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Flow Cytometry Basics
James MarvinDirector, Flow Cytometry Core FacilityUniversity of Utah Health Sciences CenterOffice 801-585-7382Lab 801-581-8641jmarvin@cores.utah.edu
New Instrumentation
iCyte/Sony Eclipse-4 lasers-5 color detection-Electronic volume
BD FacsCanto-4 lasers-8 color detection
Seventeen-colour flow cytometry: unravelling the immune systemNature Reviews Immunology, 2004
“This ain’t your grandma’s flow cytometer”
# of colors12345678910
# of plots1136101521283645
Flow CytometryApplications
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 Phagocytosis Microparticle analysis
The uses of flow in research has boomed since the mid-1980s, and is now the gold standard for a variety of applications
Section I
Background Information on Flow Cytometry
Experimental Design “One on One”
Instrumentation“Flow Basics”
Analysis“Data Analysis”
Presentation“Data Analysis”
• Sample Procurement• Sample preparation• Fix/Perm• Which Fluorophore• Controls• Isotype?• Single color• FMO
• Appropriate Lasers• Appropriate Filters• Instrument Settings• Lin vs Log• Time• A, W, H• Doublet
discrimination
• Interpretation• Mean, Median• % +• CV• SD• Signal/Noise• Gating
• Histogram• Dot Plot• Density Plot• Overlay• Bar Graph
Many components to a successful assay
What Is Flow Cytometry?
Flow ~ motionCyto ~ cellMetry ~ measure
Measuring both intrinsic and extrinsic properties of cells while in a moving fluid stream
Cytometry vs. Flow CytometryCytometry/Microscopy
Localization of antigen is possible
Poor enumeration of cell subtypes
Limiting number of simultaneous measurements
Flow Cytometry. No ability to determine
localization (traditional flow cytometer)
Can analyze many cells in a short time frame. (30k/sec)
Can look at numerous parameters at once (>20 parameters)
Section II
The 4 Main Components of a Flow Cytometer
What Happens in a Flow Cytometer?
Cells in suspension flow single filethrough a focused laser where they scatter
light and emit fluorescence that is filtered, measured, then converted to digitized
values that are stored in a file which can then be analyzed and interpreted
within specialized software.
Interrogation
Fluidics
Electronics
Interpretation
The Fluidics System“Cells in suspension flow single file”
Cells must flow one-by-one into the cytometer to do single cell analysis
Accomplished through a pressurized laminar flow system.
The sample is injected into a sheath fluid as it passes through a small orifice (50um-300um)
Sheath and Core
Sheath
Core
Hydrodynamic Focusing
V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3
Notice how the ink is focused into a tight stream as it is drawn into the tube under laminar flow conditions.
PBS/Sheath
Sample/cells/core
Laminar flowHydrodynamicFocusing
Laminar flow occurs when a fluid flows in parallel layers, with no disruption between the layers
Particle Orientation and Deformation
a: Native human erythrocytes near the margin of the core stream of a short tube (orifice). The cells are uniformly oriented and elongated by the hydrodynamic forces of the inlet flow.
b: In the turbulent flow near the tube wall, the cells are deformed and disoriented in a very individual way. v>3 m/s.
V. Kachel, et al. - MLM Chapt. 3
What Happens in a Flow Cytometer (Simplified)
Cell flash.swf
Flow Cell- the place where hydrodynamically focused cells are delivered to the focused light source
Incoming Laser
Sample
Sheath Sheath
SampleCore
Stream
Low Differential High Differential or “turbulent flow”
Laser Focal Point
Gaussian- A “bell curved” normal distribution where the values and shape falls off quickly as you move away from central, most maximum point.
Low pressure
High pressure
FL30 1024 2048 3072 4096
Cou
nt
300
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140
120
100
80
60
40
20
0
68.70 19.16 9.56
G0/G1 CV= 2.42
G2/M
S phaseG0/G1
FL3
0 1024 2048 3072 4096
Cou
nt340
320
300
280
260
240
220
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0
74.85 9.12 15.84
GO/G1 CV= 7.79
74.85 9.12 15.84
GO/G1 CV= 7.79
Fluidics Recap
Purpose is to have cells flow one-by-one past a light source.
Cells are “focused” due to hydrodynamic focusing and laminar flow.
Turbulent flow, caused by clogs or fluidic instability can cause imprecise data
What Happens in a Flow Cytometer?
Cells in suspension flow single filethrough a focused laser where they scatter
light and emit fluorescence that is filtered, measured, and converted to digitized values
that are stored in a file Which can then be read by specialized
software.
Interrogation
Fluidics
Electronics
Interpretation
InterrogationLight source needs to be focused on the
same point where cells are focused.
Light source 99%=Lasers
LasersLight amplification by stimulated emission of radiation
Lasers provide a single wavelength of light (monochromatic)
They can provide milliwatts to watts of power Low divergence Provide coherent light Gas, dye, or solid state
Coherent: all emmiting photons have same wavelength, phase and direction as stimulation photons
Light collection
Collected photons are the product of laser light scattering or bouncing off cells
488nm
Information associated with physical attributes of cells (size, granularity, refractive index)
Scatter FluorescenceVS
Collected photons are product of excitation with subsequent emission determined by fluorophore
350nm-800nm
Readout of intrinsic (autofluorescence) or extrinsic fluorescence (intentional cell labeling)
Forward Scatter
FSCDetector
Laser Beam
Original from Purdue University Cytometry Laboratories
.50-80
Forward Scatter
The intensity of forward scatter signal is often attributed to cell size, but is very complex and also reflects refractive index (membrane permeability), among other things
Forward Scatter=FSC=FALS=LALS FSC
Side Scatter
FSCDetector
CollectionLens
SSCDetector
Laser Beam
Original from Purdue University Cytometry Laboratories
Side Scatter
Laser light that is scattered at 90 degrees to the axis of the laser path is detected in the Side Scatter Channel
The intensity of this signal is proportional to the amount of cytosolic structure in the cell (eg. granules, cell inclusions, drug delivery nanoparticles.)
Side Scatter=SSC=RALS=90 degree Scatter
Why Look at FSC v. SSC Since FSC ~ size and SSC ~ internal structure, a
correlated measurement between them can allow for differentiation of cell types in a heterogenous cell population
FSC
SS
C
Lymphocytes
Monocytes
Granulocytes
RBCs, Debris,Dead Cells
LIVE
Dead
FluorescenceE
nerg
y
Absorbed exciting light
Emitted fluorescence
S0
Ground State
Excited higher energy states
S1
S2
S3
•As the laser interrogates the cell, fluorochromes on/in the cell (intrinsic or extrinsic) may absorb some of the light and become excited•As those fluorochromes leave their excited state, they release energy in the form of a photon with a specific wavelength, longer than the excitation wavelength
Stokes shift- the difference in wavelength between the excitation and the emission
Optical Filters Many wavelengths of light will be emitted 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 Filters
Transmit all wavelengths greater than specified wavelength Example: 500LP will transmit all wavelengths
greater than 500nm
400nm 500nm 600nm 700nm
Tra
nsm
ittan
ce
Short Pass Filter
Transmits all wavelengths less than specified wavelength Example: 600SP will transmit all wavelengths
less than 600nm.
400nm 500nm 600nm 700nm
Tra
nsm
ittan
ce
Original from Cytomation Training Manual, Modified by James Marvin
Band Pass Filter
Transmits a specific band of wavelengths Example: 550/20BP Filter will transmit
wavelengths of light between 540nm and 560nm (550/20 = 550+/-10, not 550+/-20)
400nm 500nm 600nm 700nm
Tra
nsm
ittan
ce
Dichroic Filters
Can be a long pass or short pass filter Depending on the specs of the filter, some of the
light is reflected and part of the light is transmitted and continues on.
DichroicFilter
Detector 1
Detector 2
Spectra of Common Fluorochromes with Typical Filters
Compensation
Fluorochromes typically fluoresce over a large part of the spectrum (100nm or more)
A detector may “see” fluorescence from more than 1 fluorochrome. (referred to as bleed over)
You need to “compensate” for this bleed over so that 1 detector reports signal from only 1 fluorochrome
Compensation-Practical Eg.
compensationexample.swf
Multi-laser Instruments and pinholes
Implications--Can separate completely overlapping emission profilesif originating off different lasers-Significantly reduces compensation
Spatial separation
Blue Laser Excitation
Blue and YellowLaser Excitation
585/
20 Y
ello
w
585/
20 B
lue
530/20 Blue 530/20 Blue
Interrogation Recap
A focused light source (laser) interrogates a cell and scatters light
That scattered light travels down a channel to a detector FSC ~ size and cell membrane integrity SSC ~ internal cytosolic structure Fluorochromes on/in the cell will become excited by the
laser and emit photons These photons travel down channels and are steered and
split by dichroic (LP/SP) filters
What Happens in a Flow Cytometer?
Cells in suspension flow single file Through a focused laser where they scatter
light and emit fluorescence that is filtered, measured
and converted to digitized values that are stored in a file
Which can then be read by specialized software.
Interrogation
Fluidics
Electronics
Interpretation
Electronics
Detectors basically collect photons of light and convert them to an electrical current
The “electronics” must process that light signal and convert the current to a digitized value/# that the computer can graph
DetectorsThere are two main types of photo detectors
used in flow cytometry Photodiodes
Used for strong signals, when saturation is a potential problem (eg. FSC detector)
Photomultiplier tubes (PMT) Used for detecting small amounts of fluorescence
emitted from fluorochromes. Incredible Gain (amplification-up to 10million
times) Low noise
Detector names
Photoelectric Effect
Einstein- Nobel Prize 1921
pmt.swf
Photons -> Photoelectrons -> Electrons
cellflashelectronics.swf
Electric pulse generation
Measurements of the Pulse
Pulse Height
Pulse Width
Pulse Area
Time
Mea
sure
d C
urre
nt a
t det
ecto
r
0
10
(Vol
ts)
Rel
ativ
e B
righ
tnes
s6.21 volts
1.23 volts
3.54 volts
101
102
103
104
1
ADCAnalog to Digital Conversion
Count
Does voltage setting matter?
Voltage=362 292 272 252 522
-Voltage doesn’t change sensitivity or laser power-All your doing is changing the amplification of the signal
-Caveat- there is large “sweet spot” of PMT voltage, outside of this range you may run the risk of non linear amplification
FSC SSC FITC PE APC APC-Cy7FCS Fileor
List Mode File
Electronics Recap Photons ElectronsVoltage pulseDigital # The varying number of photons reaching the
detector are converted to a proportional number of electrons
The number of electrons exiting a PMT can be multiplied by making more electrons available to the detector (increase Voltage input)
The current generated goes to a log or linear amplifier where it is amplified (if desired) and is converted to a voltage pulse
The voltage pulse goes to the ADC to be digitized The values are placed into a List Mode File
What Happens in a Flow Cytometer?
Cells in suspension flow single file pasta focused laser where they scatter light and
emit fluorescence that is collected, filtered and converted to digitized values that are
stored in a file Which can then be read by specialized
software.
Interrogation
Fluidics
Electronics
Interpretation
See you at Data Analysis
May 2nd 10am
AntibodyAntigen binding site
Immunophenotyping
roGFPRedox senstitive biosensor
Ca2+ Flux
Indo-1 Ca2+ freeEmission=500nm
Indo-1 Ca2+ boundEmission=395nm
Apoptosis
Live
Apoptotic
Annexin V
MT
R
PI
FL
ICA
Cell cycle
Sorting
Last attached droplet
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