Basic Principles in Flow
Cytometry
Prepared by Hector Nolla
Manager CRL Flow Cytometry Lab
University of California, Berkeley
Flow Cytometry
Flow Cytometry is the technical process that
allows for the individual measurements of
cell fluorescence and light scattering. This
process is performed at rates of thousands
of cells per second.
This information can be used to individually
sort or separate subpopulations of cells.
History
Flow cytometry developed from microscopy. Thus
Leeuwenhoek is often cited in any discussion regarding
its history.
F.T. Gucker (1947)build the first apparatus for detecting
bacteria in a LAMINAR SHEATH stream of air.
L. Kamentsky (IBM Labs), and M. Fulwyler (Los Alamos
Nat. Lab.) experimented with fluidic switching and
electrostatic cell sorters respectively. Both described cell
sorters in 1965.
M. Fulwyler utilized Pulse Height Analyzers to
accumulate distributions from a Coulter counter. This
feature allowed him to apply statistical analysis to
samples analyzed by flow.
History
In 1972 L. Herzenberg (Stanford Univ.), developed a cell
sorter that separated cells stained with fluorescent
antibodies.The Herzenberg group coined the term
Fluorescence Activated Cell Sorter (FACS).
Fluorescence Activation Process
(or Immunofluorescence)
Antibodies recognize specific
Antibodies are artificially
molecules in the surface of
conjugated to fluorochromes
some cells
Fluorochromes
FITC FITC
Antibodies
FITC When the cells are analyzed by flow
cytometry the cells expressing the marker
FITC
for which the antibody is specific will
manifest fluorescence. Cells who lack the
marker will not manifest fluorescence
But not others
Cellular Parameters Measured by Flow
Intrinsic Extrinsic
No reagents or probes Reagents are required.
required (Structural) Structural
Cell size(Forward Light DNA content
Scatter)
DNA base ratios
Cytoplasmic grabularity(90
degree Light Scatter) RNA content
Photsynthetic pigments Functional
Surface and intracellular
receptors(Immunofluorescence).
DNA synthesis
DNA degradation (apoptosis)
Cytoplasmic Ca++
Gene expression
Flow Cytometry Applications
Immunofluorescence
Cell Cycle Kinetics
Cell Kinetics
Genetics
Molecular Biology
Animal Husbandry (and Human as well)
Microbiology
Biological Oceanography
Parasitology
Bioterrorism
Flow cytometry integrates electronics,
fluidics, and optics.
Electronics are involved in signal processing,
computer display and analysis.
Fluidics are applied to sample processing and
cell sorting.
Optics component are involved in fluorescence
detection.
Flow Cytometry
Flow Cytometry and sorting Optics
Electronics
Fluidics
Laser
Cell sorting
Deflection
Plates
Computer Display
Fluidics
Flow Chamber:
Cells in the sample are
hydrodynamically focused
(See next Slide)
Differential Sample
pressure Regulator
Flow Chamber Vacuum Pump
Compressed
Air
Compressed
Air
Laser
1X PBS Sample
Sheath tube
Reservoir
Waste
Sheath pressure Regulator
Compressor
Compressed
Air
Sample
Y Cells are presented
Z
Sheath
to the laser using
principles of
hydrodynamic
X focusing
Flow
chamber
Y Z
Laser optics
X
Laser Beam
Electronics
Photomultiplier Tube (PMT) PMTs convert light into electrical signals
++++++ Anode Voltage output or signal
The higher the voltage applied to each PMT the
++++ more electrons are generated. Thus, the greater
the amplification of the light coming in (gain).
Dynodes
**Increasing the voltage does increase sensitivity.
+++ Instrument sensitivity is determined by the optics
Dynodes
Dynodes Electrons flow from dynode to Each dynode has a
dynode. Each dynode potential voltage more
++ generates a secondary positive than the
Dynodes emission of more electrons. preceding dynode
+ Electron flow
Photocathode
Light sensitive. When photons hit it,
it generates electrons
(photoelectrons).
Fluorescence Light In
Derived from Practical Flow Cytometry, 4tth edition By
Howard Shapiro
Electronics
Signal Peak
Signal from PMTs Pre-amplifiers Detectors
Analog to Digital Pulse
Converters (ADCs) Integrators
Each Pulse (or event), for each
parameter, is given a numerical Pulse Width
value. This value is then plotted. Circuitry
These numerical values are
proportional to the light
scattering and/or fluorescence
intensity of the event.
Optical Bench
Schematic FL3
Sensor FL4
FL2 620BP Sensor
Sensor 675BP
575BP
FL1
Sensor
525BP
SS
Sensor
645DL
Fluorescence
Pickup Lens
600DL
550DL
Laser
Beam
488BK
488DL
Flow
Cell
FS
Sensor
Optical Filters
Band Pass Optical Filter
525/30nm Band Pass
Long Pass Optical Filter
550nm Long Pass
Dichroic Long Pass Optical Filter
Positioned at a 45 degree
angle.
530 Dichroic Long Pass
Light Absorption
Quantum mechanics requires that molecules
absorb energy as quanta (photons)
Absorption of a photon raises the energy of
the molecule from a ground state to an
excited state
As molecules relax to a lower energy state,
light is released
From: J.Paul Robinson, Purdue University
Fluorescence
Chromophores are the components of molecules which
absorb light, they are generally aromatic rings.
Fluorescence lifetimes can be measured in
femptoseconds
Quantum Yield measures the efficiency between photons
absorbed and photons emitted
J.Paul Robinson, Purdue University
Jablonski diagram
Fluorescence
Emission Electronic Energy Levels
Rotational, Vibrational
Levels
S2
Internal Nonradiative Transitions
Conversion (Heat)
S1
Absorption
Fluorescence
S0 (Ground State)
FITC
PE
PE-Cy5
488 500nm 600nm 700nm
Sample
Y Cells are presented
Z
Sheath
to the laser using
principles of
hydrodynamic
X focusing
Flow
chamber
Y Z
Laser optics
X
Laser Beam
Laminar Fluidic Sheath
Core
Laser Sheath
PE FL
Outer
FITC FL Sheath
488nm Sct
Each cell generates a quanta of fluorescence
Photomultiplier Tubes
(PMTs)
PE FL FITC FL 488nm Sct
Discriminating
Filters
Forward
Dichroic Lenses Fluorescence Light
Pick-up Lens Scattering
Detector
Negative cells are also detected
PE FL FITC FL 488nm Sct
Forward
Dichroic Lenses Light
Pickup Lens Scatter
How Signals are Generated
Voltage
Amplitude
Electrical Pulse
Single Cells
Laser
5-10us
From Fluorescence to Computer Display
Individual cell fluorescence quanta is picked up by the
various detectors(PMTs).
PMTs convert light into electrical pulses.
These electrical signals are amplified and digitized using
Analog to Digital Converters (ADCs).
Each event is designated a channel number (based on
the fluorescence intensity as originally detected by the
PMTs) on a 1 Parameter Histogram or 2 Parameter
Histogram.
All events are individually correlated for all the
parameters collected.
Light Scattering, 2 Parameter Histogram
Bigger
Apoptotic
Cells
90 degree Bigger
Light Scatter Cells
Dead
Cells More
Y Axis
Granular
X Axis
Forward Light Scatter (FLS) Live Cells
1 Parameter Histogram
Positive
Negative
Count Dimmer Brighter
1 2 3 4 6 7 150 160 170 .. 190
Channel Number
Fluorescence picked up from the FITC
PMT
2 Parameter Histogram
Single
Positive PI Double Positive
Population Population
PE FL
Negative
Population
Single Positive
FITC FL
FITC
Population
Gating and Statistics
Data generated in flow cytometry is displayed using
Multiparamater Acquisition and Display software
platforms.
Histograms corresponding to each of the parameters of
interest can be analyzed using statistical tools to
calculate percentage of cells manifesting specific
fluorescence, and fluorescence intensity.
This information can be used to look at fluorescence
expression within subpopulations of cells in a sample
(gating).
Flow Cytometry Data
Smaller
Region,
Live cells
mostly
Larger Region
includes all cells
Running Samples
Prepare samples.
One sample should be completely negative. This sample
should be analyzed first. Adjust the Forward Light
Scatter and Side Scatter amplification.This sample is
also used for adjusting the Fluorescence PMTs
amplification voltage.
Adjust the PMT Voltage until you can see a population
peak in the first decade of your 1 parameter histogram
and or your two parameter plot.
Once the instrument settings are optimized, run samples
and collect data.
If you are analyzing 2 or more fluorescence parameters
you have to prepare Single Color samples for each of
your fluorochromes.
Sorting
Lord Rayleigh, liquid stream emerging from an orifice
becomes unstable, and breaks up into droplets
(1800s)
If a vibration is applied to a stream (emerging
from an orifice) the droplet formation becomes
stable
R. Sweet develops the drop charging and deflection
technique for ink-jet printing (1965)
In cell sorters, an electromagnetic tunable transducer
is incorporated in the flow chamber. This causes the
fluid stream to break-off into individual droplets
The stream behaves like a wavelength, drops are
spaced one wavelength apart
Sorting
The resulting droplet pattern can be described using the
wavelength equation :
v=f
v is the velocity of the stream
The droplet pattern is most f is the vibration frequency
stable when the break-off
point is closest to the is the wavelength or droplet
orifice spacing
This is achieved when the
wavelength is 4.5 stream
diameters
Substituting for 4.5 stream
diameter
v=f(4.5d)
From Howard Shapiro, Practical
Flow Cytometry, fourth edition
Sorting
Putting it together: v=f(4.5d)
For a 75uM orifice and
a stream velocity of
f=(20m/s)/(75 X 4.5)
20m/s:
f=(2.0e7)/(337.5)
f=59,259cycles /s or hertz
The ideal frequency for a 75um flow chamber nozzle is 59,259Hz
From Howard Shapiro, Practical
Flow Cytometry, fourth edition
Sorting
Droplet charging circuitry
When a cell meets a pre-determined criteria to
be sorted, a voltage is applied to the stream
The applied charge will travel down the
stream to the last attached drop
The droplet charge is delayed to coincide with
the arrival of the cell to the precise position in
the stream of the last attached drop
Charged droplets are physically deflected as they pass
through a set of two deflecting plates with opposite
polarities.
From Howard Shapiro, Practical
Flow Cytometry, fourth edition
Orifice (75um)
Observation point
Droplet
Laminar
Sorting
Fluid
S
Sheath
Electrical
Charge Break-off point
(+/-)
Individual
droplets Charged
(-) Deflection
plate
Charged (+)
droplet
Waste
Collector
Collection tube
