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Flow Cytometry
Rajan Paudel
M.Sc. Medical Microbiology
Contents
• Introduction
• History
• Principle
• Flow cytometer
• Data analysis
• Applications
Introduction
• Flow Cytometry is a technique used to detect and measure physical
and chemical characteristics of a population of cells or particles.
• A sample containing cells or particles is suspended in a fluid and
injected into the flow cytometer instrument.
• The sample is focused to ideally flow one cell at a time through a laser
beam and the light scattered is characteristic to the cells and their
components.
• Cells are often labeled with fluorescent markers so that light is first
absorbed and then emitted in a band of wavelengths.
• By utilizing highly specific antibodies labeled with fluorescent
conjugates, FACS analysis allows us to simultaneously collect data on,
and sort a biological sample by a nearly limitless number of different
parameters.
History
• In 1879 Lord Rayleigh observed that fluid emerging from an orifice
breaks into a series of droplets.
• In 1949, Wallace Coulter filed, “Means for Counting Particles
Suspended in a Fluid”.
• In 1965, Mack J. Fulwyler reported the first flow cytometry
instrument capable of sorting cells.
• In 1968 Wolfgang Göhde designed a fluorescence based flow
cytometer.
• Leonard Herzenberg, an immunologist at Stanford, coined the term
‘FACS’ – Fluorescence Activated Cell Sorter.
• Becton Dickinson (BD) owns the FACS trade name and launched the
first commercial instrument, FACS-1 in the early 1970’s.
• The first label-free high-frequency impedance flow cytometer, Ampha
Z30, was introduced by Amphasys (2012).
• Flow cytometric immunophenotyping (FCI) first appeared in clinical
laboratories in the 1980s, in the wake of the AIDS epidemic.
• Initially utilized to assess CD4 T-cells, the technique was soon applied
to lymphoid and eventually myeloid neoplasms.
Principle
• Fluorescent dyes may bind or intercalate with different cellular
components such as DNA or RNA. Additionally, antibodies conjugated
to fluorescent dyes can bind specific proteins on cell membranes or
inside cells.
• When labeled cells are passed by a light source, the fluorescent
molecules are excited to a higher energy state.
• Upon returning to their resting states, the fluorochromes emit light
energy at higher wavelengths.
• The use of multiple fluorochromes, each with similar excitation
wavelengths and different emission wavelengths (or “colors”), allows
several cell properties to be measured simultaneously.
The Flow Cytometer
• Three main systems make up the flow cytometer instrument and
these are
• the fluidics system,
• the optics system and
• the electronics system.
Flow cytometry
Flow cytometry
Flow cytometry
1. Fluidics – Transport cells in a stream to the laser beam for
interrogation.
2. Optics – Consist of lasers to illuminate the cells in the sample stream
and optical filters to direct the resulting light signals to the appropriate
detectors.
3. Electronics – Converts the detected light signals into electrical signals
that can be processed by computer.
Fluorochromes
Gating
• Gating – It is a procedure to selectively visualize the cells of interest
while eliminating results from unwanted cells and debris.
• The data from the flow cytometers is plotted in a single dimension ,
two or even three dimension.
• The plots can then be divided into regions depending on the intensity
of the fluorescence, to create a series of subset extractions called
gates.
Flow cytometry
Applications
• Cell Sorting (FACS)
• Apoptosis
• Features of apoptotic cascade can be observed.
• Cell Cycle Study
• Measurement of DNA content
• Immuno-phenotyping Analysis
Clinical Applications of Flow Cytometry
• Diagnosis of Hematologic Malignancies
• Lymphocyte Subset Enumeration (HIV)
• Efficacy of Cancer Chemotherapy
• Cell Function Analysis
• Application in Organ Transplantation
• Detection of Minimal Residual Disease
• Reticulocyte Enumeration
• Detecting microorganisms
• Protein engineering detection
• Biomarker detection
THANK YOU !!!

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Flow cytometry

  • 1. Flow Cytometry Rajan Paudel M.Sc. Medical Microbiology
  • 2. Contents • Introduction • History • Principle • Flow cytometer • Data analysis • Applications
  • 3. Introduction • Flow Cytometry is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. • A sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. • The sample is focused to ideally flow one cell at a time through a laser beam and the light scattered is characteristic to the cells and their components.
  • 4. • Cells are often labeled with fluorescent markers so that light is first absorbed and then emitted in a band of wavelengths. • By utilizing highly specific antibodies labeled with fluorescent conjugates, FACS analysis allows us to simultaneously collect data on, and sort a biological sample by a nearly limitless number of different parameters.
  • 5. History • In 1879 Lord Rayleigh observed that fluid emerging from an orifice breaks into a series of droplets. • In 1949, Wallace Coulter filed, “Means for Counting Particles Suspended in a Fluid”. • In 1965, Mack J. Fulwyler reported the first flow cytometry instrument capable of sorting cells. • In 1968 Wolfgang Göhde designed a fluorescence based flow cytometer.
  • 6. • Leonard Herzenberg, an immunologist at Stanford, coined the term ‘FACS’ – Fluorescence Activated Cell Sorter. • Becton Dickinson (BD) owns the FACS trade name and launched the first commercial instrument, FACS-1 in the early 1970’s. • The first label-free high-frequency impedance flow cytometer, Ampha Z30, was introduced by Amphasys (2012).
  • 7. • Flow cytometric immunophenotyping (FCI) first appeared in clinical laboratories in the 1980s, in the wake of the AIDS epidemic. • Initially utilized to assess CD4 T-cells, the technique was soon applied to lymphoid and eventually myeloid neoplasms.
  • 8. Principle • Fluorescent dyes may bind or intercalate with different cellular components such as DNA or RNA. Additionally, antibodies conjugated to fluorescent dyes can bind specific proteins on cell membranes or inside cells. • When labeled cells are passed by a light source, the fluorescent molecules are excited to a higher energy state. • Upon returning to their resting states, the fluorochromes emit light energy at higher wavelengths. • The use of multiple fluorochromes, each with similar excitation wavelengths and different emission wavelengths (or “colors”), allows several cell properties to be measured simultaneously.
  • 9. The Flow Cytometer • Three main systems make up the flow cytometer instrument and these are • the fluidics system, • the optics system and • the electronics system.
  • 13. 1. Fluidics – Transport cells in a stream to the laser beam for interrogation. 2. Optics – Consist of lasers to illuminate the cells in the sample stream and optical filters to direct the resulting light signals to the appropriate detectors. 3. Electronics – Converts the detected light signals into electrical signals that can be processed by computer.
  • 15. Gating • Gating – It is a procedure to selectively visualize the cells of interest while eliminating results from unwanted cells and debris. • The data from the flow cytometers is plotted in a single dimension , two or even three dimension. • The plots can then be divided into regions depending on the intensity of the fluorescence, to create a series of subset extractions called gates.
  • 17. Applications • Cell Sorting (FACS) • Apoptosis • Features of apoptotic cascade can be observed. • Cell Cycle Study • Measurement of DNA content • Immuno-phenotyping Analysis
  • 18. Clinical Applications of Flow Cytometry • Diagnosis of Hematologic Malignancies • Lymphocyte Subset Enumeration (HIV) • Efficacy of Cancer Chemotherapy • Cell Function Analysis • Application in Organ Transplantation • Detection of Minimal Residual Disease • Reticulocyte Enumeration
  • 19. • Detecting microorganisms • Protein engineering detection • Biomarker detection