Zeiss LSM 880, Operating Manual

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PossibilitiestoimproveSNR
Pinhole diameters providing a resolution probability below
90% may still yield useful images if one uses a longer pixel
time or employs the signal averaging function. In the for-
mer case, additional photons are collected at each pixel;
in the latter case, each line of the image, or the image as
a whole, is scanned repeatedly, with the intensities being
accumulated or averaged. The influence of shot noise on
image quality decreases as the number of photons detected
increases. As fluorescence images in a confocal LSM tend
to be shot-noise-limited, the increase in image quality by
the methods described is obvious.
Furthermore, detector noise, same as laser noise at high
signal levels, is reduced. The figures on the right show the
influence of pixel time (figure 18) and the influence of the
number of signal acquisitions (figure 19) on SNR in [dB].
The linearity apparent in the semilogarithmic plot applies
to shot-noise-limited signals only. (As a rule, signals are
shot-noise-limited if the PMT high vol tage needed for signal
amplification is greater than 500 V).
A doubling of pixel time, same as a doubling of the number
of signal acquisitions, improves SNR by a factor of about
 2 (3 dB). The advantage of the averaging method is the
lower laser load on the specimen, as the exposure time per
25
pixel remains constant. Photon statistics are improved by
the addition of photons from several scanning runs (SNR
=

n·N

; N = constant, n = number of scans averaged). By
comparison, a longer pixel time directly improves the pho-
ton statistics by a greater number N of photons detected
per pixel (SNR =
 N , N = variable), but there is a greater
probability of photobleaching or saturation effects of the
fluorophores (see also details “Fluorescence”).
Figures 18 and 19 Improvement of the signal-
to-noise ratio. In figure 18 (top), pixel time is
varied, while the number of signal acquisitions
(scans averaged) is constant.
In figure 19 (bottom), pixel time is constant,
while the number of signal acquisitions is varied.
The ordinate indicates SNR in [dB], the abscissa
the free parameter (pixel time, scans averaged).
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1 10
Variation of pixel time
Pixel time [ s]
SNR
[dB]
2 3 4
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Variation of averages
Number of averages
SNR
[dB]
2 3 4
Variation of pixel time
Pixel time [µs]
Fig. 18
SNR
[dB]
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1 10
Variation of pixel time
Pixel time
[ s]
SNR
[dB]
2 3 4
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1 10
Variation of averages
Number of averages
SNR
[dB]
2 3 4
Variation of averages
Number of averages
Fig. 19
SNR
[dB]
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PART 2
SignalProcessing