20
The main types of noise important in a confocal
LSM system are detector noise (dark noise, sec-
ondary emission noise), laser noise, and shot noise
of the light (
see Details “Sources of Noise”
). As a
rule, these sources of noise are of a statistical
nature. Periodic noise rarely occurs, and if it does,
it tends to correlate with defective devices or
mechanical vibration in the setup; therefore it has
been left out of consideration here.
As the graphs in figure 15 show, the number of
photons hitting the PMT depends not only on the
intensity of the fluorescence signal (
see Details
“Fluorescence”
), but also on the diameter of the
pinhole. The graph shows the intensity distribu-
tion of a two-point object resulting behind the
pinhole, in normalized (left) and non-normalized
form (right). The pinhole diameter was varied
between 2 AU and 0.05 AU. At a diameter of 1 AU
the pinhole just equals the size of the Airy disk, so
that there is only a slight loss in intensity. The gain
in resolution, is minimum in this case.
With a pinhole diameter <1AU, resolution improves
(better point separation thanks to a deeper dip),
which is penalized by a drastic loss in energy.
Moreover, it should be considered that it depends
on the signal level wich noise source dominates.
With high-amplitude signals (number of detected
photons > 10,000), laser noise is the dominating
effect, whereas the quality of low signals (number
of detected photons < 1000) is limited by the shot
noise of the light.
Therefore, laser noise tends to be the decisive
noise factor in observations in the reflection mode,
while shot noise dominates in the fluorescence
mode. With recent PMT models (e.g., from Hama-
matsu), detector dark noise is extremely low, same
as secondary emission noise, and both can be neg-
lected in most practical applications (
see Details
“Sources of Noise”
).
Therefore, the explanations below are focused on
the influence of shot noise on lateral resolution.
Fig. 15 As shown in Part 1, small pinhole diameters lead to improved resolution
(smaller FWHM, deeper dip – see normalized graph on the left). The graph on the right
shows, however, that constricting the pinhole is connected with a drastic reduction in
signal level. The drop in intensity is significant from PH <1 AU.
Noise
0.2
0.4
0.6
0.8
1.0
0.2
0.4
0.6
0.8
1.0
0.5
1
1.5
2
0.5
1
1.5
2
[AU]
[AU]
d = 0.05 AU
d = 0.50 AU
d = 1.00 AU
d = 0.25 AU
d = 2.00 AU
d = 0.05 AU
d = 0.50 AU
d = 1.00 AU
d = 0.25 AU
d = 2.00 AU
Relative intensity
337_Zeiss_Grundlagen_e 25.09.2003 16:16 Uhr Seite 23
