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 Dittrich & Gohde  1969 
Impulscytophotometer
(ICP) used ethidium
bromide for a DNA stain and a high NA objective used as a condenser and
collection lens

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 Coulter Electronics manufactured the TPS1 (Two parameter sorter) in
1975 which could measure forward scatter and fluorescence using a 35mW
argon laser.

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 197778 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.

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 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.
 1979  NIH scientists had added a
krypton laser at 568 nm to excite Texas Red fluorescence at 568 nm and
emit at 590630 nm. Argon (488 nm FITC was measured simultaneously
without signal crosstalk  thus the FACS IV was developed (BD).

12

 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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 Energy
 joules, radiant flux (energy/unit time)
 watts (1 watt=1 joule/second)
 Angles
 steradians  sphere radius r 
circumference is 2pr^{2};
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 4pr^{2}. One steradian is defined as the
solid angle which intercepts as area equal; to r^{2} on the
sphere surface

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 Side scatter, forward angle scatter, cell volume, coulter volume:
 Understand light scattering concepts; intrinsic and extrinsic parameters
 Photometry:
 Light  what is it  wavelengths we can see 400750 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 pr^{2}; one
steradian is defined as that solid angle which intercepts an area equal
to r^{2} on the surface.
 Mole  contains Avogadro's number of molecules (6.02 x 10^{23})
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 10^{8} m/s).
The energy (joules) of a photon is
 E=hn and E=hn/l [nfrequency, lwavelength, hPlanck's constant 6.63 x
10^{34} jouleseconds]
 Energy  higher at short wavelengths  lower at longer wavelengths.

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 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

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 One photon from a 488 nm argon laser has an energy of
 E= 6.63x10^{34}
jouleseconds x 3x10^{8}
 To get 1 joule out of a 488 nm laser you need 2.45 x 10^{18}
photons
 1 watt (W) = 1 joule/second a 10 mW laser at 488 nm is putting out
2.45x10^{16} photons/sec

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 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

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 Materials scatter light at wavelengths at which they do not absorb
 If we consider the visible spectrum to be 350850 nm then small
particles (< 1/10 l) scatter rather than absorb light
 For small particles (molecular up to sub micron) the Rayleigh scatter
intensity at 0^{o} and 180^{o} 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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 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

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 Snell’s Law: The angle of reflection (Ø_{r}) is equal to the
angle of incidence (Ø_{i}) regardless of the surface material
 The angle of the transmitted beam (Ø_{t}) is dependent upon the composition
of the material

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 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 90^{o}
 thus Ø_{r} + Ø_{t}
= 90^{o}
 since sin (90x) = cos x
 Snell’s provides (sin Ø_{i}
/ cos Ø_{i } )
= n_{2}/n_{1}
 Ø_{r} is
Brewster’s angle

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 History of Flow
 Principles of light and matter
 Basic Optics
 Essentials of lasers
