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Introduction To Flowcytometer

The document describes the key components and functioning of a flow cytometer. A flow cytometer uses fluidics, optical, and electronics systems to measure physical characteristics of cells as they flow in a fluid stream through a laser. The fluidics system transports cells to the laser for interrogation. The optical system includes lasers that illuminate cells and filters that direct resulting light signals to detectors. The electronics convert light signals to digital data that can be analyzed on a computer.

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Sooridarsan Krishnan
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68 views11 pages

Introduction To Flowcytometer

The document describes the key components and functioning of a flow cytometer. A flow cytometer uses fluidics, optical, and electronics systems to measure physical characteristics of cells as they flow in a fluid stream through a laser. The fluidics system transports cells to the laser for interrogation. The optical system includes lasers that illuminate cells and filters that direct resulting light signals to detectors. The electronics convert light signals to digital data that can be analyzed on a computer.

Uploaded by

Sooridarsan Krishnan
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPT, PDF, TXT or read online on Scribd

1.

Overview
2. Fluidics System
3. Optical system
4. Electronics System
 What is Flowcytometer?

Flow :(of a liquid, gas, or electricity) move steadily


and continuously in a current or stream
Cyto :Cell
Meter :Measurement

Flowcytometer is a technology that simultaneously


measures and analyzes multiple physical
characteristic of single particles, usually cells, as
they flow in a fluid stream through a beam of light
 The properties measured include a particle’s size,
relative granulity and fluorescence intensity.
 These characteristics are determined using an
optical-to-electronic coupling system.

 A Flowcytometer is made up of 3 main systems:


1. The Fluidics:
The fluidics system transports particles in a stream
to the laser beam for interrogation
2. The Optics
The optics system consists of laser to illuminate
the particles in the sample stream and optical
filters to direct the resulting light signals to the
appropriate detectors.
3. The Electronics
The electronics system converts the detected light
signals into electronic signals that can be
processed by the computer.
 The purpose of the fluidics system is to
transport particles in a fluid stream to the
laser beam for interrogation
 Interrogation is the contact point between the

particle and the laser.


 The optical system consist of the excitation
optics and the collection optics.
 The excitation optics consist of laser beam

and several lenses to shape and focus the


laser beam.
 Collection optics consist of a collection lenses

to collect light emmited from the particle,


laser beam interaction.
 Light Scattering occurs when a particle deflects incident laser light.
 This scattering depends on the physical property of the cell namely
its size and internal complexity.
 Forward Scatter (FSC):
FSC is the measurement of the size of the particle
 Side Scatter (SSC)
SSC is the measurement of the granularity of the particle.

Side Scatter

Light Source Forward Scatter


 A fluorescence compound absorbs light energy
over a range of wavelengths that is characteristic
for the compound.
 This absorption of light causes an electron in the
fluorescent compound to be raised to a higher
energy level .
 The excited electron quickly decay to its ground
state, emiting the excess energy as a photon of
light. This is fluorescence
Optical bench diagram of the FACS Calibur
Benchtop Flowcytometer
 Individual cell fluorescence quanta is picked up by
the various detectors(PMT’s).
 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.
Single
Positive PI Double Positive
Population Population

PE FL

Negative
Population

Single Positive
FITC FL
FITC
Population

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