21
10.2.
Z-attenuation compensation
As images are collected deeper in to the sample there can be significant loss of signal.
This can be caused but refractive index mismatch, light scattering and absorption.
This signal loss can be compensated for collecting the attenuated slices with higher gains
and/or with higher laser intensity.
Two reference points are set, one near the top where the signal is the brightest, and one
near the bottom where there is still significant signal but it has been attenuated.
The laser intensity and gains are set for each reference slice (the bottom reference slice
having higher gain/laser). As the software acquires the z-series, it gradually steps up the
gain and laser as it approaches the bottom reference slice.
1. In the
Scan Control: Z-settings
window activate the
Auto Z.Corr
.
2. While in
Fast XY
scan
mode, focus to near the
top, of the sample where it
is brightest. You should
have already set the detector
and laser settings for this
slice.
If not go the
Scan Control:
Channels
window and set
the detector gains and laser
intensity to get adequate
signal at this focus position.
3. In the
Scan Control: Z-settings
window click
Set A
.
4. While in
Fast XY
scan, manually focus to the near the bottom of the z-series which
still has significant signal.
5. Go to the
Scan Control:
Channels
window and set
the detector gains and laser
intensity to get adequate
signal at this focus position.
6. Go back to the
Scan Control: Z-settings
window and click the
Set B
button.
7. Now start the Z-series acquisition by clicking
Start
.
