Transcript BMS 631 - Lecture 2

BMS 631 - Lecture 2
Who’s and Why’s of Flow Cytometry
The History of Flow Cytometry:
An introduction to the early beginnings of flow cytometers;
the rationale for early investigations; a summary of the
state-of-the-art; the events that led to modern cytometry;
early fluorescent dyes; image analysis; DNA cytology
J.Paul Robinson, PhD
Professor of Immunopharmacology and Bioengineering
References: (Shapiro 3rd ed. pp43-71)
Note: these slides were converted to web slides by Microsoft
PowerPoint directly. Microsoft made such a bad job of this
process that all text boxes had to be eliminated because they
did not translate at all – so forgive the problems – they are mostly
Bad Microsoft programming - - - thanks Bill!
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Herzenberg
Lou Herzenberg - 1969 - sorter based on fluorescence (arc lamp) built after working
with one of Kamentsky’s RCS systems where they built an instrument they called the
Fluorescence Activated Cell Sorter (FACS)
Photos ©2000 – J.P. Robinson
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Dittrich & Göhde
Dittrich & Gohde - 1969 - Impulscytophotometer (ICP)- used
ethidium bromide for a DNA stain and a high NA objective used
as a condenser and collection lens
Laerum, Göhde, Darzynkiewicz (1998)
Göhde and Laerum (1998)
Photos ©2000 – J.P. Robinson
© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
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Kamentsky
Kamentsky - Bio/Physics Systems - 1970 commercial cytometer - the “Cytograph”
He-Ne laser system at 633 nm for scatter (and extinction) - supposedly the first
commercial instrument incorporating a laser. It could separate live and dead cells by
uptake of Trypan blue. A fluorescence version called the “Cytofluorograph” followed
using an air cooled argon laser at 488 nm excitation
1970 Cytograph presently at the Purdue University Cytometry Laboratories
Photo ©2000 – J.P. Robinson
© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
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History
Phywe AG of Gottingen (1970) - produced a commercial version of the ICP built around a
Zeiss fluorescent microscope
Don’t have photo….
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Herzenberg & Becton Dickinson
Herzenberg -1972 - Argon laser flow sorter - placed an argon laser onto their sorter and
successfully did high speed sorting - Coined the term Fluorescence Activated Cell Sorting
(FACS) This instrument could detect weak fluorescence with rhodamine and fluorescein
tagged antibodies. A commercial version was distributed by B-D in 1974 and could collect
forward scatter and fluorescence above 530 nm.
Photo ©2000 – J.P. Robinson
© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
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Mack Fulwyler
• Coulter Electronics manufactured the TPS-1 (Two parameter sorter) in
1975 which could measure forward scatter and fluorescence using a
35mW argon laser.
This photo (©2000 – J.P.
Robinson) is one of only
one or two surviving TPS
Instruments. It is very
similar to the Coulter
Counter of the day.
Photo ©2000 – J.P. Robinson
© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
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Shapiro
Shapiro and the Block instruments (1973-76) - a series of multibeam flow cytometers
that did differentials and multiple fluorescence excitation and emission
Photos ©2000 – J.P. Robinson
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Hemalog D
Technicon - Hemalog D - 1974 - first commercial differential flow cytometer - light scatter
and absorption at different wavelengths - chromogenic enzyme substrates were used to
identify neutrophils and eosinophils by peroxidase and monocytes by esterase, basophils
were identified by the presence of glycosaminoglycans using Alcian Blue - the excitation for
all measurements was a tungsten-halogen lamp
Insert photos on
page 60
Image from Shapiro
“Practical Flow
Cytometry”, Wiley-Liss,
1995
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Coulter Electronics
• 1977-78 developed the Epics series of instruments which were
essentially 5 watt argon ion laser instruments, complete with a
multiparameter data analysis system, floppy drive and graphics printer.
Photo ©2000 – J.P. Robinson
Epics V front end (left) and MDADS (right)currently at
Purdue University
© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
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Biophysics -Ortho
• Ortho Diagnostics (Johnson and Johnson) purchased Biophysics in
1976 and in 1977 the System 50 Cytofluorograph was developed - this
was a droplet sorter, with a flat sided flow cell, forward and orthogonal
scatter, extinction, 2 fluorescence parameters, multibeam excitation,
computer analysis option.
Photo ©2000 – J.P. Robinson
• 1979 - NIH scientists had added a krypton laser at 568 nm to excite
Texas Red fluorescence at 568 nm and emit at 590-630 nm. Argon
(488 nm FITC was measured simultaneously without signal cross-talk
- thus the FACS IV was developed (B-D).
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Stuart Schlossman
• Schlossman at the Farber Institute in Boston, began to
make monoclonal antibodies to white blood cell antigens
in 1978. Eventually he collaborated with Ortho
Diagnostics who distributed the famous “OK T4” etc.,
Mabs
• Coulter Immunology also distributed his antibodies
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Introductory Terms and Concepts
• Parameter/Variable
• Light Scatter- Forward (FALS), narrow (FS)
- Side, Wide, 90 deg, orthogonal
• Fluorescence - Spectral range
• Absorption
• Time
• Count
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Concepts
Scatter:
Size, shape, granularity, polarized
scatter (birefringence)
Fluorescence:
Intrinsic: Endogenous pyridines and flavins
Extrinsic: All other fluorescence profiles
Absorption: Loss of light (blocked)
Time:
Useful for kinetics, QC
Count:
Always part of any collection
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Instrument Components
Electronics: Control, pulse collection, pulse analysis,
triggering, time delay, data display, gating, sort control,
light and detector control
Optics: Light source(s), detectors, spectral separation
Fluidics: Specimen, sorting, rate of data collection
Data Analysis: Data display & analysis,
multivariate/simultaneous solutions, identification of sort
populations, quantitation
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Fundamentals of a Flow Cytometer
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Data Analysis Concepts
Gating
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Single parameter
Dual parameter
Multiple parameter
Back Gating
Note: these terms are introduced here, but will be discussed in more
detail in later lectures
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Data Presentation Formats
• Histogram
• Dot plot
• Contour plot
• 3D plots
• Dot plot with projection
• Overviews (multiple histograms)
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Light and Matter
J.Paul Robinson
Professor of Immunopharmacology
School of Veterinary Medicine, Purdue University
Hansen Hall, B050
Purdue University
Office: 494 0757
Fax 494 0517
email\; [email protected]
WEB http://www.cyto.purdue.edu
Shapiro p 75-93
© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
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Light and Matter
• Energy
– joules, radiant flux (energy/unit time)
– watts (1 watt=1 joule/second)
• Angles
– steradians - sphere radius r - circumference is 2r2; the
angle that intercepts an arc r along the circumference is
defined as 1 radian. (57.3 degrees) a sphere of radius r
has a surface area of 4r2. One steradian is defined as
the solid angle which intercepts as area equal; to r2 on the
sphere surface
3rd Ed - Shapiro p 75
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Terms
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Side scatter, forward angle scatter, cell volume, coulter volume:
Understand light scattering concepts; intrinsic and extrinsic parameters
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Photometry:
Light - what is it - wavelengths we can see 400-750 nm, most sensitive around 550 nm.
Below 400 nm essentially measuring radiant energy. Joules (energy) radiant flux
(energy per unit time) is measured in watts (1 watt=1 joule/second).
Steradian (sphere radius r has surface area of 4 r2; one steradian is defined as that
solid angle which intercepts an area equal to r2 on the surface.
Mole - contains Avogadro's number of molecules (6.02 x 1023) and contains a mass in
grams = molecular weight. Photons - light particles - waves - Photons are particles
which have no rest mass - pure electromagnetic energy - these are absorbed and
emitted by atoms and molecules as they gain or release energy. This process is
quantized, is a discrete process involving photons of the same energy for a given
molecule or atom. The sum total of this energy gain or loss is electromagnetic
radiation propagating at the speed of light (3 x 108 m/s). The energy (joules) of a
photon is
E=hn and E=hn/l [n-frequency, l-wavelength, h-Planck's constant 6.63 x 10-34 jouleseconds]
Energy - higher at short wavelengths - lower at longer wavelengths.
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Photons and Quantum Theory
• Photons
– particles have no rest mass - composed of pure electromagnetic energy
- the absorption and emission of photons by atoms and molecules is the
only mechanism for atoms and molecules can gain or lose energy
• Quantum mechanics
– absorption and emission are quantized - i.e. discrete process of gaining
or losing energy in strict units of energy - i.e. photons of the same
energy (multiple units are referred to as electromagnetic radiation)
• Energy of a photon
– can be computed from its frequency (n)
in hertz (Hz) or its wavelength (l) in meters from
E=hn and E=hc/
 = wavelength
h = Planck’s constant
(6.63 x 10-34 joule-seconds
c = speed of light (3x108 m/s)
3rd Ed Shapiro p 76
© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
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Laser power
E=hn and E=hc/
• One photon from a 488 nm argon laser has an
energy of
E=
6.63x10-34 joule-seconds x 3x108
488 x 10-3
= 4.08x10-19 J
• To get 1 joule out of a 488 nm laser you need
2.45 x 1018 photons
• 1 watt (W) = 1 joule/second a 10 mW laser at
488 nm is putting out 2.45x1016 photons/sec
3rd Ed. Shapiro p 77
© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
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What about a UV laser?
E=
6.63x10-34 joule-seconds x 3x108
325 x 10-3
= 6.12 x 10-19 J so 1 Joule at 325 nm = 1.63x1018 photons
What about a He-Ne laser?
E=
6.63x10-34 joule-seconds x 3x108
633 x 10-3
= 3.14 x 10-19 J so 1 Joule at 633 nm = 3.18x1018 photons
3rd Ed. Shapiro p 77
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Polarization and Phase: Interference
• Electric and magnetic fields are
vectors - i.e. they have both
magnitude and direction
• The inverse of the period
(wavelength) is the frequency in
Hz
3rd Ed. Shapiro p 78
© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
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Interference
0o 90o 180o 270o 360o
Wavelength
Amplitude
A+B
A
Constructive
Interference
B
C+D
C
D
The frequency does
not change, but the
amplitude is doubled
Here we have a phase difference of
180o (2 radians) so the waves
cancel each other out
Destructive
Interference
Figure modified from Shapiro “Practical Flow
Cytometry” Wiley-Liss, p79
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Light Scatter
• Materials scatter light at wavelengths at which they do not absorb
• If we consider the visible spectrum to be 350-850 nm then small
particles (< 1/10 ) scatter rather than absorb light
• For small particles (molecular up to sub micron) the Rayleigh
scatter intensity at 0o and 180o are about the same
• For larger particles (i.e. size from 1/4 to tens of wavelengths) larger
amounts of scatter occur in the forward not the side scatter
direction - this is called Mie Scatter (after Gustav Mie) - this is how
we come up with forward scatter be related to size
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Shapiro
p 79
© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Rayleigh Scatter
• Molecules and very small particles do not
absorb, but scatter light in the visible
region (same freq as excitation)
• Rayleigh scattering is directly proportional
to the electric dipole and inversely
proportional to the 4th power of the
wavelength of the incident light
the sky looks blue because the gas molecules scatter more
light at shorter (blue) rather than longer wavelengths (red)
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Reflection and Refraction
• Snell’s Law: The angle of
reflection (Ør) is equal to the
Transmitted
angle of incidence (Øi)
(refracted)Beam
regardless of the surface
material
t
• The angle of the transmitted
beam (Øt) is dependent upon
the composition of the
material
Incident Beam
i
r
Reflected Beam
n1 sin Øi = n2 sin Øt
The velocity of light in a material
of refractive index n is c/n
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Shapiro
p 81
© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Refraction & Dispersion
rac
Short wavelengths are “bent”
more than long wavelengths
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Brewster’s Angle
• Brewster’s angle is the angle at which the reflected light is
linearly polarized normal to the plane incidence
• At the end of the plasma tube, light can leave through a
particular angle (Brewster’s angle) and essentially be highly
polarized
• Maximum polarization occurs when the angle between reflected
and transmitted light is 90o
thus Ør + Øt = 90o
since sin (90-x) = cos x
Snell’s provides (sin Øi / cos Øi ) = n2/n1
Ør is Brewster’s angle
Ør = tan -1 (n2/n1)
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Shapiro
p 82
© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Brewster’s Angle
Photo ©2000 – J.P. Robinson
Photo ©2000 – J.P. Robinson
Photo ©2000 – J.P. Robinson
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt
Lecture Summary
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History of Flow
Principles of light and matter
Basic Optics
Essentials of lasers
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© 2002 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture002.ppt