Transcript Flow Cytometry and Sorting, Part 1

‫بنام خداوند بخشنده مهربان‬
Flow Cytometry
Principles & practice of “Fluorescence
Spectroscopy in Biological Diagnosis &
Research”
M.Farhadi.M.D
Definitions

Flow Cytometry
– Measuring properties of cells in flow

Flow Sorting
– Sorting (separating) cells based on properties
measured in flow
– Also called Fluorescence-Activated Cell Sorting
(FACS)
Flow Cytometry
 Flow Cytometry is the technological 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 it’s
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.
Basics of Flow Cytometry
•Cells in suspension
Fluidics
•flow in single-file through
•an illuminated volume where they
Optics
•scatter light and emit fluorescence
•that is collected, filtered and
Electronics
•converted to digital values
•that are stored on a computer
Fluidics

Need to have cells in suspension flow in single
file through an illuminated volume
 In most instruments, accomplished by injecting
sample into a sheath fluid as it passes through
a small (50-300 µm) orifice
Flow Cell
Injector
Tip
Fluorescence
signals
Focused laser
beam
Sheath
fluid
Fluidics

When conditions are right, sample fluid flows
in a central core that does not mix with the
sheath fluid
 This is termed Laminar flow
Fluidics

The introduction of a large volume into a small
volume in such a way that it becomes
“focused” along an axis is called
Hydrodynamic Focusing
Fluidics - Differential Pressure
System

Use air (or other gas) to pressurize sample and
sheath containers
 Use pressure regulators to control pressure on
each container separately
Fluidics - Differential Pressure
System

Sheath pressure will set the sheath volume
flow rate (assuming sample flow is negligible)
 Difference in pressure between sample and
sheath will control sample volume flow rate
 Control is not absolute - changes in friction
cause changes in sample volume flow rate
Fluidics - Differential Pressure System
C. Göttlinger, B. Mechtold, and A. Radbruch
Fluidics - 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
Fluidics - Flow Chambers
Flow
through
cuvette
(sense in
quartz)
H.B. Steen - MLM Chapt. 2
Flow Cell
Injector
Tip
Fluorescence
signals
Focused laser
beam
Sheath
fluid
Optics

Need to have a light source focused on the
same point where cells have been focused (the
illumination volume)
 Two types of light sources
– Lasers
– Arc-lamps
Optics - Light Sources

Lasers
– can provide a single wavelength of light (a laser
line) or (more rarely) a mixture of wavelengths
– can provide from milliwatts to watts of light
– can be inexpensive, air-cooled units or expensive,
water-cooled units
– provide coherent light
Optics - Light Sources

Arc-lamps
– provide mixture of wavelengths that must be
filtered to select desired wavelengths
– provide milliwatts of light
– inexpensive, air-cooled units
– provide incoherent light
Optics - Forward Scatter Channel

When a laser light source is used, the amount
of light scattered in the forward direction
(along the same axis that the laser light is
traveling) is detected in the forward scatter
channel
 The intensity of forward scatter is proportional
to the size, shape and optical homogeneity of
cells (or other particles)
Forward Angle Light Scatter
Laser
FALS Sensor
Optics - Side Scatter Channel

When a laser light source is used, the amount
of light scattered to the side (perpendicular to
the axis that the laser light is traveling) is
detected in the side or 90o scatter channel
 The intensity of side scatter is proportional to
the size, shape and optical homogeneity of
cells (or other particles)
90 Degree Light Scatter
Laser
FALS Sensor
90LS Sensor
Optics - Light Scatter

Forward scatter tends to be more sensitive to
surface properties of particles (e.g., cell
ruffling) than side scatter
– can be used to distinguish live from dead cells

Side scatter tends to be more sensitive to
inclusions within cells than forward scatter
– can be used to distinguish granulated cells from
non-granulated cells
Fluorescence Detectors
Laser
Freq
FALS Sensor
Fluorescence
Fluorescence detector
(PMT3, PMT4 etc.)
Light Scattering, 2 Parameter Histogram
Bigger
Apoptotic
Cells
90 degree
Light Scatter
Y Axis
Dead
Cells
X Axis
Forward Light Scatter (FLS)
Bigger
Cells
More
Granular
Live Cells
1 Parameter Histogram
Positive
Negative
Count
Brighter
Dimmer
6
4
1
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
Population
Double Positive
Population
PE FL
Negative
Population
FITC FL
Single Positive
FITC
Population
Optics - Filter Properties

Long pass filters transmit wavelengths above
a cut-on wavelength
 Short pass filters transmit wavelengths below
a cut-off wavelength
 Band pass filters transmit wavelengths in a
narrow range around a specified wavelength
– Band width can be specified
Optical Filters
Standard Long Pass
Filters
Light Source
520 nm Long Pass Filter
Transmitted Light
>520 nm
Light
Standard Short Pass Filters
Light Source
575 nm Short Pass Filter
Transmitted Light
<575 nm
Light
Standard Band Pass Filters
630 nm BandPass Filter
White Light Source
Transmitted Light
620 -640 nm Light
Optics - Filter Properties
When a filter is placed at a 45o angle to a light
source, light which would have been
transmitted by that filter is still transmitted but
light that would have been blocked is reflected
(at a 90o angle)
 Used this way, a filter is called a dichroic
filter or dichroic mirror

Dichroic Filter/Mirror
Filter placed at 45o
Light Source
Transmitted Light
Reflected light
Optics - Filter Layout

To simultaneously measure more than one
scatter or fluorescence from each cell, we
typically use multiple channels (multiple
detectors)
 Design of multiple channel layout must
consider
– spectral properties of fluorochromes being used
– proper order of filters and mirrors
Common
Laser
Lines
350
300 nm
457 488 514
400 nm
500 nm
610 632
600 nm
700 nm
PE-TR Conj.
Texas Red
PI
Ethidium
PE
FITC
cis-Parinaric acid
Compensation
• A very
important
function of
the
electronics
system is to
perform
compensation

There is some overlap between the colors emitted by different
fluorescent markers, therefore mathematical compensation is
used to reduce overlapping results
http://www.bdbiosciences.com
Example Channel Layout for
PMT
Laser-based Flow
Cytometry
4
Flow cell
PMT
Dichroic
Filters
3
PMT
2
Bandpass
Filters
PMT
1
Laser
Optics - Detectors

Two common detector types
– Photodiode
 used for strong signals when saturation is a potential
problem (e.g., forward scatter detector)
– Photomultiplier tube (PMT)
 more sensitive than photodiode but can be destroyed by
exposure to too much light
Photomultipliers and
Photodiodes


http://www.bdbiosciences.com
These are the
two types of
detectors that
convert
photons into
electrical
signals
They control
sensitivity by
adjusting
voltage
Summary of Part 1
•Cells in suspension
Fluidics
•flow in single-file through
•an illuminated volume where they
Optics
•scatter light and emit fluorescence
•that is collected, filtered and
Electronics
•converted to digital values
•that are stored on a computer
Typical Research Cytometer
(Coulter 753) (1980s)
$200-300,000
Detectors
Lasers
Fluidics
Computers
Laser Power Supply
Typical Clinical
Cytometer
Computer System
Detector &
Mechanical Fluidics
$90-120,000
Clinical Applications Of
Flow Cytometric Analysis
FlowCytometric(immunophenotypic)
Classification Of Leukemias
Immunophenotyping
CD4
CD # = cluster designation number
CD2
Lymphocyte
Immunophenotyping
Peripheral White Blood Cells
CD45+
Monocytes
Granulocytes
Lymphocytes
Monocytes
B
T
CD3+
T
Helper
CD3+
CD4+
T
Cytotoxi
c
CD3+
CD8+
CD3CD19+
Neutrophils
N
K
CD3CD16+
CD56+
Basophils
Eosinophils
0
0
3
0
Count
7
0
1
1
0
1
5
Immunophenotyping
.1
1
10
Log FITC
100
1000
CD4/CD8 Quadstats
100
1
2
45%
1
10
2%
27% 3
4
.1
26%
.1
1
10
100
Log FITC Fluorescence (CD8)
1000
Light Scatter Gating
1000
Side Scatter Projection
Forward Scatter Projection
Neutrophils
800
1000
100
50
600
200
40
20
Monocytes
400
30
15
Lymphocytes
0
8
200
Forward Scatter
Scale
0
200
400
600
800
90 Degree Scatter
1000
The Cell Cycle
G2
M
G1
S
G0
Quiescent cells
DNA Analysis
Ethanol
Deterge
nt
Nucleic Acid Dye
DNA Dye
Propidium
NH2
Ethidium
NH2
NH2
N+
C2H5
C2H2)3
N+
CH3
C2H5
NH2
N+
C2H5
Normal Cell Cycle
G2
M
G0
DNA Analysis
G1
G0G1
s
C
o
u
n
t
G2 M
s
0
200
400
600
4N
2N
DNA content
800
1000
300
225
DNA index 1.21
150
Aneuploid peak
0
75
Counts
DNA Analysis
0
200
400
600
PI Fluorescence
800
1000
112
112
150
150
Reticulocyte Analysis
RMI = 34
Count7 5
Coun7t5
RMI = 0
R4 R3 R2 R1
0
0
37
37
R4 R3 R2 R1
.1
1
10
100
log Thiazole Orange
1000
.1
1
10
100
1000
log Thiazole Orange
Intracellular Analysis
Permeabilizin
g solution
Fixation
solution
• Cytokine
• Enzyme
• signal
transduction
molecule
• …etc.
Cytokine Detection
Picture From www.fredonia.edu
LPS
Ionomycin
PHA
T cell
CD28
Con A
CD3
To enhance the accumulation of intracellular cytokines.
Stimulation
Secretion
stop
Monensin: Cytokines accumulate in the
ER
Brefeldin A: in Golgi complex. (Brefeldin A or Monensin)
Only in vitro
To maintain structural integrity.
Formaldehyde or glutaraldehyde
Keep the protein structure and doesn't change the
(accessibility of the) epitopes too much
Intracellular Staining
Permeabilisation
Saponin (permeablisation buffer).
Fixation
Combination of Cell
Surface and Cytoplasmic
Staining
Cytokine Profiles of Memory T Cell Subsets
Signal Transduction
Intracellular Staining in
Activated Lysed Whole
Blood
Apoptosis (Sub G1)
Cell Function Analysis
• Membrane Potential (DiOC6, JC-1)
• Oxidative Metabolism (Free Radicals)
• Intracellular PH Value (Snarf-1)
• Ca++ Influx (Fluo-4/Fura Red, Indo-1)
• Phagocytosis
• Cell Proliferation (PI, BrdU, Intracellular Cyclins)
• Apoptosis (Annexin V, active Caspase-3)
Annexin V Assay
Annexin V/PI Double Staining
Early
Apoptosis
Mitochondria Membrane Potential (JC-1)
Proliferation Assay--BrdU
Cytometric Beads Array (CBA)
Single
Step
Incubation
or
Two-Step
Incubation
Cytometry Beads Array (CBA)