Forensic Biology & Its biological significance.pdf
Flow cytometry.pptx
1.
2. • Cytometer (Cyto:cells; meter: measuring
device) is a device used to count the
different types of cells.
• The hemocytometer is the most basic
device used to count the different types of
cells, preferably blood cells.
• Hemocytometers, though still in use, are
time consuming and cannot be fully-
automated.
INTRODUCTION
3. • Then in 1950s, discovery of flow cytometry, changed the way the
cells were analysed. It is a much rapid process, that can be fully
automated.
• Flow cytometer -used to measure and analyze various characteristics
of particles, like cells, as they flow through a beam of laser.
• The cells or particles scatter the incident laser beam.
• This scattered light is detected and determined using optical-to-
electronic coupling system.
FLOW CYTOMETER
4. • The flow cytometers on an average can be used to analyze particles at
a rate of up to 20,000 cells per second (some devices are even faster).
• The different characteristics that the flow cytometer can analyze are
the size, granularity and fluorescence intensity of the particles. It can
also sort the particles based on these characteristics.
6. 1. The fluidics system:
• The fluidics system is used to align the
sample particles in a single line.
• It consists of a central core, through which
the sample fluid is injected, enclosed by a
tube, through which the outer sheath fluid
is passed.
• The narrow inner sample tube ends after a
short distance and allows sheath fluid to
interact with the sample.
7. • The sheath fluid flows under high pressure in the outer tube.
• At optimum pressure, the outer fluid pulls the inner sample fluid
along, without mixing, in a way that sample is narrowed down to
create a thin stream, allowing single line of particles. This
phenomenon is called hydrodynamic focusing.
• Few examples of the sheath fluids used in the flow cytometer are
phosphate buffered saline, Hepes-buffered saline and water with
2-phenoxyethanol (0.1%).
8. 2. Optics:
• The optics involves the incident light and scattering.
• The most preferred light source for the flow cytometer
is the laser.
Lasers:
• Lasers are the most commonly used light sources in
modern flow cytometers. They emit light which is
coherent (synchronized) and monochromatic (single
wavelength). Flow cytometers may have one or more
lasers, with a range of options of wavelength, from
ultraviolet to far red, to be chosen.
• The laser beam interacts with the particles in the
sample and causes scattering of light. It may cause
fluorescence emission if the sample has been labeled
with a fluorophore. The pattern of light scattering or
fluorescence by the particles provide information
about the particles
9. • Scattering of laser beam:
There are two types of scattering occurring when the laser beam
interacts with it.
• 1. Forward scatter (FSC)
• 2. Side scatter (SSC)
10. 1. Forward scatter (FSC)
This is the light scattered in the forward direction when the particle pass
through the beam of laser light.
The forward light is collected by a photodetector known as forward
scatter channel (20 ͦ angle).
The forward scatter gives information about the particle’s size. The larger
cells refract more light than smaller ones.
11. 2. Side scatter (SSC):
• The side scatter is the amount of light scattered at around right angle
(see fig 6). It is measured at an approximately 90° angle to the laser
beam. The side scatter gives information about the granularity and
internal structures.
• Each particle in the heterogenous mixture will have a unique
combination of FSC and SSC. This unique combination can be used to
differentiate between different types of particles in a heterogenous
sample, eg. blood.
• The FSC and SSC are effected by various factors like the sample type
and sample preparation procedure. Therefore, these factors can
effect the results considerably and fluorescent labeling is preferred.
