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Flow Cytometry

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4 views

Flow Cytometry

Uploaded by

minasadia2004
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPTX, PDF, TXT or read online on Scribd
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FLOW CYTOMETRY

PRINCIPLE & APPLICATION


Definition
• 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.
• The use of flow cytometry has increased over the years as it provides a
rapid analysis of multiple characteristics (both qualitative and
quantitative) of the cells.
• The properties that can be measured by this process include a particle’s
size, granularity or internal complexity, and fluorescence intensity.
• These characteristics are determined using an optical-to-electronic
coupling system that detects the cells based on laser scattered by the
cells.
• A flow cytometer, despite its name, does not necessarily deal with cells;
it deals with cells quite often, but it can also deal with chromosomes or
PRINCIPLE
• The basic principle of flow cytometry is based on
the measurement of light scattered by particles
and the fluorescence observed when these
particles are passed in a stream through a laser
beam.
Light Scattering
• Light scattering results when a particle deflects incident laser light.
The extent to which this happens depends on the physical properties
of a particle, namely its size and internal complexity.
• Forward-scattered light (FSC) is proportional to the cell-surface area
or size of the cell. It is a measurement of mostly diffracted light and
detects rays that are just off the axis of the incident laser beam
dispersed in the forward direction by a photodiode.
• Side-scattered light (SSC) indicates the cell granularity or internal
complexity of the cells. SSC is a measurement of mostly refracted and
reflected light that occurs at any interface within the cell where there
is a change in the refractive index.
• The measurements of FSC and SSC are used for the differentiation of
cell types in a heterogeneous cell population.
Fluorescence
• Fluorescent markers used to detect the expression of cellular molecules such as
proteins or nucleic acids in a system.
• The fluorescent compound absorbs light energy over a range of wavelengths that
is characteristic of that compound.
• This absorption of light causes an electron in the fluorescent compound to be
raised to a higher energy level.
• The excited electron quickly decays to its ground state, emitting the excess energy
in the form of fluorescence which is then collected by detectors.
• In a mixed population of cells, different fluorochromes can be used to distinguish
separate subpopulations.
• The fluorescence pattern of each subpopulation, combined with FSC and SSC data,
can be used to identify which cells are present in a sample and to count their
relative percentages.
• The electronics system then converts the detected light signals into electronic
signals that can be processed by the computer.
Instrumentation/Parts of Flow
Cytometry
• A flow cytometer is made up of three main systems:
1. Fluidics system
2. Optics system
3. Electronics system
Types of Flow Cytometry
There are different types of flow cytometers based on the purpose and
precision of the process:

• Traditional flow cytometers


• Acoustic Focusing Cytometers
• Cell sorters
• Imaging flow cytometer
Applications
Flow Cytometry is used in several fields including molecular
biology, pathology, immunology, virology, plant biology, and marine biology. Some of
the common application include:
• It is used in clinical labs for the detection of malignancy in bodily fluids like
leukemia.
• Cytometers like cell sorters can be used to separate the cells of interest in separate
collection tubes physically.
• It can be used for the detection of the content of DNA by using fluorescent markers.
• Flow cytometers allow the analysis of replication cells by using fluorescent dye for
four different stages of the cell cycle.
• Acoustic flow cytometers are used in the study of multi-drug resistant bacteria in
the blood and other samples.
• The different stages of cell death, apoptosis, and necrosis can be detected by flow
cytometers based on the differences in the morphological and biochemical changes.
Limitations
• This process doesn’t provide information on the intracellular
location or distribution of proteins.
• Over time, debris is aggregated, which might result in false
results.
• The pre-treatment associated with sample preparation and
staining is a time-consuming process.
• Flow cytometry is an expensive process that requires highly
qualified technicians.

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