49
ALIGNMENT
First is that of alignment of the flow cytometer. This usually requires very stable particles that
may have properties very different from the biological particles which are ultimately to be
analyzed. Alignment is usually done using plastic beads of uniform size and very high levels
of fluorescence over a wide spectral range. The necessity for a brightly fluorescent particle
arises because, if the flow cytometer is seriously out of alignment, it is necessary to see the
weak signal from the bright particle in order to begin the process of alignment. As flow
cytometers have improved, this step of alignment has become less important. The stability of
the particles is important in order to detect changes from day to day that may reveal gross
instrument malfunction. However, since the particles may not resemble biological particles
and are usually very bright, they may not reveal subtle malfunctions. For example, if their
emission spectrum is not the same as that of the biological particles they may not reveal
defects in optical filters.
SENSITIVITY AND LINEARITY
Second is the determination of the sensitivity and linearity of the now optimally aligned flow
cytometer. This is best done with particles that fluoresce with a known intensity. U suall y
multiple particles of differing intensities are used. They must be stable, but again need not
bear any relation in terms of fluorochrome or emission spectrum with the biological samples
to be analyzed. Perhaps the greatest difficulty with the determination of sensitivity is the need
to determine the "noise" level which ultimately limits sensitivity. This is usually done with
a "non-fluorescent" particle which, when run through the instrument gives a signal
corresponding to "noise". The determination that a particle is non-fluorescent is not easy. In
addition, in the absence of fluorescence, there may nevertheless be a signal due to light scatter
from the particle which penetrates the barrier filter(s). Thus the determination of instrument
noise is complex and includes both true noise, i.e. noise in amplifiers and photomultiplier
tubes, as well as noise due to photon statistics, and aberrant or irrelevant signals that are
operationally included with the noise.
One widely used set of particles for determination of sensitivity and linearity are offered
commercially by the Flow Cytometry Standards Corporation in Research Triangle Park, North
Carolina U.S.A. They offer a set of five beads, including "non-fluorescent" beads and four
others of known fluorescence intensity using fluorescein as the fluorochrome. Beads
containing other fluorochromes may also be available from this source. These beads are run
on the flow cytometer and a regression of the known mean fluorescence for each bead versus
ALIGNMENT
First is that of alignment of the flow cytometer. This usually requires very stable particles that
may have properties very different from the biological particles which are ultimately to be
analyzed. Alignment is usually done using plastic beads of uniform size and very high levels
of fluorescence over a wide spectral range. The necessity for a brightly fluorescent particle
arises because, if the flow cytometer is seriously out of alignment, it is necessary to see the
weak signal from the bright particle in order to begin the process of alignment. As flow
cytometers have improved, this step of alignment has become less important. The stability of
the particles is important in order to detect changes from day to day that may reveal gross
instrument malfunction. However, since the particles may not resemble biological particles
and are usually very bright, they may not reveal subtle malfunctions. For example, if their
emission spectrum is not the same as that of the biological particles they may not reveal
defects in optical filters.
SENSITIVITY AND LINEARITY
Second is the determination of the sensitivity and linearity of the now optimally aligned flow
cytometer. This is best done with particles that fluoresce with a known intensity. U suall y
multiple particles of differing intensities are used. They must be stable, but again need not
bear any relation in terms of fluorochrome or emission spectrum with the biological samples
to be analyzed. Perhaps the greatest difficulty with the determination of sensitivity is the need
to determine the "noise" level which ultimately limits sensitivity. This is usually done with
a "non-fluorescent" particle which, when run through the instrument gives a signal
corresponding to "noise". The determination that a particle is non-fluorescent is not easy. In
addition, in the absence of fluorescence, there may nevertheless be a signal due to light scatter
from the particle which penetrates the barrier filter(s). Thus the determination of instrument
noise is complex and includes both true noise, i.e. noise in amplifiers and photomultiplier
tubes, as well as noise due to photon statistics, and aberrant or irrelevant signals that are
operationally included with the noise.
One widely used set of particles for determination of sensitivity and linearity are offered
commercially by the Flow Cytometry Standards Corporation in Research Triangle Park, North
Carolina U.S.A. They offer a set of five beads, including "non-fluorescent" beads and four
others of known fluorescence intensity using fluorescein as the fluorochrome. Beads
containing other fluorochromes may also be available from this source. These beads are run
on the flow cytometer and a regression of the known mean fluorescence for each bead versus