Yan Deng (X4-5225), [email protected]Gerald Denis (X4-1371), [email protected]Mike Xie (X4-5225), [email protected]for users of the Flow Cytometry Core Facility at BUMC 21 September 2010 Introduction to the Principles of Flow Cytometry John Meyers (X8-7543), [email protected]
Introduction to the Principles of Flow Cytometry. for users of the Flow Cytometry Core Facility at BUMC 21 September 2010. Mike Xie (X4-5225), [email protected]. Yan Deng (X4-5225), [email protected]. John Meyers (X8-7543), [email protected]. Gerald Denis (X4-1371), [email protected]. - PowerPoint PPT Presentation
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1. Transport particles in a fluid stream to the laser beam to be interrogated
2. Position the sample core in the center of the laser beam
sheath fluid
samplefluid
‘hydrodynamic focusing’
single file particles
● low flow rate
● narrow sample core
● high resolution
● high flow rate
● wide sample core
● low resolution
Always filter your samples to remove aggregates.
Fluidics
When conditions are right (i.e. when turbulence is minimal):
sample fluid flows in a central core
does not mix with the sheath fluid
This is termed ‘laminar flow’
optics
“SSC”
“FSC”
ele
ctro
nic
s
Forward and side scatter of leukemic cellsMalignant, large B cellsNormal B cells
FSC
SSC
The intensity of forward scatter light is proportional to size and cross-sectional area of the cells.The intensity of side scatter light is proportional tosize, shape and internal structure/irregularity of the cells.
Photomultiplier (PMT) detectors convert photons (selected by mirrors and filters) to electrical
pulsesPeak Height (volts)
Peak Width (time)
Peak Area
The higher the PMT voltage (user controllable), the greater the output magnitude for a given photon. At higher PMT voltages, the level of noise will also increase.
Adjusting the voltageof the PMT helps tooptimize captureof desired populations
Photomultiplier Tubes (PMTs)
FSC: forward scatter(size; cross-sectional area)
SSC: side scatter (granularity, internalor surface structurethat scatters light)
A dotplot represents two properties of a single cell
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
SS
C-A
FSC
SSC
A histogram represents the distribution of a single parameter across many cells
0 102 103 104 105
APC-A: pH2AX
0
20
40
60
80
100
% o
f Ma
x
Control Condition Experimental10,000 cells each!
1 cell
1 cell
1 cell1 cell
Electronic processing of emission signals
Amplifiers are of two types: linear or logarithmic
Linear amplification is typically used with scatter.
Logarithmic amplification is typically used with fluorescence.
DNA content (Linear detection)
DNA content (Log detection)
Gating
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
SS
C-A
0 102 103 104 105
APC-A: pH2AX
0
50
100
150
# C
ells
Gating allows one to select populations based on computer or human-derived criteria and further gate or display the included
cells
Backgating – don’t lose your bearings!
Backgating allows one to determine if a gating strategy is all-inclusive of a desired cell type.
In the above example, some cells are missed! What are they?Many investigators overlook the importance of verification by backgating!
FLUORESCENCE
Excitation wavelength and emission wavelength areunique properties of each specific molecular structure
(FITC)
blue laser
Stokes Fluorescence
Excitation Emission
Stokes shift
Fluorescein (FITC)
Hoechst 33258 Texas Red
Propidium iodide (PI)
Laser light must overlap with excitation wavelength
yes
no
488
488
488
488
ex
ex ex
ex
em
em em
em
But different lasers are available to excite other molecules (LSR II)
Isotype control antibodies should be used at the same concentration to stain cells at the same cell density as the experimental, but they give fluorescent signals that define a negative result.
How do you knowit’s real?
isotype control test
Resolution sensitivity
Resolution sensitivity, the abilityto resolve a faint signal frombackground) depends on thedifference D between the positiveand background peaks and the spread of the backgroundpeak W
and avoid spillover of brightcell populations into detectorchannels that require highsensitivity for rare signals
Problems in Emission Fluorescence
Spectral overlap
Excitation Emission
Optical solutions to spectral overlap: Filters
Filters resolve overlapping wavelengths of emitted light
Longpass filter: transmits light of longer than or equal toa specific wavelength
Shortpass filter: transmits light of shorter than or equal toa specific wavelength
Bandpass filter: transmits light only within a narrow rangeof wavelengths
Examples of optical filters in flow cytometry
Optical detector configurations
octagon
660/20
APC
735 LP
780/60
APC-Cy7
red trigon
bandpass
bandpasslongpass
EMISSION
two bandpass filters
Electronic solutions to spectral overlap: Compensation
To correct for emission spillover of FITC signal (normally detected in the FL1 channel) into the FL2 channel (which detects PE), it is necessary to use filters or electronic compensation or both.
Uncompensated Optimal
COMPENSATION
Before After
Multicolor immunophenotyping
No antibody. Autofluorescence only. No compensation applied.
CD4-PE. No compensation applied.
CD4-PE. Correct compensation applied.
1.4% PE subtracted from FITC PMT, 6.5% PE subtracted from APC PMT.
CD8-FITC. No compensation applied.
CD8-FITC. Correct compensation applied.
12.5% FITC subtracted from PE PMT.
CD4-PE + CD8-FITC. Streptavidin-APC alone.
CD4-PE + CD8-FITC. CD3-biotin + Streptavidin-APC
3 COLORS, CORRECTLY COMPENSATED
Spectral overlap of some fluorochrome combinationscannot be compensated easily or at all
Cy5APC
Therefore, avoid such combinations
Contour plots provide more accurate data representation than dot plots