Leica TCS SP8 SMD, Руководство пользователя

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183
F(L)CS Data Acquisition
Setup
tab and enter
1
(
Figure 159, item 2
). This will automatically take a new image after
photobleaching all points.
11. Set the laser used for photobleaching to 100 %.
12. Under Measurement Duration , enter a suitable bleaching period; this will typically be a
few seconds (
Figure 159, item 3
).
13. Start the bleaching series from LAS AF by clicking the image acquisition button Run FCS .
14. In the image acquired after photobleaching, compare the markings with the actual
bleach points.
15. To archive your measurement, you can create a snapshot of the image with your bleach
points. To do so, right-click this image and select Snapshot .
If the actual bleach points are not exactly what you want, repeat the calibration
(
Chapter 18.1.2 ).
18.1.4 Adjusting the Correction Ring on the Objective
To guarantee the correct form of the detection volume, the correction ring at the objective
must be adjusted to the thickness of the specimen slide below the specimen. You must
always make this adjustment when you change the specimen carrier, even if it comes from
the same batch. You should also repeat this adjustment if the temperature changes. Follow
these steps:
1. Start LAS AF.
2. Use the objective 63x Plan APO 1.2W CORR CS 0.14 to 0.18 . Add immersion water to the
objective and position the FCS specimen.
3. When you open the Setup Corr-ring step in the FCS Wizard, a preconfigured setting of
the beam path is loaded. Adjust the settings as follows:
• Select scan mode xz-y (z-Galvo).
• Set the format to 512 x 512.
• Set the zoom to the smallest possible setting.
• Select the laser line at 488 nm.
• Use bidirectional scan.
• Set the AOBS to reflection.
• Enable a SP PMT (no HyD and no SP FLIM PMT).
• Adjust the detector range to detect the laser line as well.
• Select Glow (O&U) in the color look-up table (LUT).
4. Click the Live image acquisition button.
A live image of the reflection line is displayed, which you can now optimize.