Leica TCS SP8 SMD, User Manual

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F(L)CS Data Acquisition
Repeat the correction ring adjustment whenever you insert a new FCS specimen.
18.1.5 Setting the Reference Position
When FCS measurements are taken in solutions, the measuring point must always be
defined with the aid of a reference point. The reflection that is generated at the transition
between cover slip and specimen serves as reference.
1. To do this, proceed as described in
Chapter 18.1.4
. Make sure that you obtain two
reflections. On the inverted microscope, the upper reflection appears at the transition
between the immersion and the cover slip; the lower reflection appears at the transition
between the cover slip and specimen (
Figure 161 ).
2. On the inverted microscope, place the reflection from the cover slip/specimen-transition
approximately in the middle of the image.
3. Set the z-position to 0 on the stand.
Observe the user manuals for external lasers
Please refer to the information from the documents provided by the laser
manufacturer for the external lasers. Pay particular attention to the laser
manufacturer's notes!
4. Move the focus on the stand about 10-20 μ m towards the specimen (positive numbers on
the inverted stand). The reflection on the inverted stand should move towards the top
edge of the image (
Figure 164
).
Figure 164: Selection of the FCS measurement position for measurements in solutions: The reflection
between the cover slip and the solution must be above the middle of the image.
18.1.6 Acquiring a Reference Image
For measurements in cells or other structures, you will need an image of your specimen. You
can acquire this image in any step of the FCS Wizard. However, you can change the settings
for image acquisition only in the
Setup Imaging step.