Flow CytometryFlow 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 ?What is Flow Cytometer ?

A technology that simultaneously measures A technology that simultaneously measures multiple characteristics of single cells at a multiple characteristics of single cells at a rapid rate.rapid rate.

A Modern Cytometer Needs a Combined System of:

FluidicsFluidics

To introduce and focus the cells for interrogationTo introduce and focus the cells for interrogation

OpticsOptics

To generate and collect the light signalsTo generate and collect the light signals

ElectronicsElectronics

To convert the optical signals to proportional To convert the optical signals to proportional electronic signals and digitize them for computer analysiselectronic signals and digitize them for computer analysis

ComputerComputer

Basic PrincipleBasic Principle

Flow Cell

Injector Tip

FluorescenceFluorescencesignalssignals

Focused laserFocused laserbeambeam

Sheath fluid

Forward Angle Light Scatter

FALS Sensor

Laser

90 Degree Light Scatter

FALS Sensor

90LS Sensor

Laser

Dot plot

Laser

Fluorescence Detectors

Fre

q

Fluorescence

FALS Sensor

Fluorescence detector(PMT3, PMT4 etc.)

488 nm laser

+-

Fluorescence Activated Cell Sorting

Charged Plates

Single cells sortedinto test tubes

FALS Sensor

Fluorescence detector

Optical Filters

Dichroic Filter/Mirror at 45 deg

Reflected light

Transmitted LightLight Source

Standard Band Pass Filters

Transmitted LightWhite Light Source

630 nm BandPass Filter

620 -640 nm Light

Standard Long Pass FiltersStandard Long Pass Filters

Transmitted LightTransmitted LightLight SourceLight Source 520 nm Long Pass Filter520 nm Long Pass Filter

>520 nm Light>520 nm Light

Transmitted LightTransmitted LightLight SourceLight Source575 nm Short Pass Filter575 nm Short Pass Filter

<575 nm Light<575 nm Light

Standard Short Pass FiltersStandard Short Pass Filters

PMT

PMT

PMT

PMT

DichroicFilters

BandpassFilters

Flow Cytometry Optics

Laser

1

2

3

4

Flow cell

EthidiumEthidium

PEPE

cis-Parinaric acidcis-Parinaric acid

Texas RedTexas Red

PE-TR Conj.PE-TR Conj.

PIPI

FITCFITC

600 nm300 nm 500 nm 700 nm400 nm457350 514 610 632488 Common Laser LineCommon Laser Line

Propidium Iodide

400 nm 500 nm 600 nm 700 nm

Re

lativ

e In

ten

sity

PI

DNADNA

Excitation Emisson300 nm 400 nm 500 nm 600 nm 700 nm

Fluorescein (FITC)

400 nm 500 nm 600 nm 700 nm

Re

lativ

e In

ten

sity

Wavelength

ProteinProtein

ExcitationExcitation EmissonEmisson300 nm 400 nm 500 nm 600 nm 700 nm

ExcitationExcitation EmissonEmisson300 nm 400 nm 500 nm 600 nm 700 nm

Phycoerytherin (PE)

Protein

Allophycocyanin (APC)ProteinProtein 632.5 nm (HeNe632.5 nm (HeNe))

ExcitationExcitation EmissonEmisson

300 nm 400 nm 500 nm 600 nm 700 nm

Basics of Flow CytometryBasics 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

FluidicsFluidics

OpticsOptics

ElectronicsElectronics

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