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209
Summarized manual for FCS or other point measurements
19 Summarized manual for FCS or other point
measurements
19.1 Prerequisites
The following description assumes that the following prerequisites are met:
• SymPhoTime is started and a workspace is loaded.
• LAS AF was started.
• The right objective has been chosen and the correction ring adjusted to the specimen
used ( Chapter 18.1.1 and Chapter 18.1.4 ).
• Your specimen is already placed onto the microscope and brought into focus. If you are
measuring in solution, it is assumed that the laser is focused into the solution (see
Chapter 18.1.5
).
• The selected excitation lasers are switched on and activated ( Chapter 18.2.2 and
Chapter 18.3.2
).
• The correct emission filter(s) has/have been placed in your detector unit ( Chapter 23 ).
• For beam splitter systems: The correct beam splitter for your wavelength has been
selected in LAS AF.
• With AOBS systems: The acousto-optical beam splitter is in fluorescence mode.
19.1.1 Choosing the Location for the FCS Measurement
1. Start the FCS Wizard in LAS AF.
2. Scan an image. Also use the Setup Imaging step in the FCS Wizard.
3. Select the position at which you want to run a point measurement (
Chapter 18.2
).
4. Start the FCS test mode in LAS AF by selecting the image acquisition button Run FCS Test
under the
Setup FCS
step in the FCS Wizard (
Chapter 18.3
).
Normally, the SymPhoTime software will start the data acquisition automatically.
However, you can also start the FCS data acquisition by selecting the
Acquire Point
Measurements button in the SymPhoTime software.
The Measurement Preview dialog in SymPhoTime opens on the FCS tab ( Figure 182 , left).
5. The display of the cross-trace and the two autocorrelation traces can be selected using
the check boxes above the trace.
6. The update time can be set in the
Refresh
field. Together with the average count rate,
the G(0) value and the molecular brightness ( Counts/Molec. ) are shown for the individual
channels.
The molecular brightness is calculated by G(0) times the average count rate and displays
the mean fluorescence count rate per dye molecule. The system is optimized (for example
by adjusting the laser intensity and the objective collar) when maximum molecular
brightness is attained.
In the Time Trace tab ( Figure 182 , right) the changes of the count rate over time can be
observed. The binning ( resolution ) should be set to 1 ms.