, immunoelectrophoresis, competitive and non-competitive radioimmunoassays/ELISAs, direct and indirect immunofluorescence, and complement fixation.
This document summarizes autoantibody patterns, associated diseases, and diagnostic testing strategies for various autoimmune conditions. It lists common autoantibodies found in systemic lupus erythematosus, mixed connective tissue disease, progressive systemic sclerosis, primary Sjögren's syndrome, autoimmune hepatitis, primary biliary cirrhosis, drug-induced lupus, and other conditions. It also provides information on screening tests, specific assays, and interpretation guidelines to help clinicians evaluate patients for autoimmune diseases.
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This document provides an overview of flow cytometry, including its history, principles, components, applications, and quality control. Flow cytometry involves measuring physical and chemical properties of cells or particles as they pass through a fluid stream. Key developments included Moldavan's early work in 1934 and the coinage of the term "flow cytometry" in the mid-1970s. The three main systems of a flow cytometer are fluidics to transport particles, optics like lasers and detectors, and electronics to convert light signals to data. Applications include clinical uses like detection of bacteria and characterization of cells and particles across many fields.
This document discusses flow cytometry, which measures properties of cells as they flow through a fluid stream. It describes the principles and components of a flow cytometer, including the flow system that orders cells into a single-file stream, the optical system that illuminates cells and detects light scattering/fluorescence, and the electronic system that converts signals to digital data. The document outlines how flow cytometry is used to analyze physical/antigen characteristics of cells and identifies different cell types. It provides examples of clinical applications like leukemia diagnosis and CD4 counting in HIV/AIDS.
Flow cytometry is a technique that uses lasers and fluorescence to count, examine, and sort cells suspended in fluid based on their optical and physical properties. Cells are stained with fluorescent markers and passed in a stream through a laser beam, which causes them to emit light at different wavelengths. Detectors then measure the light scattered and emitted to identify cell characteristics like size, granularity, and marker expression. Data is analyzed using software to identify cell populations and phenotypes. Flow cytometry has many applications in fields like immunology, hematology, and stem cell research.
Flow cytometry is a technique that uses lasers and fluorescence to count, examine, and sort cells suspended in fluid based on their optical and physical properties. Cells are stained with fluorescent markers and passed in a stream through a laser beam, which causes them to emit light at different wavelengths. Detectors then measure the light scattered and emitted to identify cell characteristics like size, granularity, and marker expression. Data is analyzed using software to identify cell populations and phenotypes. Flow cytometry has many applications in fields like immunology, hematology, and oncology for analyzing blood, tissue, and other cell samples.
Flow cytometry is a technique that uses laser light scattering and fluorescence to detect and measure physical and chemical characteristics of cells or particles in a fluid mixture. A flow cytometer passes cells in a fluid stream through a laser beam, measuring light scattering and fluorescence to identify cell types and count cell populations. Measurements of forward-scattered light, side-scattered light, and fluorescence emissions are used to distinguish cell types and detect the expression of cellular molecules like proteins or nucleic acids.
Flow cytometry is a technique used in biotechnology that uses lasers to measure cells labeled with fluorescent antibodies as they flow in a liquid stream. It allows for cell counting, sorting, biomarker detection, and analysis of cell properties. In preclinical studies, flow cytometry is used to measure immune cells, tissue culture cells, and antibodies binding to immune molecules. It utilizes multiple fluorochrome-conjugated antibodies that emit photons at distinct wavelengths when excited by lasers, enabling detection and analysis of specific cell populations. Working with flow cytometry experts can help propel preclinical research programs forward more quickly through specialized sample analysis.
Immunoelectron microscopy combines electron microscopy with antibody labeling to localize antigens within cells and tissues at the nanoscale level. It works by labeling samples with antibodies attached to electron-dense gold particles, which are then visible under the electron microscope. This allows researchers to determine the precise subcellular localization of proteins and study biological processes and structures at high resolution. Key applications include analyzing host-pathogen interactions, plant virus diagnosis, and identifying protein localization in neurons.
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
Principle and applications of flow cytometryDinesh Gangoda
Flow cytometry is a technique used to analyze physical and chemical characteristics of cells or particles in suspension as they flow in a fluid stream past a laser. It works by fluorescently labeling cells and components, then passing them in single file through a laser which detects scattered and fluorescent light. This allows for quantitative and qualitative analysis of cell populations. Properties like size, granularity, and fluorescence intensity can be measured. Main applications include immunophenotyping, cell sorting, cell cycle analysis, apoptosis analysis, and measuring intracellular calcium flux and cell proliferation in response to stimuli.
Flow cytometry is a laser-based technique used to analyze physical and chemical properties of cells/particles as they flow in a fluid stream through a laser beam. It allows for rapid, multiparameter analysis of individual cells. The key components of a flow cytometer are fluidics, optics, and electronics systems. Lasers illuminate cells and light is scattered and detected to analyze properties like size, granularity, and fluorescence. Applications in food microbiology include quantifying bacteria in foods and beverages, monitoring dairy starters, and studying probiotics under stress conditions.
This presentation shows a basic overview of all aspects of Fluorescence Microscopy including its description, history, mechanism, applications, advantages, limitations, and some examples of studies that used this technique.
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 is a technique that uses laser-based technology to count, sort, and profile cells in a fluid mixture. A flow cytometer passes cells in single file through a laser, and measures light scatter and fluorescence to obtain quantifiable data on physical and chemical characteristics of cells. Key components include a fluidic system to hydrodynamically focus cells through the laser, an optics system using lasers and detectors to measure light signals, and an electronics system to convert these signals into electronic data that can be analyzed. Common applications include immunophenotyping, apoptosis measurement, and cell sorting.
Introduction
Definition
Basic mechanism
Prerequisite of flow cytometer
Components of flow cytometry
Flow system
Optics system
Concept of scattering
Advantage
Limitation
Application
Conclusion
References
Flow cytometry is a technique used in cell biology that allows for the analysis of physical and chemical characteristics of cells as they flow in a fluid stream through a beam of light. It provides rapid multi-parametric analysis of cells based on light scattering, fluorescence, and other optical properties. Flow cytometry gives information about cell size, granularity, and the expression of cell surface markers or intracellular proteins through the use of fluorescent probes. It has many applications in fields like immunology, cancer research, and infectious disease.
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.
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 discusses several common microbiological techniques used to study microbes. It describes microscopy techniques like brightfield and phase contrast microscopy used to view microbes. It also discusses gel electrophoresis to separate DNA, RNA, and proteins by size and charge. Flow cytometry is described as a method to analyze physical and chemical properties of particles using fluorescence. Additional techniques covered include cell counting, spectrophotometry, plating, and polymerase chain reaction (PCR) to amplify DNA segments.
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There is increasing confidence that cell therapies will soon play a role in the treatment of autoimmune disorders, but the extent of this impact remains to be seen. Early readouts on autologous CAR-Ts in lupus are encouraging, but manufacturing and cost limitations are likely to restrict access to highly refractory patients. Allogeneic CAR-Ts have the potential to broaden access to earlier lines of treatment due to their inherent cost benefits, however they will need to demonstrate comparable or improved efficacy to established modalities.
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2. Immunofluorescence
• Immunofluorescence : Immunofluorescence is a powerful
technique that utilizes fluorescent-labeled antibodies to
detect specific target antigens..
Fluorescein is a dye which emits greenish fluorescence under
UV light. It can be tagged to immunoglobulin molecules.
• This technique is sometimes used to make viral plaques more
readily visible to the human eye.
• Immunofluorescent labeled tissue sections are studied using a
fluorescence microscope. 2
3. • Immunofluorescence (IF) microscopy is a particularly robust and broadly
applicable method generally used by researchers to assess both the
localization and endogenous expression levels of proteins of interest.
• Immunofluorescence microscopy is a widely used example of
immunostaining and is a form of immunohistochemistry based on the use
of fluorophores to visualize the location of bound antibodies.
• The effective application of this method comprises several considerations,
including the nature of the antigen, specificity and sensitivity of the
primary antibody, properties of the fluorescent label, permeabilization
and fixation technique of the sample, and fluorescence imaging of the
cell.
• Immunofluorescence can be used on tissue sections, cultured cells or
individual cells that are fixed by a variety of methods. Antibodies can be
used in this method to analyze the distribution of proteins, glycoproteins
and other antigen targets including small biological and non-biological
molecules.
3
4. Examples Of Fluorescent Dyes
Fluorescein Rhodamine
4
Confocal image to detect
phosphorylated AKT (green)
in cardiomyocytes infected with
adenovirus
6. • There are two ways of doing IF staining
• Direct immunofluorescence
• Indirect immunofluorescence
1.Direct immunofluorescence
• It’s just a simple & a very common procedure in this regard.
• Ag is fixed on the slide
• Fluorescein labeled Ab’s are layered over it
• Slide is washed to remove unattached Ab’s
• Examined under UV light in an fluorescent microscope
• The site where the Ab attaches to its specific Ag will show apple
green fluorescence
• Use: Direct detection of Pathogens or their Ag’s in tissues or in
pathological samples.
6
7. 2. Indirect immunofluorescence:
• Indirect test is a double-layer technique
• The unlabelled antibody is applied directly to the tissue substrate
• Treated with a fluorochrome-conjugated anti-immunoglobulin
serum.
7
Advantage over direct IF
Since several fluorescent anti-
immunoglobulins can bind to
each antibody present in the first
layer, the fluorescence is brighter
than the direct test.
It is also more time-efficient
since it is only one signal
labelled reagent, the anti-
immunoglobulin, is prepared
during the lengthy conjugation
process.
8. What Immunoflouroscence Does
Immunoflourescence is a Microscopic-based technique, used clinically to
diagnose certain cutaneous diseases ( e.g; Lyme Disease) by the
detection of AG:AB Complexes.
Techniques including DIF, IDIF & Salt-split Skin are utilized depending on
clinical scenario.
DIF is performed on patient’s skin using flourophore-labeled antibodies
that directly bind to pathogenic autoantibody-antigen complexes in the
skin.
• IDIF techniques are used in Dermatology primarily to detect circulating
pathogenic autoantibodies.
LIMITATIONS
• Fluorescence signals depend on the quality & Concentration of
antibodies, proper handling of specimen & detection with appropriate
secondary antibodies.
8
9. Part 1: Tissue preparation
1. Fixation
Fresh unfixed, fixed, or formalin fixation and paraffin embedding
2. Sectioning
3. Whole Mount Preparation
Part 2: pretreatment
1. Antigen retrieval : Proteolytic enzyme method and Heat-induced method
2. Inhibition of endogenous tissue components: 3% H2O2, 0.01% avidin
3. Blocking of nonspecific sites : 10% normal serum
Part 3: staining
Make a selection based on the type of specimen, the primary antibody, the
degree of sensitivity and the processing time required. 9
Immunohistochemistry Protocol
10. • Same as a conventional light
microscope (CM) with added features
to enhance its capabilities.
• CM - visible light (400-700
nanometers)
• FM- higher intensity light source which
excites a fluorescent species in a
sample.
• This fluorescent species in turn emits a
lower energy light of a longer
wavelength that produces the
magnified image instead of the original
light source.
Fluorescence microscope
10
11. Fluorescence microscope
Applications
Imaging structural components of small specimens,
(cells)
viability studies on cell populations (alive or dead)
Imaging the genetic material within a cell (DNA and RNA)
Viewing specific cells within a larger population with
techniques such as FISH
11
14. Types of Fluorescent Microscopes
Immunofluoresence microscopy can be used in several
microscope designs for analysis of
immunofluorescence samples.
•epifluorescence microscope.
•confocal microscopy is widely used, newer designs of
super resolution microscopes, such as
•STED (Stimulated Emission Depletion) microscopy,
allow for nanoscopy - much higher resolution.
14
15. What is flow cytometry?
Flow cytometry is a method of measuring multiple physical and
chemical characteristics of particles by optical means.
Such as Peripheral blood, Bone marrow cells, Bacteria, Yeast
Applications of Flow Cytometry.
•Cell size.
• Cytoplasmic granularity.
• Cell surface antigens (phenotyping).
• Apoptosis.
• Intracellular cytokine production.
• Intracellular signalling.
• Gene reporter (GFP).
• Cell cycle, DNA content, composition, synthesis.
• Bound and free calcium.
• Cell proliferation (BRDU and CFSE)
• Cell sorting, single cell cloning (clonecyt)
Flow cytometry
15
16. A suspension of single cells or other particles
in a suitable buffer, usually PBS.
Typical density : 105
- 107
cells / ml
+
Incubate Acquire
Phenotyping, Size and granularity detection:
Sample requirements:
16
17. FACS - Flow cytometry
Flow cytometry (abbreviated: FCM) is a technique for counting
and examining microscopic particles, such as cells and
chromosomes, by suspending them in a stream of fluid and
passing them by an electronic detection apparatus.
It allows simultaneous multi - parametric analysis of the
physical and/or chemical characteristics of up to thousands of
particles per second.
17
18. FACS - Flow cytometry
Fluorescence-activated cell sorting is a specialized type of
flow cytometry. It provides a method for sorting a
heterogeneous mixture of biological cells into two or more
containers, one cell at a time, based upon the specific light
scattering and fluorescent characteristics of each cell.
It is a useful scientific instrument, as it provides fast,
objective and quantitative recording of fluorescent signals
from individual cells as well as physical separation of cells
of particular interest.
FACS is a trademark of Becton Dickinson Immunocytometry
Systems (BDIS). All FACS instruments are BDIS systems, but
not all cytometers are FACS.
18
19. 19
•The cell suspension is entrained in the center of a narrow,
rapidly flowing stream of liquid. The flow is arranged so that
there is a large separation between cells relative to their
diameter.
•A vibrating mechanism causes the stream of cells to break into
individual droplets. The system is adjusted so that there is a low
probability of more than one cell per droplet.
•Just before the stream breaks into droplets, the flow passes
through a fluorescence measuring station where the
fluorescent character of interest of each cell is measured.
FACS – Working principle
20. • An electrical charging ring is placed just at the point where
the stream breaks into droplets. A charge is placed on the
ring based on the immediately prior fluorescence intensity
measurement, and the opposite charge is trapped on the
droplet as it breaks from the stream.
• The charged droplets then fall through an electrostatic
deflection system that diverts droplets into containers
based upon their charge.
• In some systems, the charge is applied directly to the
stream, and the droplet breaking off retains charge of the
same sign as the stream. The stream is then returned to
neutral after the droplet breaks off. 20
FACS – Working principle
21. FACS - Flow cytometry
• Flow cytometry integrates electronics, fluidics, computer, optics,
software, and laser technologies in a single platform.
21
Ultrasonic
Transducer
488nm Formard Light Scatter Detector
Collimated Light Path Through
Dichroic and Band Pass Filters
SS FL2
FL1
FL4
FL3
Pulse Height
(0-10Volts)
Time(useconds)
Pressurized
1X
PBS(Sheath)
Pressurized Cell
Sample
Analog Data
PMTs
Video Tutorial:
Thermo Scientific
Flow Cytometer
27. From Fluorescence to Computer Display
• Individual cell fluorescence quanta is picked up by the
various detectors(PMT’s-photo multiplier tubes as
detectors).
• PMT’s convert light into electrical pulses.
• These electrical signals are amplified and digitized using
Analog to Digital Converters (ADC’s).
• Each event is designated a channel number (based on the
fluorescence intensity as originally detected by the PMT’s)
on a 1 Parameter Histogram or 2 Parameter Histogram.
• All events are individually correlated for all the parameters
collected.
27
28. Light Scattering, 2 Parameter Histogram
28
Forward Light Scatter (FLS)
90 degree
Light Scatter
Bigger
More
Granular
Live Cells
Bigger
Cells
Dead
Cells
Apoptotic
Cells
X Axis
Y Axis
29. 2 Parameter Histogram
29
FITC FL
PE FL
Negative
Population
Single Positive
FITC
Population
Single
Positive PI
Population
Double Positive
Population
30. 1 Parameter Histogram
30
1 2 3 4 6 7 150 160 170 .. 190
Channel Number
Positive
Negative
Brighter
Dimmer
Count
1
4
6
Fluorescence picked up from the FITC
PMT
31. Representative - Flow Cytometry Data
31
Smaller
Region,
Live cells
mostly
Larger Region
includes all cells