Download 2012 Flow Cytometry Basics

Flow Cytometry Basics
James Marvin
Director, Flow Cytometry Core Facility
University of Utah Health Sciences Center
Office 801-585-7382
Lab 801-581-8641
[email protected]
New Instrumentation
BD FacsCanto
-4 lasers
-8 color detection
iCyte/Sony Eclipse
-4 lasers
-5 color detection
-Electronic volume
Seventeen-colour flow cytometry: unravelling the immune system
Nature Reviews Immunology, 2004
“This ain’t your
grandma’s flow
cytometer”
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The uses of flow in research has
boomed since the mid-1980s, and
is now the gold standard for a
variety of applications
Flow Cytometry
Applications
Medline Publications citing "Flow
Cytometry"
5000
4000
3000
2000
1000
0
19
1960
1965
1970
1975
1980
1985
1990
2095
00

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
Publications

Year
Section I
Background Information on Flow Cytometry
Many components to a
successful assay
Experimental Design
“One on One”
Instrumentation
Analysis
Presentation
“Flow Basics”
“Data Analysis”
“Data Analysis”
•Sample Procurement •Appropriate Lasers
•Sample preparation •Appropriate Filters
•Fix/Perm
Settings
•Which Fluorophore •Instrument
•Lin vs Log
•Controls
•Time
•Isotype?
•A, W, H
•Single color
•Doublet
•FMO
discrimination
•Interpretation
•Mean, Median
•% +
•CV
•SD
•Signal/Noise
•Gating
•Histogram
•Dot Plot
•Density Plot
•Overlay
•Bar Graph
What Is Flow Cytometry?

Flow ~ motion
 Cyto ~ cell
 Metry ~ measure
Measuring both intrinsic and extrinsic
properties of cells while in a moving fluid
stream
Cytometry vs. Flow Cytometry



Cytometry/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 file
 through 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.
Fluidics
Interrogation
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
Core
Sheath
Hydrodynamic
Focusing
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
Hydrodynamic
Focusing
Laminar flow
Laminar flow occurs when a fluid flows in parallel layers, with no
disruption between the layers
V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3
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)
Flow Cell- the place where hydrodynamically
focused cells are delivered to the focused
light source
Cell
flash.
swf
Gaussian- A “bell
Sample
curved” normal distribution
where the values and shape
falls off quickly as you
move away from central,
most maximum point.
Sheath
Sheath
Laser
Focal Point
Sample
Core
Stream
Incoming
Laser
Low Differential
High Differential or “turbulent flow”
300
280
G0/G1 CV= 2.42
Low pressure
Count
260
240
220
200
180
68.70
19.16
9.56
160
140
120
100
80
S phase
G0/G1
60
40
G2/M
20
0
High pressure
Count
0
340
320
300
280
260
240
220
200
180
160
140
120
100
80
60
40
20
0
1024
2048
FL3
3072
4096
GO/G1 CV= 7.79
74.85
0
1024
2048
FL3
9.12
3072
15.84
4096
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?

Fluidics
Cells in suspension flow single file
 through a focused laser where they scatter
Interrogation
light and emit fluorescence that is filtered,
 measured, and converted to digitized values
Electronics
that are stored in a file
 Which can then be read by specialized
Interpretation
software.
Interrogation

Light source needs to be focused on the
same point where cells are focused.

Light source
 99%=Lasers
Lasers
Light 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
Scatter
Fluorescence
VS

Collected photons are the
product of laser light
scattering or bouncing off
cells

Collected photons are
product of excitation with
subsequent emission
determined by fluorophore

488nm

350nm-800nm

Information associated
with physical attributes of
cells (size, granularity,
refractive index)

Readout of intrinsic
(autofluorescence) or
extrinsic fluorescence
(intentional cell labeling)
Forward Scatter
Laser Beam
.50-80
Original from Purdue University Cytometry Laboratories
FSC
Detector
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
Laser Beam
FSC
Detector
Collection
Lens
SSC
Detector
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
Granulocytes
Dead
SSC
Lymphocytes
LIVE
Monocytes
RBCs, Debris,
Dead Cells
FSC
Fluorescence
S3
Excited higher
energy states
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
Energy
S2
•
S1
Absorbed
exciting light
•As the laser interrogates the cell,
Emitted
fluorescence
S0
Ground State
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
Transmittance

400nm
500nm
600nm
700nm
Short Pass Filter

Transmits all wavelengths less than
specified wavelength
600SP will transmit all wavelengths
less than 600nm.
Transmittance
 Example:
400nm
500nm
600nm
700nm
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)
Transmittance

400nm
500nm
600nm
700nm
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.
Detector 1
Detector 2
Dichroic
Filter
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.
Multi-laser Instruments and
pinholes
Implications-Can separate completely
overlapping emission profiles
if originating off different lasers
-Significantly reduces compensation
Blue Laser
Excitation
530/20 Blue
585/20 Yellow
585/20 Blue
Spatial separation
Blue and Yellow
Laser Excitation
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?

Fluidics
Cells in suspension flow single file
 Through a focused laser where they scatter
Interrogation
light and emit fluorescence that is filtered,
measured
 and converted to digitized values that are
Electronics
stored in a file
Interpretation
 Which can then be read by specialized
software.
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
Detectors

There 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
 Used
tubes (PMT)
for detecting small amounts of fluorescence
emitted from fluorochromes.
 Incredible Gain (amplification-up to 10million
times)
 Low noise
Detector names
Photons -> Photoelectrons -> Electrons
Photoelectric
Effect
Einstein- Nobel Prize 1921
Electric pulse
generation
Measurements of the Pulse
Measured Current at detector
Pulse Area
Pulse Height
Pulse Width
Time
10
Analog to Digital
Conversion
6.21 volts
103
(Volts)
3.54 volts
104
1.23 volts
102
101
Relative Brightness
ADC
0
1
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
FCS File
or
List Mode File
FSC
SSC
FITC
PE
APC
APC-Cy7
Electronics Recap






Photons ElectronsVoltage pulseDigital #
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 past
 a 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.
Fluidics
Interrogation
Electronics
Interpretation
See you at Data Analysis
May 2nd 10am
Antibody
Antigen
binding site
Immunophenotyping
roGFP
Redox senstitive biosensor
Ca2+ Flux
Indo-1 Ca2+ free
Emission=500nm
Indo-1 Ca2+ bound
Emission=395nm
Apoptosis
Apoptotic
Annexin V
FLICA
MTR
Live
PI
Cell cycle
Sorting
Last attached
droplet