FLOW CYTOMETRY

Dr. MOHAMMED H SAIEMA LDAHRKAAU

FACULTY OF APPLIED MEDICAL SCIENCESMEDICAL 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, 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

FluorescenceFluorescencesignalssignals

Focused laserFocused laserbeambeam

Sheathfluid

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

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

FALS Sensor

Laser