FLOW CYTOMETRY

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FLOW CYTOMETRY

• Dr. MOHAMMED H SAIEMA LDAHR
• KAAU
• FACULTY OF APPLIED MEDICAL SCIENCES
• MEDICAL TECHNOLOGY DEPT.
• 2ND YEAR MT
• INSTROMINTATION
• EXT 21060

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WHAT IS A FLOW CYTOMETER

• IT IS A MACHINE

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FCM
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FLOW CYTOMETRY
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FLOW CYTOMETRY
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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.

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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.

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WHAT IS THE COMPONANTS OF THE FLOW CYTOMETER ?

• Instrument Components are,
• Fluidics
• Optics
• Electronics
• Data Analysis

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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.

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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.
  

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FACScan FLUIDICS SYSTEM speed of the entry of
the sample combined with the back pressure
generated by the exit orifice results in sample
turbulence.
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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

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OPTICS

• LASER Light Amplification by Stimulated Emission
  of Radiation

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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.

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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.

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OPTICS (FSC)