6
as well as that of the fluorescence), the light scattering is strongly dependent on scattering
angle (See Fig.6). N. is a complex function of the size and shape of the cell, its composition,
and the refractive index of its various constituents, as described by the theory of light
scattering. It also depends on the excitation wavelength.
Each cell emits typically many thousands of photons of fluorescence and scattered light as it
passes through the excitation focus. Only a relatively small fraction of these photons fall
within the aperture of the fluorescence collecting optics. The number of fluorescence photons,
nf' that reach the light detector depends on the numerical aperture, NA, of this optics and the
optical transmission, T, of the lenses and filters in the optical path (Eq. 2):
nf = const2 NA T Nf
(2)
The light detector, which is usually a photomultiplier tube (PMT), converts the light pulses,
each of which usually consists of many photons, into equivalent electrical pulses. The
sensitivity of the PMT depends primarily on the photoelectron quantum yield, ¢e, of its
photocathode, that is the probability that a photon shall cause emission of a photoelectron. ¢e,
which varies with wavelength, usually does not exceed 0.2. Thus the average number of
photoelectrons, ne, produced by nf photons is:
(3)
The electrons released from the photocathode are multiplied as they pass through the PMT,
the multiplying factor increasing with the high voltage across the tube, so that the gain of the
PMT may be varied typically from 10 2 to 10 7 depending on high voltage.
The noise of the signal is of two principally different kinds: optical noise and electronic noise.
At least in principle, the latter can be reduced to insignificant levels by the use of appropriate
electronics. In contrast, optical noise is inherent in all kinds of emission of light and cannot
be reduced below a certain level. This noise stems from the fact that the emission of light is
a stochastic process, which implies that photons are emitted randomly in time. The result is
as well as that of the fluorescence), the light scattering is strongly dependent on scattering
angle (See Fig.6). N. is a complex function of the size and shape of the cell, its composition,
and the refractive index of its various constituents, as described by the theory of light
scattering. It also depends on the excitation wavelength.
Each cell emits typically many thousands of photons of fluorescence and scattered light as it
passes through the excitation focus. Only a relatively small fraction of these photons fall
within the aperture of the fluorescence collecting optics. The number of fluorescence photons,
nf' that reach the light detector depends on the numerical aperture, NA, of this optics and the
optical transmission, T, of the lenses and filters in the optical path (Eq. 2):
nf = const2 NA T Nf
(2)
The light detector, which is usually a photomultiplier tube (PMT), converts the light pulses,
each of which usually consists of many photons, into equivalent electrical pulses. The
sensitivity of the PMT depends primarily on the photoelectron quantum yield, ¢e, of its
photocathode, that is the probability that a photon shall cause emission of a photoelectron. ¢e,
which varies with wavelength, usually does not exceed 0.2. Thus the average number of
photoelectrons, ne, produced by nf photons is:
(3)
The electrons released from the photocathode are multiplied as they pass through the PMT,
the multiplying factor increasing with the high voltage across the tube, so that the gain of the
PMT may be varied typically from 10 2 to 10 7 depending on high voltage.
The noise of the signal is of two principally different kinds: optical noise and electronic noise.
At least in principle, the latter can be reduced to insignificant levels by the use of appropriate
electronics. In contrast, optical noise is inherent in all kinds of emission of light and cannot
be reduced below a certain level. This noise stems from the fact that the emission of light is
a stochastic process, which implies that photons are emitted randomly in time. The result is