10
Dichroic mirrors and band filters are employed to separate scattered light from the
fluorescence as well as to separate different spectral components of the fluorescence. The low
angle scattering is measured by a detector situated close to the forward direction of the laser
beam. Hence, such an instrument has three independent, perpendicular axis which must be
aligned so as to intersect in a common point to within a few micrometers. This makes the
laser-based instruments susceptible to mechanical shock. The alignment is often a tricky and
time consuming task, especially since none of the axis can be visualized directly with
appropriate magnification.
A main advantage of the laser-based instruments is the very high excitation intensities that can
be achieved, partly because of the very powerful lasers which are available, but also because
laser light can be focused into a very small and correspondingly intense focus. This facilitates
a small measuring volume and low background provided appropriate spatial filtering is
employed (see below). Some laser-based flow cytometers have two lasers which are focused
to separate foci so that cells can be excited sequentially by two different wavelengths. This
device makes it possible to measure two dyes independently in the same cell without
interference from spectral overlap and excitation energy transfer.
The near-parallel excitation light beam of laser-based flow cytometers facilitates light
scattering measurement at almost any scattering angle. Several workers have developed
instruments with light scattering detectors at several different scattering angles and have
demonstrated an improved ability to distinguish different types of cells (for review see
Salzman et al., 1990). However, commercial instruments only have detectors for low angle
and 90° scattering. The disadvantage of the near parallel excitation light of laser instruments
is that it produces cell orientation dependent artifacts in the measurement of non-spherical
cells (Pinkel et al., 1985).
Laser light is usually polarized, and the orthogonal configuration of laser-based instruments
facilitates polarization measurements (Arndt-Jovin et al., 1976; Lindmo and Steen, 1977)
which may be useful, for example, to study the binding of fluorochromes. Flow cytometric
measurement of the polarization of the light scattering of biological cells has interesting
possibilities which have not been exploited.
Dichroic mirrors and band filters are employed to separate scattered light from the
fluorescence as well as to separate different spectral components of the fluorescence. The low
angle scattering is measured by a detector situated close to the forward direction of the laser
beam. Hence, such an instrument has three independent, perpendicular axis which must be
aligned so as to intersect in a common point to within a few micrometers. This makes the
laser-based instruments susceptible to mechanical shock. The alignment is often a tricky and
time consuming task, especially since none of the axis can be visualized directly with
appropriate magnification.
A main advantage of the laser-based instruments is the very high excitation intensities that can
be achieved, partly because of the very powerful lasers which are available, but also because
laser light can be focused into a very small and correspondingly intense focus. This facilitates
a small measuring volume and low background provided appropriate spatial filtering is
employed (see below). Some laser-based flow cytometers have two lasers which are focused
to separate foci so that cells can be excited sequentially by two different wavelengths. This
device makes it possible to measure two dyes independently in the same cell without
interference from spectral overlap and excitation energy transfer.
The near-parallel excitation light beam of laser-based flow cytometers facilitates light
scattering measurement at almost any scattering angle. Several workers have developed
instruments with light scattering detectors at several different scattering angles and have
demonstrated an improved ability to distinguish different types of cells (for review see
Salzman et al., 1990). However, commercial instruments only have detectors for low angle
and 90° scattering. The disadvantage of the near parallel excitation light of laser instruments
is that it produces cell orientation dependent artifacts in the measurement of non-spherical
cells (Pinkel et al., 1985).
Laser light is usually polarized, and the orthogonal configuration of laser-based instruments
facilitates polarization measurements (Arndt-Jovin et al., 1976; Lindmo and Steen, 1977)
which may be useful, for example, to study the binding of fluorochromes. Flow cytometric
measurement of the polarization of the light scattering of biological cells has interesting
possibilities which have not been exploited.