Orifice = 50 to 400 µ Principles of Flow Cytometry Quartz nozzle Fluorescence signals Focalized...
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Transcript of Orifice = 50 to 400 µ Principles of Flow Cytometry Quartz nozzle Fluorescence signals Focalized...
Orifice = 50 to 400 µ
Principles of Flow Cytometry
Quartznozzle
Fluorescence signalsFluorescence signals
Focalized laser beamFocalized laser beam
Injection of cells
Freq
Fluorescence
Photodiode
Light can be measured at 90° : Side scatter + Fluorescence
Side scatter reflects the cell content
LaserLaser
Fluorescence intensity
FITC
FIT
C
101 104103102
Relative fluorescence intensity
Nu
mb
er o
f E
ven
ts
FITC
FIT
CFITC
FITC
FITC
FITC
FIT
C
FITC
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
The automated Microscope
Waste
Detector& Counter
Sample
This primitive diagram shows the principle: Cells are passing the microscope objective, and an electronic circuit decides whether the cells is fluorescent or not. This is how a flow cytometer works!
1
Hydrodynamic focussing in the cuvette
SheathSample
SheathSample
Sample pressure low, small core stream. Good for DNA analysis
High sample pressure, broader core stream.Bad for DNA analysis
LOW HIGH
• Pressure (= Sheath Pressure) drives the sheath buffer through
the cuvette, and the higher pressure in the sample tube
(= Sample Differential) delivers the sample to the cuvette.
• In the cuvette the principle of hydrodynamic focussing
arranges the cells like pearls on a string before they arrive at
the laser interception point for analysis
• Hydrodynamic focussing cannot separate cell aggregates!
Flow cytrometry is a technique that requires single cell
suspensions
Summary
Basic opticsc
A system of prisms and lenses directs the laser light to the interrogation point in the cuvette
Laser delay
SheathSample
•Umožňuje cross beam kompenzaci
•Vyžaduje stabilní fluidics
Summary
• Excitation light is steered with prisms and lenses to the interception point
• Emitted light is collected using lenses and is split up with dichroic mirrors and filters
Tasks for the electronical system
Convert the optical signals into electonic signals (voltage pulses)
Digitise the data
Analyse Height (H), Width (W) and Area (A) of the pulse
Send the data to the analysis computer
How a voltage pulse from the PMT is generated
Voltage
LaserLaser
LaserLaser
LaserLaser
t
t
t
Voltage
Voltage
1.
2.
3.
Height, Area, and Width
Time (µs)
Volt
age
Pulse area(A)
Puls
e H
eig
ht
(H)
Pulse Width (W)
400
Threshold
The threshold defines the minimal signal intensity which has to be surpassed on a certain channel. All signals with a lower intensity are not displayed and not recorded for later analysis.
Summary
During passing the laser voltage pulses are generated at the PMT
Amplifiers enhance the signals Only signals passing the desired threshold(s) are
analysed and recorded The data are finally passed to the analysis
computer connected to the cytometer
An overview
YearInstrument
IntroducedMost Frequently
Heard Comments
1976 FACS II When can I get one?
1991 FACS Vantage Do you really need 5 colors?
1998 FACS Vantage SE Do you really need 6 colors?
2000 FACS DiVa Do you really need 8 colors?
1980 FACS IV/440 Do you really need 4 colors?
2003 FACS Aria When can I get one?
Why always more colours?• More informations from Cell Phenotyping (Cell Surface Antigens)
– around 300 CD Cell Surface Antigens
– Many functional populations require 5 or more surface markers to be fully distinguished
• Functional Assays
– Cell Cycle (PI, BrdU, Intracellular Cyclins)
– Apoptosis (Annexin-V, Active Caspase-3)
– Ca++ Flux [Indo-1, FuraRed, Fluoro-4]
• Cytokine Production
• Intracellular Signaling (Rb phosphorylation)
• Gene Reporter [Molecular] Assays
– GFP, BFP, YFP, CFP Expression
– LacZ Expression
What are the advantages / disadvantages?
• Advantages
• Save Time and Samples
• (1) 6-color stain = (15) 2-color stains
• Exponential increase in information
• Data from (1) 6-color stain » (15) 2-color stains
• Identify new/rare populations (<0.05%)
• Internal controls
• Problems
• Must carefully choose combinations of fluorochrome conjugates
• Not all reagents are available in all colors
• Greater potential for errors in compensation
• Proper controls required
Excitation- and Emission spectra of dyes for the blue laser
•Stejná excitace různá emise
•Překryv spekter(overlap)
•Excitace jiným laserem?
Compensation
www.bdbiosciences.com/spectra /
How much compensation is correct?
PE
PE
Importance of ACCURATE Compensation
RPCI
LFC
n = negativesd = dim positivesb = bright positives
10 1 10 2 10 3 10 4
FL3-Height -->
101
102
103
104
FL4
-->
PE-CY5-CD8
APC CD45
10 1 10 2 10 3 10 4
FL3-Height -->
101
102
103
104
FL4
-->
n d b n d b
10 1 10 2 10 3 10 4
FL3-Height -->
101
102
103
104
FL4
-->
Uncompensated Compensated Over Compensated
bn
Where is the CD8 dull population?!
Which marker for compensation?
Small errors in compensation of a dim control (A) can result in large compensation errors with bright reagents (B & C). Use bright markers to setup proper compensation.
Hardware Compensation
How to set compensation on the instrument
Setting compensation
• Prepare single stained controls that have both a positive and negative population.
• Adjust the PMT voltages so that the negative population is off the axis in every channel.
• Align the centers of the positive and negative cell populations by matching the median fluorescence.
-Run unstained cells
-Adjust the PMT voltages so that the negative population is off the axis in every channel.
Setting compensation- PMT Voltage
FL1-no stain
FL
2-no
sta
in
Uncompensated Compensated
Median values both = ~3.2
Setting compensation - FITC single stain
FL1-FITC CD3
FL
2-no
sta
in
FL
2-no
sta
in
-Run single stained control (FITC stained only)
-Adjust the compensation value so that positive and negative population have the same FL2 median fluorescence intensity.
FL1-FITC CD3
Setting compensation - PE single stain-Run single stained control (PE stained only)
-Adjust the compensation value so that positive and negative population have the same FL1 median fluorescence intensity.
Compensated
Median values both = ~2.5
FL
2-P
E C
D4
FL1-no stain
Compensation Controls
Single Stain Controls
Does not matter as long as:
• The autofluorescence is the same in the negative and positive populations you are lining up.
– eg, Pre-gate on lymphocytes if you are using CD8 FITC as a single stain control
• The compensation values will be valid for ALL cell types, regardless of which type of cell is used to calculate the values.
– The compensation is specific for the fluorochrome, not the cell type
Single Stain Controls - Which cells?
Use the same reagent (Ab-fluorochrome conjugate) as used in the experimental sample…
OR
A different antibody may be substituted, as long as it is conjugated to the same fluorochrome.
However…
Single Stain Controls - which reagents?
Caveats for substituting reagents:
– Controls should be as bright as possible• As bright or brighter than the experimental stains
– GFP, CFSE, and FITC are NOT the same fluorochrome • even though they are all green!
– With tandem dyes (Cy5PE/Cy7PE etc.) it is necessary to use the exact same reagent
• spillover varies from reagent to reagent
Single Stain Controls - which reagents?
Compensation of tandem-conjugates can differ from lot to
lot
• Use same reagent as experimental sample• Lots positive• Small CV, bright• Some reagents won’t work (IgL, EMA/PI)
– can mix with regular comps
Using Antibody Capture Beadsas single stained controls
Software Compensation
Automated Tools for Setting Compensation
Compensation Tools
•Must have single stained controls•Software calculated compensation for you!•Easy, accurate and quick.•Makes MULTI- Color compensation possible
Software Compensation Tools
•Available on new generation machines•DakoCytomation’s Summit (version 4)•Coulter FC500 •BD Diva•Others
•Post-acquisition software•FCS Express•FCS Press•WinList •FlowJo•Others
Compensation Matrix
FL1 FL2 FL3
FL1 Comp 3.96 0
FL2 Comp 27.35 5.15
FL3 Comp 0 11.18
Take Away Lessons
• Proper CONTROLS are essential
• DON’T compensate by eye– Use Median to adjust the populations if you
must do it manually
• TRUST the software to do it for you– It does it quicker and more accurately
Polychromatická cytometriePolychromatická cytometrie
Design experimentu a analýzaDesign experimentu a analýza
Childhood Leukemia Investigation Prague -Childhood Leukemia Investigation Prague -
Ústav imunologie, Ústav imunologie, Klinika dětské hematologie a onkologie, Klinika dětské hematologie a onkologie, UK 2.LF a FN MotolUK 2.LF a FN MotolPrahaPraha
Which fluorochrome to use?
•Major Factors
• Fluorochrome brightness
• PerCP ≈ APC-Cy7 ≈ FITC << PerCP-Cy5.5 < PE-Cy7 < APC =
PE-Cy5 < PE
• Antigen density
• Background staining of mAb
• Inherent background (stickiness) of mAb
• Antibody strength (Avidity)
• Less antibody needed = less background
• Amount of compensation required between conjugates
• Single or multiple laser
Comparison of the dye intensity for the same marker
Baumgarth, Roederer, JIM, 2000, A practical approach to multicolor flow cytometry for immunophenotyping
Spektra fluorochromů
www.bdbiosciences.com/spectra /
Which fluorochrome for which marker?
In general, try to use brighter fluorochrome conjugates for
duller antibodies or lower density antigens (e.g. activation
antigens such as CD80, CD86, CD25, or CD28)
Use brighter reagents for staining cell populations with high
autofluorescent backgrounds (e.g. granulocytes, monocytes, or
activated lymphocytes)
Use duller conjugates (FITC or PerCP) for antigens expressed at
high levels (e.g. B220 or CD4)
Zkreslení vlivem kompenzací
PE
FITC
PE
PE-TxRed
Přesvit (spilover, spectral overlap)z PE do FITC je malý = malá kompenzacez PE do PE-TxRed je velký = velká komp.
PE-TxRed – PE = 65%
Grafické řešení„Loglinear transformation“„Biexponencial display“
Zkreslení vlivem kompenzacíJe třeba promyslet odkud se dívat
Obvyklé problémy: PE vs PE-TxRed, PE-Cy5 vs APC
Nelze použít vždy histogram Nelze vždy použít čtverce či kvadrantyJe třeba promyslet jak postavit gate (kontroly FMO)V silně komp. kanálech je menší rozlišení a horší kvantifikace
Design experimentu
Na interpretaci dat myslet PŘEDEM
Bez správných kontrol někdy interpretovat nelze
Jak naložit se zkreslením komp. dat?
Sestavit design experimentu tak, aby se předešlo potížím při analýze
PE PE-TxRed
•u CD4 PE pos. buněk CD8 PE-TxRed není
Jak naložit se zkreslením?
„donor“ „akceptor“
Na donor pozitivních buňkách se akceptor pozitivní znak nevyskytuje/nehodnotí
CD4 PE
CD
8 P
E-T
xRed
PE PE-TxRed
•znak s nízkou int. do PE
Jak naložit se zkreslením?
„donor“ „akceptor“
Nižší intenzita donoru = nižší rozptyl akceptoruCD3 PE
PE
-TxR
ed
PE PE-TxRed
vysoce exprimovaný znak do PE-TxRed
Jak naložit se zkreslením?
„donor“ „akceptor“
CD19 PE
CD
10 P
E-T
xRed
PE PE-TxRed
•kvalitativní znak do PE-TxRed
Jak naložit se zkreslením?
„donor“ „akceptor“
CD3 PE
CD
8 P
E-T
xRed
780/6
0735 LP
A
575/26
556 LP
D
488/10
-
F
H
695/
40
655
LP
B
610/20
595 LP
C
530/30
502 LP
E
G
PE-Cy7, PE-Alexa 750
PerCP-Cy5.5PerCP, PC5, Tricolor
Cy-Chrome PE-Dyomics647
PE-Texas RedECDPE –
Dyomics590
PE
FITC
Alexa 488
SSC
488nm Blue laser octagon
720/4
0680 LP
A
660/
20
-
B
C
Alexa 680
APC
Alexa 633
Alexa 647
Dyomics 647
633nm Red laser trigon
530/3
0502 LP
A
450/
40
-
B
C
Alexa Fluor 430
DAPI
Hoechst
Alexa 405
Pacific Blue
407nm Violet laser trigon