Flow Cytometry and Cell Sorting

1
Flow Cytometry and Cell Sorting

• Adapted by Albert D. Donnenberg, Ph.D. from
• Fluorescence Spectroscopy in Biological
  Research
• Robert F. Murphy, Ph.D. Carnegie Mellon University

2
Sources

• Flow Cytometry and Sorting, 2nd ed. (M.R.
  Melamed, T. Lindmo, M.L. Mendelsohn, eds.),
  Wiley-Liss, New York, 1990 - referred to here as
  MLM
• Flow Cytometry Instrumentation and Data Analysis
  (M.A. Van Dilla, P.N. Dean, O.D. Laerum, M.R.
  Melamed, eds.), Academic Press, London, 1985 -
  VDLM

3
Sources (continued)
4
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)

5
Basics of Flow Cytometry
Cells in suspension flow in single-file
through an illuminated volume where they scatter
light and emit fluorescence that is collected,
filtered and converted to digital values that are
stored on a computer
Fluidics
Optics
Electronics
6
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

7
Flow Cell
Injector Tip
Sheath fluid
Fluorescence
signals
Focused laser
beam
Purdue University Cytometry Laboratories
8
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

9
Fluidics - Laminar Flow

• Whether flow will be laminar can be determined
  from the Reynolds number
• When Re
• When Re 2300, flow can be turbulent

10
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

11
Fluidics
The figure shows the mapping between the flow
lines outside and inside of a narrow tube as
fluid undergoes laminar flow (from left to
right). The fluid passing through cross section
A outside the tube is focused to cross section a
inside.
V. Kachel, H. Fellner-Feldegg E. Menke - MLM
Chapt. 3
12
Fluidics
Notice how the ink is focused into a tight stream
as it is drawn into the tube under laminar flow
conditions.
Notice also how the position of the inner ink
stream is influenced by the position of the ink
source.
V. Kachel, H. Fellner-Feldegg E. Menke - MLM
Chapt. 3
13
Fluidics
Notice how the ink is focused into a tight stream
as it is drawn into the tube under laminar flow
conditions.
Notice also how the position of the inner ink
stream is influenced by the position of the ink
source.
V. Kachel, H. Fellner-Feldegg E. Menke - MLM
Chapt. 3
14
Fluidics

• How do we accomplish sample injection and
  regulate sample flow rate?
• Differential pressure
• Volumetric injection

15
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

16
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

17
Fluidics - Differential Pressure System
C. Göttlinger, B. Mechtold, and A. Radbruch
18
Fluidics - Volumetric Injection System

• Use air (or other gas) pressure to set sheath
  volume flow rate
• Use syringe pump (motor connected to piston of
  syringe) to inject sample
• Sample volume flow rate can be changed by
  changing speed of motor
• Control is absolute (under normal conditions)

19
Volumetric Injection System
H.B. Steen - MLM Chapt. 2
20
Fluidics - Particle Orientation and Deformation

• As cells are hydrodynamically focused, they
  experience shear stresses on different points on
  their surfaces (an in different locations in the
  stream)
• These cause cells to orient with their long axis
  (if any) along the axis of flow
• The shear stresses can also cause cells to deform
  (e.g., become more cigar-shaped)

21
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. v3 m/s.
V. Kachel, et al. - MLM Chapt. 3
22
Fluidics - Flow Chambers

• The flow chamber
• Defines the axis and dimensions of sheath and
  sample flow
• Defines the point of optimal hydrodynamic
  focusing
• Can also serve as the interrogation point (the
  illumination volume)

23
Fluidics - Flow Chambers

• Four basic flow chamber types
• Jet-in-air
• best for sorting, inferior optical properties
• Flow-through cuvette
• excellent optical properties, can be used for
  sorting
• Closed cross flow
• best optical properties, cant sort
• Open flow across surface
• best optical properties, cant sort

24
Fluidics - Flow Chambers
Jet-in-air nozzle (sense in air)
H.B. Steen - MLM Chapt. 2
25
Fluidics - Flow Chambers
Flow through cuvette (sense in quartz)
H.B. Steen - MLM Chapt. 2
26
Fluidics - Flow Chambers
Closed cross flow chamber
H.B. Steen - MLM Chapt. 2
27
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

28
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

29
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

30
Optics - Optical Channels

• An optical channel is a path that light can
  follow from the illuminated volume to a detector
• Optical elements provide separation of channels
  and wavelength selection

31
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 most
  influenced by the size of cells (or other
  particles)

32
Forward Angle Light Scatter
Purdue University Cytometry Laboratories
33
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 most influenced
  by the shape and optical homogeneity of cells

34
90 Degree Light Scatter
Purdue University Cytometry Laboratories
35
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

36
Optics - Fluorescence Channels

• The fluorescence emitted by each fluorochrome is
  usually detected in a unique fluorescence channel
• The specificity of detection is controlled by the
  wavelength selectivity of optical filters and
  mirrors

37
Fluorescence Detectors
Laser
Purdue University Cytometry Laboratories
38
Optics - Filter Properties

• Optical filters are constructed from materials
  that absorb certain wavelengths (while
  transmitting others)
• Transitions between absorbance and transmission
  are not perfect the sharpness can be specified
  during filter design

39
Optics - Filter Properties

• Filters must have very sharp cutons and cutoffs
  since scattered laser light is several orders of
  magnitude greater than emitted fluorescence
• Filters are designed to reject light to specific
  tolerances (e.g., reject 488 nm light at 10-6
  level only 0.0001 of incident light at 488 nm
  gets through)

40
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

41
Standard Long Pass Filters
520 nm Long Pass Filter
Light Source
Transmitted Light
520 nm Light
Standard Short Pass Filters
575 nm Short Pass Filter
Light Source
Transmitted Light
Purdue University Cytometry Laboratories
42
Standard Band Pass Filters
630 nm BandPass Filter
White Light Source
Transmitted Light
620 -640 nm Light
Purdue University Cytometry Laboratories
43
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

44
Dichroic Filter/Mirror
Filter placed at 45o
Transmitted Light
Light Source
Purdue University Cytometry Laboratories,
modified by R.F. Murphy
Reflected light
45
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

46
Common Laser Lines
PE-TR Conj.
Texas Red
PI
Ethidium
PE
FITC
cis-Parinaric acid
Purdue University Cytometry Laboratories
47
Channel Layout for Laser-based Flow Cytometry
PMT
4
PMT
Dichroic
3
Filters
Flow cell
PMT
2
Bandpass
Filters
PMT
1
Laser
Purdue University Cytometry Laboratories,
modified by R.F. Murphy
48
Channel Layout for Arc Lamp-based Flow Cytometry

• (Overhead 10)

H.B. Steen - MLM Chapt. 2
49
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

50
Wavelength Dependence of Photomultipliers
We should consider the properties of PMTs when
designing an optical layout knowledge of PMT
types on a particular instrument allows optimum
use of available fluorescence channels
H.B. Steen - MLM Chapt. 2
51
Electronics

• Processing of signals from detectors
• Preamplification
• Strengthen signals so that they can travel from
  remote detectors to central electronics
• Amplification
• Adjust signal intensity
• Linear or Logarithmic
• Log transformation can also be performed after
  digitization using a look-up table

52
Comparison of linear and logarithmic amplification
P. N. Dean - MLM Chapt. 22
53
Electronics

• Processing of signals from detectors
• Generation of Integral or Pulse Width
• Gated Peak-Sense-And-Hold
• Timing Adjustment
• Necessary for multiparameter systems
• Analog-Digital Conversion

54
Signal Processing
In non-colinear multi-laser flow cytometers each
cell passes through each measurement station at
different times. The figure shows how the
information obtained at each station is aligned
in time so that it is all recorded at one time
for each cell.
R.D. Hiebert R.G. Sweet - VDLM Chapt. 4
55
Data Acquisition

• Each measurement from each detector is referred
  to as a parameter
• Data are acquired as a list of the values for
  each parameter (variable) for each event
  (cell)

56
Listmode Data Acquisition
57
Single parameter histograms
P. N. Dean - MLM Chapt. 22
58
Bivariate Histograms
Two ways of displaying the correlation between
two parameters a perspective (or projection)
plot and a contour map.
P. N. Dean - MLM Chapt. 22
59
Gating
Sub-cellular debris with low light scatter
obscures the fluorescence signals of two cell
populations. Limiting analysis within particular
boundaries of forward and side scatter (B)
reveals the fluorescence of the cells within that
gate (C).
P. N. Dean - MLM Chapt. 22
60
Basics of Flow Sorting

• Droplet formation
• Timing
• Coincidence - Purity and Efficiency

61
Fluorescence Activated Cell Sorting
FALS Sensor
488 nm laser
Fluorescence detector
-
Charged Plates
Single cells sorted into test tubes
Purdue University Cytometry Laboratories
62
Droplet formation
As liquid is ejected into air, it will form
droplets. By vibrating the nozzle at a defined
frequency, the size of these droplets and the
position along the stream where they form can be
controlled with great precision.
T. Lindmo, D.C. Peters R.G Sweet - MLM Chapt. 8
63
Timing
T. Lindmo, D.C. Peters R.G Sweet - MLM Chapt. 8
64
Coincidence - Purity

• As droplets form, they can contain wanted cells
  as well as unwanted cells. If all droplets
  containing a wanted cell are sorted (regardless
  of whether they also contain unwanted cells), the
  purity of the sorted sample will be reduced.

65
Coincidence - Purity

• The purity can be improved by checking for
  coincidence events and not sorting any wanted
  cell that occurs too close to an unwanted cell.
• This causes an increase in purity but a reduction
  in sorting efficiency.

66
Coincidence - Efficiency
The efficiency of sorting (with coincidence
checking) for three-droplet sorting (solid lines)
and one-droplet sorting (broken line) is shown as
a function of event rate.
T. Lindmo, D.C. Peters R.G Sweet - MLM Chapt. 8
67
Cell Cycle Analysis

• One of the earliest applications of flow
  cytometry was the analysis of cell cycle position
  by quantifying cellular DNA.
• Flow cytometry is still the method of choice for
  fast, accurate determination of cell cycle
  distributions.

68
Univariate Cell Cycle Methods

• In the simplest method, cellular DNA is detected
  using a fluorescent dye that binds preferentially
  to DNA.
• Propidium iodide is most commonly used. It
  undergoes a dramatic increase in fluorescence
  upon binding DNA. It requires permeabilization
  of the plasma membrane.
• Hoechst 33342 can be used where labeling of
  unpermeabilized (live) cells is desired.

69
Univariate Cell Cycle Methods

• When the amount of DNA per cell is measured on a
  sample from an asynchronously growing cell
  culture, cells with various amounts of DNA from
  the 2N (G0/G1) amount to the 4N (G2/M) amount are
  observed. A histogram reveals the fraction of
  cells in the various cell cycle phases.

70
Normal Cell Cycle
M
G0
G2
DNA Analysis
G1
s
Count
s
0
200
400
600
800
1000
DNA content
Purdue University Cytometry Laboratories
71
DNA Analysis
0
200
400
600
800
1000
4N
2N
PI Fluorescence
Purdue University Cytometry Laboratories
72
Cell cycle progression of synchronized cells
J.W. Gray, F. Dolbeare M.G. Pallavicini - MLM
Chapt. 23
73
DNA Analysis
Aneuploid peak
0
200
400
600
800
1000
PI Fluorescence
Purdue University Cytometry Laboratories
74
Bivariate Cell Cycle Analysis

• To aid in the detection of cells in S-phase, a
  brief pulse of a marked nucleotide can be used.
  The most common such nucleotide is
  bromodeoxyuridine (BrdU) which is incorporated
  into DNA in place of thymidine. The incorporated
  BrdU can be detected with an antibody,
  identifying those cells that synthesized DNA
  during the pulse.

75
Detection of incorporated BrdU
J.W. Gray, F. Dolbeare M.G. Pallavicini - MLM
Chapt. 23
76
J.W. Gray, F. Dolbeare M.G. Pallavicini - MLM
Chapt. 23
77
Chromosome Analysis and Sorting

• Individual chromosomes can be analyzed in flow
  after appropriate preservation and isolation.
  The most common method is to use two different
  DNA dyes, one (Hoechst 33258) that binds
  preferentially to AT-rich DNA and one
  (chromomycin A3) that binds preferentially to
  GC-rich DNA.

78
Two-color chromosome analysis
J.W. Gray L.S. Cram - MLM Chapt. 25
79
Normal human
Normal hamster
Human X hamster
Normal mouse
J.W. Gray L.S. Cram - MLM Chapt. 25
80
Immunofluorescence Analysis

• A major application of flow cytometry is the
  analysis (and sorting) of subsets of blood cells
  using surface markers.
• A useful feature is that the major blood cell
  types show distinct forward and side scatter
  profiles.

81
Light Scatter Gating
Side Scatter Projection
Neutrophils
Scale
1000
200
100
50
40
Monocytes
30
20
15
Lymphocytes
8
200
400
600
800
1000
0
90 Degree Scatter
Purdue University Cytometry Laboratories