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F(L)CS Data Acquisition
18.3 Setup FCS Step – Optimizing FCS Measurement Conditions
1. Open the
Setup FCS
step in the FCS Wizard.
2. Now set the instrument parameters for an FCS measurement.
18.3.1 Selecting APDs
The APDs are external detectors. For FCS, they do not have to be activated specially in the
software.
1. Switch the APDs on electrically.
2. If you do not receive a signal after starting a test measurement, check that the external
port (
X1 Port
) is set to
---
.
Activation/deactivation of individual detectors in
Beam Path Settings
and settings for the
spectral sliders for the photomultipliers are not relevant for the FCS measurement.
18.3.2 Selecting Laser Lines for FCS
Typically, FCS measurements are carried out using continuous wave VIS laser lines. UV
laser lines are not specified and are also not recommended due to the reduced quantum
efficiency of the APDs in the blue spectrum.
Furthermore, MP excitation is not specified for FCS. Pulsed visible lasers (diode lasers,
white light lasers) can be used for time-resolved FCS measurements, like FLCS or gated FCS.
18.3.2.1 Using Continuous Wave VIS Lasers
1. Choose a suitable laser line and laser intensity by setting the AOTF slider.
2. The acousto-optical beam splitter must be set to fluorescence.
3. In front of the argon laser, there is an FCS filter wheel for additional clean up of selected
laser lines and for further attenuation. When selecting a laser line by the AOTF setting,
the FCS filter wheel is automatically moved to the right position. You can check the
position in the pull-down menu from
Filter Wheel
in
Beam Path Settings
(
Figure 172
).
Other laser lines are not affected by the FCS filter wheel.
Do not change Acquisition mode any more
Do not make any more changes to the
Acquisition mode
(xyz or xz) used
in the last image. The positioning of the FCS point must be set in the same
mode as the most recently acquired image.