2015

What Is Flow CytometryWhat Is Flow Cytometry??

Flow ~ cells in motion- זרימהCyto ~ cell- תאיםMetry ~ measure- מדידהMeasuring properties of cells while in

a fluid stream

Cytometry vs. Flow Cytometry vs. Flow CytometryCytometryCytometryLocalization of antigen

is possiblePoor enumeration of

cell subtypesLimiting number of

simultaneous measurements

Flow Cytometry.Cannot tell you

where antigen is.Can analyze many

cells in a short time frame.

Can look at numerous parameters at once.

FACSFACSאפליקציות בשימוש ה-אפליקציות בשימוש ה- It can be used for…

Immunophenotyping DNA cell cycle/tumor

ploidy Membrane potential Ion flux Cell viability Intracellular protein

staining pH changes Cell tracking and

proliferation Sorting Redox state Chromatin structure Total protein Lipids Surface charge Membrane fusion/runover Enzyme activity Oxidative metabolism Sulfhydryl

groups/glutathione DNA synthesis DNA degradation Gene expression

Medline Publications citing "Flow Cytometry"

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The use of flow in research has boomed since the mid-1980s

גודל יחסי של התא

גרגור יחסי של התא

עוצמת פלואורסנציה יחסיתבתא

מורכבת משלוש מערכות:FACSפעילות מכשיר ה- -זרםאופטיקהאלקטרוניקה

What Happens in a Flow What Happens in a Flow CytometerCytometer

Fluidics

optics

electronics

electronics

Flow Cytometryהגדרת המושג FACSהגדרת הדוגמאות המתאימות למכשיר ה-

הגדרת המאפיינים שהשיטה מודדת.FACSהגדרת שלוש המערכות שעובדות במכשיר ה

How The Flow Cell WorksHow The Flow Cell WorksThe cells from the sample tube are

injected into the sheath streamFlow in a flow cell is laminar.Hydrodynamic focusing pushes the

cells to line up single file along their long axis.

The shape of the flow cell provides the means for hydrodynamic focusing.

Interrogation point

The Flow CellThe Flow Cell

The introduction of a large volume into a small volume in such a way that it becomes “focused” along an axis is called Hydrodynamic Focusing.Hydrodynamic Focusing.

Sample Stream

Cell

Sheath

Incoming Laser

Sample

Sheath Sheath

Sheath

Sample

SampleCore

Stream

Low Differential High Differential

Laser Focal Point

Sample DifferentialSample Differential

10 psi

10.2 psi

10 psi

10.4 psi

10 psi

10.8 psi

Low pressure

High pressure

FL30 1024 2048 3072 4096

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68.70 19.16 9.56

G0/G1 CV= 2.42

G2/M

S phaseG0/G1

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74.85 9.12 15.84

GO/G1 CV= 7.79

74.85 9.12 15.84

GO/G1 CV= 7.79

Cell cycle

מה תפקיד מערכת הזרימהhydrodynamic focusingמהו

כיצד קצב הריצה של הדוגמא והלחץ במערכת משפיעים על התוצאה.

Excitation

CollectionOptics system

LLight ight AAmplification by mplification by SStimulated timulated EEmission mission of of RRadiationadiation

FiltersFiltersMany wavelengths of light will be

scattered from a cell, we need a way to split the light into its specific wavelengths in order to detect them independently. This is done with filters

Optical filters are designed such that they absorb or reflect some wavelengths of light, while transmitting other.

3 types of filters Long Pass filter Short Pass filter Band Pass filter

Long Pass FiltersLong Pass FiltersTransmit all wavelengths greater

than specified wavelength Example: 500LP will transmit all

wavelengths greater than 500nm

400nm400nm 500nm500nm 600nm600nm 700nm700nm

Tra

nsm

itta

nce

Tra

nsm

itta

nce

Short Pass FilterShort Pass FilterTransmits all wavelengths less

than specified wavelengthExample: 600SP will transmit all

wavelengths less than 600nm.

400nm400nm 500nm500nm 600nm600nm 700nm700nm

Tra

nsm

itta

nce

Tra

nsm

itta

nce

Band Pass FilterBand Pass FilterTransmits a specific band of

wavelengthsExample: 550/20BP Filter will

transmit wavelengths of light between 540nm and 560nm (550/20 = 550+/-10, not 550+/-20)

400nm400nm 500nm500nm 600nm600nm 700nm700nm

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nsm

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Dichroic FiltersDichroic FiltersCan be a long pass or short pass filterFilter is placed at a 45º angle to the

incident lightPart of the light is reflected at 90º to

the incident light, and part of the light is transmitted and continues on.

DichroicDichroicFilterFilter

Detector 1Detector 1

Detector 2Detector 2

750-810nm

<670nm

564-606nm

515-545nm

483-493nm

Electronics SystemElectronics System•Converts light signals into numerical data•Eliminate small signals

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PhotodetectorsPhotodetectorsPhotodiode (FSC)PMT- photomultiplier tube

How Do PMTs Work?

Laser DelayLaser Delay

TresholdTreshold

The PulseThe Pulse

TimeTime

P

hoto

ns /

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ctor

Ph

ot o

ns /

Dete

ctor

(V)

(V)

What are doublet & clumps

Doublet: Two cells stuck together

Clumps: More than two cells stuck cells stuck together

Why do we need to gate out doublets?

GFP+ GFP+GFP+

GFP+GFP- GFP- GFP-GFP-

G1 G2M