Zeiss LSM 880 Operating Manual page 676

a)
b)
c)
26
The pictures on the left demonstrate the influence of pixel
time and averaging on SNR; object details can be made
out much better if the pixel time increases or averaging is
employed.
Another sizeable factor influencing the SNR of an image
is the efficiency of the detection beam path. This can be
directly influenced by the user through the selection of ap-
propriate filters and dichroic beamsplitters. The SNR of a
FITC fluorescence image, for example, can be improved
by a factor of about 2 (6dB) if the element separating the
excitation and emission beam paths is not a neutral 80/20
beamsplitter but a dichroic beamsplitter optimized for the
1
particular fluorescence.
The difficult problem of quantifying the interaction between
resolution and noise in a confocal LSM is solved by way of
the concept of resolution proba bility; i.e. the unrestricted
validity of the findings described in Part 1 is always de-
pendent on a sufficient number of photons reaching the
detector.
Therefore, most applications of confocal fluorescence mi-
croscopy tend to demand pinhole diameters greater than
0.25 AU; a diameter of 1 AU is a typical setting.
Fig. 20 Three confocal images of the same fluorescence
specimen (mouse kidney section, glomeruli labeled with
Alexa488 in green and actin labelled with Alexa 564
phalloidin in red).
All images were recorded with the same parameters,
except pixel time and average. The respective pixel times
were 0.8 µs in a), 6.4 µs (no averaging) in b),
and 6.4 µs plus 4 times line-wise averaging in c).
1 An 80/20 beamsplitter reflects 20% of the laser light
onto the specimen and transmits 80% of the emitted
fluorescence to the detector.