Transcript FLOW CYTOMETRY

FLOW CYTOMETRY
Dr. MOHAMMED H SAIEMA LDAHR
KAAU
FACULTY OF APPLIED MEDICAL SCIENCES
MEDICAL TECHNOLOGY DEPT.
2ND YEAR MT
INSTROMINTATION
EXT 21060
WHAT IS A FLOW CYTOMETER
IT IS A MACHINE
FCM
FLOW CYTOMETRY
FLOW CYTOMETRY
WHAT IS FLOW CYTOMETRY ?
 Flow Cytometry means measuring cells in a fluid medium.
It is a technique for making rapid physical and or chemical
measurement on particles or cells as they flow in a fluid stream
one by one through sensing point .
 A technology that simultaneously measures multiple
characteristics of single cells at a rapid rate.
 Is the measurement of cellular properties as they move in a fluid
past a stationary set of detectors.
 FCM permits analysis of 105-106 cells per minutes,
simultaneously measuring physical and biological properties.
WHAT IS FLOW CYTOMETRY ?
 FCM is an automated method used to measure cells or
particles as they flow single file though a sensing area.
 Optical sensing is done with intense light source,
usually LASER or mercury arc lamp.
 The FCM measures light scatter and fluorescent signal
generated as cell pass through a light beam.
WHAT IS THE COMPONANTS OF THE FLOW CYTOMETER ?
 Instrument Components are,
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Fluidics
Optics
Electronics
Data Analysis
Fluidics
 Cell transportation
 To introduce and focus the cells for interrogation
 Most instruments utilize a laminar/sheath flow and
turbulent boundary drag techniques to confine (detain)
cells to the center of the flow stream.
 Cells enters the chamber under pressure through a
small aperture (opening) which is surrounded by sheath
fluid.
Fluidics
 The sheath fluid in the sample chamber creates a
hydrodynamic focusing effect and draw the sample
fluid into a stream.
 Turbulent boundary flow, The sample is injected into
a chamber tapering (narrowing) to a small exit orifice.
The speed of the entry of the sample combined with
the back pressure generated by the exit orifice results
in sample turbulence.
FACScan FLUIDICS SYSTEM
speed of the entry of the sample combined with the back pressure
generated by the exit orifice results in sample turbulence.
Flow Cell
Injector
Tip
Fluorescence
signals
Focused laser
beam
Sheath
fluid
OPTICS
 Excitation optics consist of:
 Laser: Light Amplification by Stimulated Emission of
Radiation
 Lenses to shape and focus the laser beam
 Collection optics consist of;
i- a collection lens to collect light emitted from the particle-laser
beam interaction
ii- a system of optical mirrors and filters to route specified
wavelengths of the collected light to designated optical detectors
OPTICS
LASER: Light Amplification by Stimulated Emission of Radiation
OPTICS
 Laser have the advantage of producing an intense beam
of monochromatic light.
The most common laser used in FCM are;
 Argon lasers, produce light between (351-528 nm)
 Krypton laser, produce light between (350-799 nm)
 Helium-neon laser produce lines at 543,594,611, and 633nm
 Helium-cadmium laser produce lines at 325 and 441 nm.
OPTICS
Forward Scatter Channel (FSC):
 A beam of laser light is projected at specific point of
the flow cell .
 As a particle passes through the laser beam light, most
of the light is scattered in the forward direction, this
forward scatter is related to the size of the particle.
OPTICS (FSC)
Forward Angle Light Scatter
Laser
FALS Sensor