5
collected by suitable optics and directed onto sensitive detectors which transform the light
pulses into equivalent electrical pulses. The scattered light, which provides information on cell
size and structure, is measured at various scattering angles by separate detectors. The
electrical pulses from the detectors are measured and digitized by appropriate electronics and
the information stored in a computer, which typically presents it in the form of histograms
showing the number of cells as a function of cellular fluorescence andlor the light scattering.
Flow cytometers have a remarkable performance. The measuring precision per cell can be
about 1 %, the detection limit around 1000 molecules per cell, and the measuring rate up to
10000 cells S-I. However, the performance depends on the instrument characteristics as well
as the application. The following discussion will show how the performance depends on some
of the basic instrument characteristics.
Sensitivity and resolution: The sensitivity of the flow cytometer is determined primarily by
two factors: the magnitude of the signal produced by a given cell or particle and the
background noise on which this signal is to be measured. Thus, the sensitivity is essentially
a measure of the signal to noise ratio, SIN, under given conditions; in order to increase
sensitivity, it is as important to reduce the noise as to increase the signal. As expressed by
Eq.1, the number of fluorescence photons emitted by a cell, N f , is proportional to the intensity
of the excitation light, I, as well as to the time spent by the cell in the excitation focus, that
is inversely proportional to the flow velocity. This holds true only to the extent that there is
no photodegradation, i.e. bleaching, of the fluorophore during the exposure. Furthermore, N r
is proportional to the overlap between the spectrum of the excitation light, P(A), and the
absorption spectrum of the fluorophore, E(A). Hence, fluorescence sensitivity may vary
significantly between applications which employ different dyes.
N f = const l 'I V-I P(A) E(A)d
(1)
The intensity of the scattered light, N I , is also proportional to the excitation intensity and
inversely proportional to the flow velocity. In a first approximation, the light scattering of
biological cells is independent of their light absorption. In contrast to the fluorescence, which
is largely isotropic (although this depends on the degree of polarization of the excitation light
collected by suitable optics and directed onto sensitive detectors which transform the light
pulses into equivalent electrical pulses. The scattered light, which provides information on cell
size and structure, is measured at various scattering angles by separate detectors. The
electrical pulses from the detectors are measured and digitized by appropriate electronics and
the information stored in a computer, which typically presents it in the form of histograms
showing the number of cells as a function of cellular fluorescence andlor the light scattering.
Flow cytometers have a remarkable performance. The measuring precision per cell can be
about 1 %, the detection limit around 1000 molecules per cell, and the measuring rate up to
10000 cells S-I. However, the performance depends on the instrument characteristics as well
as the application. The following discussion will show how the performance depends on some
of the basic instrument characteristics.
Sensitivity and resolution: The sensitivity of the flow cytometer is determined primarily by
two factors: the magnitude of the signal produced by a given cell or particle and the
background noise on which this signal is to be measured. Thus, the sensitivity is essentially
a measure of the signal to noise ratio, SIN, under given conditions; in order to increase
sensitivity, it is as important to reduce the noise as to increase the signal. As expressed by
Eq.1, the number of fluorescence photons emitted by a cell, N f , is proportional to the intensity
of the excitation light, I, as well as to the time spent by the cell in the excitation focus, that
is inversely proportional to the flow velocity. This holds true only to the extent that there is
no photodegradation, i.e. bleaching, of the fluorophore during the exposure. Furthermore, N r
is proportional to the overlap between the spectrum of the excitation light, P(A), and the
absorption spectrum of the fluorophore, E(A). Hence, fluorescence sensitivity may vary
significantly between applications which employ different dyes.
N f = const l 'I V-I P(A) E(A)d
(1)
The intensity of the scattered light, N I , is also proportional to the excitation intensity and
inversely proportional to the flow velocity. In a first approximation, the light scattering of
biological cells is independent of their light absorption. In contrast to the fluorescence, which
is largely isotropic (although this depends on the degree of polarization of the excitation light