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=46urther on the pulse width forward light scatter question.
You may eliminate a possible artifact in the fls versus tof relation by
switching from pulse height analysis to pulse integral analysis of your
scatter signals (if you did not already). Signal saturation starts already
with particles having a stretched length comparable to the height of your
laser focus if you are in pulse height mode.
Below is a quick distillation from a -home- manual of phytoplankton flow
cytometry I made some years ago. It's descriptive and you come across algae
and stuff but it may give you some clous. Email me at dubelaar@pi.net if
you want the complete text (figures and refs etc).
Best wishes,
George Dubelaar
DRIE
Zeelt 2, 2411 DE Bodegraven NL
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BASIC LIGHT SCATTERING MECHANISMS
Properties governing light scatter are particle size, form, inner
structure, absorption and refractive index, the wavelength of the incident
light and the refractive index of the surrounding medium. This brief
overview of various light scattering mechanisms starts with the smallest
particles: the electrons of an atom in an electromagnetic field experience
a shift in distribution around the nucleus. The oscillating electric field
of the incident light causes similar oscillations in these distribution
shifts (or dipoles), which generate radiation. The light is scattered more
or less uniformly in all directions (Rayleigh scattering). If a group of
atoms is excited, the scattered light of those atoms interferes, stronger
if the atoms are regularly positioned. In the case of silicium atoms in a
regular array, the interference is total in all directions but the forward
(Huygens principle). With increasing size, from molecules to small
particles like bacteria, the interference phenomena cause the scattering
pattern to change from the uniform Rayleigh distribution to a more
forwardly directed pattern (Rayleigh-Gans). With transparent, bigger
objects like phytoplankton cells, the scattered light can be regarded as
originating from two separate phenomena: diffraction and refraction. Light
entering the particle and, diminished to some extent by absorption, leaving
the particle again in various directions, is refracted light. Diffraction
is the change in the direction of light propagating along the outside
=8Ccontours=B9 of the particle. Diffraction causes an interference pattern o=
f
concentric light and dark rings in the field far behind the particle. This
scattering is very much concentrated in the forward direction (Fraunhofer
diffraction). Macroscopic particles generally do not transmit refracted
light, owing to complete absorption. With biological particles such as
phytoplankton cells, a significant part of the light incident on a cell is
transmitted (refracted), more with low absorption; less with high
absorption. On the other hand, the diffraction pattern of these microscopic
particles gets less intense, and much wider distributed, as compared with
larger objects. The diffracted light and the refracted light have
comparable intensity, and start also to interfere with each other
(anomalous diffraction), causing weird oscillations in the scattering
pattern with changes in size, absorption and refractive index. When sizes
further decrease, the two phenomena merge (back to Rayleigh-Gans
scattering). Many phytoplankton particles fall in the anomalous diffraction
domain.
=46LS VERSUS PLS AND BIG VERSUS SMALL
It has long been shown that forward light scatter (FLS) or small angle
scatter represents mainly particle size, whereas perpendicular light
scatter (PLS) or wide angle scatter is more sensitive to small structures.
The highest scatter intensities are at small scattering angles. Significant
differences exist however between small particles as for instance bacteria
and larger particles as for instance ciliates. The intensity of the light
scattered by the bacteria drops 3 orders of magnitude with increasing
angle, whereas the light scattered by the ciliates drops 6 (!) orders of
magnitude. The total amount of scattered light increases strongly with
size. For species, representative for phytoplankton, a typical forward
scatter detector lense collects well over 90% of the total scattered light.
=46or a 0.1=B5m particle this is less than 10%, owing to the angular scatter=
ing
distribution! The laser beam stop blocks a lot of forward scattered light;
this effect gets serious with the very large particles. The PLS lense may
collect only about 0.01% of the scattered light of an algal cell, but up to
3% of the scattered light of a 0.1 micron particle! Suppose for instance
a typical phytoplankton cell having a number of small, submicron
structures. Even if the micro-structures contribute less than 1% to the
total collective scattering (all directions integrated) of the cell, they
still make up 75% of the PLS signal. On the other hand, the
micro-structures may have a total scattering comparable to the cell without
contributing to the FLS more than about 5%.
ANOMALOUS DIFFRACTION AND ABSORPTION (viz. also Cytometry 8, 405-412)
Intracellular microstructures may have another effect on the forward light
scatter signal, caused by their effect on the global refractive index of
the cell. When light traverses a particle, its speed (phase velocity) is
decreased proportionally to the density of the particle, and its intensity
(amplitude) is decreased progressively by absorption. Internal structures
are local fluctuations in density (refractive index) and cause changes in
light speed. Gas vacuoles speed up the light (relatively) whereas high
density granules slow down the light. Through the mechanism of interference
with the diffracted light this turns out that the gas vacuoles make the
cell seem smaller and the granules make the cell seem bigger through the
eyes of the FLS detector. These effects may be much larger than the actual
contribution of the substructures to the cells light scattering. There is
no linear relation in this, but weird maxima and minima.
Absorption reduces total light scatter (and eliminates most of the
interference maxima and minima). The maximum theoretical reduction is about
70%; for phytoplankton about 50%, for the phytoplankton cellular size
range. The extent of this reduction may vary, depending on particle size,
more than a factor 3 for realistic phytoplankton absorption values. In this
respect it should be noticed that the real part and the imaginary part of
the refractive index are not independent. Anomalous changes in the
refractive index occur in the vicinity of an absorption band. This effect
has been called selective scattering.
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