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eliminate the majority of background signals. Laser power and pinhole width can then be used
to refine the signal morphology to ensure the error value calculation is optimised.
7.3 Enhanced focus control via Focus Flag
7.3.1 Focus Stability
Focus stability can be enhanced using the Focus Flag parameters. This can allow higher P-gain and
I-gain settings to be used with higher magnification objectives which may otherwise cause visible
oscillations when acquiring images. It can also increase focus stability for samples which exhibit poor
low signal to noise.
If, after having following the instructions in Section 6 and checking the section 10: Troubleshooting,
the focus is still unstable, the interrupt function can be used to turn off the autofocus servo (without
switching the controller into MAN mode) when the focus flag is active. By default, the focus flag is true
when the error value is <0.05.
Bring the sample into focus, ideally by switching the controller into SRV mode.
Open the Flags submenu in the main PureFocus850 GUI.
Click ‘On’ in the focus flag section of the Flags submenu.
If focus stability is still insufficient after switching on the interrupt function, the error parameter
can be increased to inactivate the servo at a higher error value.
Switch the controller into MAN FOC mode.
Slowly adjust the focus on the sample using the controller digipot. Check the error value
displayed on the main PureFocus850 GUI. Determine the maximum error value which can be
allowed whilst also maintaining focus on the sample. Increase the value into the error
parameter field in the Focus Flag submenu to this value. This will depend on the depth of focus
of the objective.
7.3.2 Low depth of focus objectives
For low depth of focus objectives, an error parameter value of 0.05 may be insufficient to keep the
sample in focus with the interrupt function switched on. Signal complexity can also lead to focus
oscillation between two adjacent focal planes. In these cases, the focal plane must be specified
stringently.
Bring the sample into focus, ideally by switching the controller into SRV mode.
Open the Flags submenu in the main PureFocus850 GUI.
Turn on the interrupt function by clicking ‘On’ in the focus flag section of the Flags submenu.
Switch the controller into MAN FOC mode.
