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
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, • 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.
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.