Flow Cytometry Becton Dickinson Asia Limited Company

Flow Cytometry Basic Principle

Basic Principle of Flow Cytometer Conception Combined System Basic Principle What Can a Flow Cytometer Tell us about a Cell ? Review

What is Flow Cytometer ? A technology that simultaneously measures multiple characteristics of single cells at a rapid rate.

A Modern Cytometer Needs a Combined System of: Fluidics To introduce and focus the cells for interrogation To introduce and focus the cells for interrogationOptics To generate and collect the light signals To generate and collect the light signalsElectronics To convert the optical signals to proportional electronic signals and digitize them for computer analysis To convert the optical signals to proportional electronic signals and digitize them for computer analysisComputer

Basic Principle

Flow Cell Injector Tip Fluorescence signals Focused laser beam Sheath fluid

Forward Angle Light Scatter FALS Sensor Laser

90 Degree Light Scatter FALS Sensor 90LS Sensor Laser

Dot plot

Laser Fluorescence Detectors Fluorescence FALS Sensor Fluorescence detector (PMT3, PMT4 etc.)

488 nm laser + - Fluorescence Activated Cell Sorting Charged Plates Single cells sorted into test tubes FALS Sensor Fluorescence detector

Optical Filters Dichroic Filter/Mirror at 45 deg Reflected light Transmitted LightLight Source

Standard Band Pass Filters Transmitted Light White Light Source 630 nm BandPass Filter nm Light

Standard Long Pass Filters Transmitted Light Light Source 520 nm Long Pass Filter >520 nm Light Transmitted Light Light Source 575 nm Short Pass Filter <575 nm Light Standard Short Pass Filters Standard Short Pass Filters

PMT Dichroic Filters Bandpass Filters Flow Cytometry Optics Laser Flow cell

Ethidium PE cis-Parinaric acid Texas Red PE-TR Conj. PI FITC 600 nm300 nm500 nm700 nm400 nm Common Laser Line

Propidium Iodide 400 nm500 nm600 nm700 nm PI DNA Excitation Emisson 300 nm 400 nm 500 nm 600 nm 700 nm

Fluorescein (FITC) 400 nm500 nm600 nm700 nm Wavelength Protein Excitation Emisson 300 nm 400 nm 500 nm 600 nm 700 nm

Excitation Emisson Phycoerytherin (PE) Protein

Allophycocyanin (APC) Protein nm (HeNe ) Excitation Emisson 300 nm 400 nm 500 nm 600 nm 700 nm

Basics of Flow Cytometry Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence that is collected, filtered and converted to digital values that are stored on a computer Fluidics Optics Electronics

What Can a Flow Cytometer Tell Us About a Cell ? Its relative size (Forward Scatter --FSC) Its relative granularity or internal complexity (Side Scatter--SSC) Its relative fluorescence intensity (FL1, FL2, FL3, FL4, and FL5, etc)

Review Created an illumination region with the excitation optics Passed the cells precisely through the illumination region using hydrodynamic focusing Routed the generated light signals to the specific detectors by the collection optics