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 A Modern Cytometer Needs a Combined System of:   Dot plot Optical Filters Standard Long Pass Filters Propidium Iodide Fluorescein (FITC) Phycoerytherin (PE) Allophycocyanin (APC) Basics of Flow Cytometry 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 * * What is Flow Cytometer ? A technology that simultaneously measures multiple characteristics of single cells at a rapid rate. Fluidics To introduce and focus the cells for interrogation Optics To generate and collect the light signals Electronics To convert the optical signals to proportional electronic signals and digitize them for computer analysis Computer 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 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 Dichroic Filter/Mirror at 45 deg Reflected light Transmitted Light Light Source Standard Band Pass Filters Transmitted Light White Light Source 630 nm BandPass Filter 620 -640 nm Light 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 PMT PMT PMT PMT Dichroic Filters Bandpass Filters Flow Cytometry Optics Laser 1 2 3 4 Flow cell Ethidium PE cis-Parinaric acid Texas Red PE-TR Conj. PI FITC 600 nm 300 nm 500 nm 700 nm 400 nm 457 350 514 610 632 488 Common Laser Line 400 nm 500 nm 600 nm 700 nm PI DNA Excitation Emisson 300 nm  400 nm  500 nm 600 nm  700 nm 400 nm 500 nm 600 nm 700 nm Wavelength Protein Excitation Emisson 300 nm  400 nm  500 nm 600 nm  700 nm Excitation Emisson 300 nm  400 nm  500 nm 600 nm  700 nm Protein Protein 632.5 nm (HeNe) Excitation Emisson 300 nm  400 nm  500 nm 600 nm  700 nm 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)  * *