flow cytometry
DESCRIPTION
FLOW CYTOMETRY. Dr. MOHAMMED H SAIEMA LDAHR KAAU FACULTY OF APPLIED MEDICAL SCIENCES MEDICAL TECHNOLOGY DEPT. 2 ND YEAR MT INSTROMINTATION EXT 21060. WHAT IS A FLOW CYTOMETER. IT IS A MACHINE. FCM. FLOW CYTOMETRY. FLOW CYTOMETRY. WHAT IS FLOW CYTOMETRY ?. - PowerPoint PPT PresentationTRANSCRIPT
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FLOW CYTOMETRY
Dr. MOHAMMED H SAIEMA LDAHRKAAU
FACULTY OF APPLIED MEDICAL SCIENCESMEDICAL 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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Flow Cell
Injector Tip
FluorescenceFluorescencesignalssignals
Focused laserFocused laserbeambeam
Sheathfluid
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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 interactionii- 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)
Forward Angle Light Scatter
FALS Sensor
Laser