1
FLOW CYTOMETRY –
o 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.
o It involves suspending a sample of cells in a fluid and injecting it into an instrument
called a flow cytometer.
o The cells are then passed one at a time through a laser beam, and the machine
measures how the light scatters and how any fluorescent dyes on the cells emit light.
o This provides information about the size, shape, and internal complexity of the cells, as
well as the presence of specific markers.
Fig : Flow Cytometer Instrument
Figure: A representation of hydrodynamic focusing in the flow cell
R. MAJHI, Lect. In Botany, L.N College, Jharsuguda
, 2
Flow Cytometry Principle –
o The basic principle of flow cytometry is based on light scattering and fluorescence.
o A sample of cells, which may be stained with fluorescent dyes, is suspended in a fluid
and passed through the flow cytometer in a single-file stream. This stream passes
through one or more laser beams. As each cell intercepts the laser, it scatters the light in
two main ways:
o Forward Scatter (FSC): Light scattered in the forward direction. This measurement is
generally proportional to the size of the cell.
o Side Scatter (SSC): Light scattered at a 90-degree angle. This measurement provides
information about the internal complexity, granularity, and nuclear structure of the cell.
o If the cells have been stained with fluorescent dyes, the laser excites these
fluorochromes, causing them to emit light at different wavelengths.
o Detectors collect both the scattered and fluorescent light, and this information is
converted into a digital signal for analysis.
Instrumentation/Parts of Flow Cytometry -
A flow cytometer is made up of three main systems: fluidics, optics system, and electronics
system.
1. Fluidics System: The fluidics system is the "plumbing" of the flow cytometer.
o Its primary purpose is to transport the cells from the sample tube to the laser beam in a
precise, single-file stream. This is achieved through a process called hydrodynamic
focusing.
o The sample, containing the cells, is injected into the center of a larger stream of "sheath
fluid" (usually a buffered saline solution).
o The sheath fluid flows at a higher pressure and rate, compressing the sample stream
into a narrow core. This forces the cells to pass through the laser's interrogation point
one by one, ensuring accurate and individual measurements.
2. Optics System: The optics system is responsible for illuminating the cells and collecting
the light signals they produce.
It consists of two main parts:
Excitation Optics: This includes the laser (or multiple lasers) which acts as the light
source. Lenses are used to focus the laser beam to a specific point, known as the
interrogation point, where the cells pass through.
R. MAJHI, Lect. In Botany, L.N College, Jharsuguda
FLOW CYTOMETRY –
o 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.
o It involves suspending a sample of cells in a fluid and injecting it into an instrument
called a flow cytometer.
o The cells are then passed one at a time through a laser beam, and the machine
measures how the light scatters and how any fluorescent dyes on the cells emit light.
o This provides information about the size, shape, and internal complexity of the cells, as
well as the presence of specific markers.
Fig : Flow Cytometer Instrument
Figure: A representation of hydrodynamic focusing in the flow cell
R. MAJHI, Lect. In Botany, L.N College, Jharsuguda
, 2
Flow Cytometry Principle –
o The basic principle of flow cytometry is based on light scattering and fluorescence.
o A sample of cells, which may be stained with fluorescent dyes, is suspended in a fluid
and passed through the flow cytometer in a single-file stream. This stream passes
through one or more laser beams. As each cell intercepts the laser, it scatters the light in
two main ways:
o Forward Scatter (FSC): Light scattered in the forward direction. This measurement is
generally proportional to the size of the cell.
o Side Scatter (SSC): Light scattered at a 90-degree angle. This measurement provides
information about the internal complexity, granularity, and nuclear structure of the cell.
o If the cells have been stained with fluorescent dyes, the laser excites these
fluorochromes, causing them to emit light at different wavelengths.
o Detectors collect both the scattered and fluorescent light, and this information is
converted into a digital signal for analysis.
Instrumentation/Parts of Flow Cytometry -
A flow cytometer is made up of three main systems: fluidics, optics system, and electronics
system.
1. Fluidics System: The fluidics system is the "plumbing" of the flow cytometer.
o Its primary purpose is to transport the cells from the sample tube to the laser beam in a
precise, single-file stream. This is achieved through a process called hydrodynamic
focusing.
o The sample, containing the cells, is injected into the center of a larger stream of "sheath
fluid" (usually a buffered saline solution).
o The sheath fluid flows at a higher pressure and rate, compressing the sample stream
into a narrow core. This forces the cells to pass through the laser's interrogation point
one by one, ensuring accurate and individual measurements.
2. Optics System: The optics system is responsible for illuminating the cells and collecting
the light signals they produce.
It consists of two main parts:
Excitation Optics: This includes the laser (or multiple lasers) which acts as the light
source. Lenses are used to focus the laser beam to a specific point, known as the
interrogation point, where the cells pass through.
R. MAJHI, Lect. In Botany, L.N College, Jharsuguda
