Focus Stabilisation may be activated in the Protocol Manager Tab to enable the Focus Stabilisation function of the system.
The Focus Seek and Lock (FSL) module, located directly below the objective lens turret carousel, operates by directing near infrared (NIR) light of a light-emitting diode (LED) towards the sample where it is back-reflected from the sample. This back-reflected NIR light is monitored by the system detector, and tracks the lowest coverslip surface relative to the objective lens. The FSL module exploits this in two ways: The Find Coverslip feature and the Focus Stabilisation feature.
When the Find Coverslip command is activated, the FSL module determines the Z-motorposition that corresponds the bottom of the coverslip position as described above, and then the focus drive is offset upwards by a pre-determined offset corresponding to the thickness of the coverslip to bring the bottom of the sample into focus.
Note: More information on Find Coverslip can be seen here: Find Coverslip. More information on supported objectives can be found here:Supported Objectives.
When the Focus Stabilisation option is activated within an imaging protocol at the beginning of the protocol the FSL module will determine the Z-motor position which corresponds to the bottom of the coverslip and references it relative to the focus position set by the user. Thereafter, the Focus Stabilisation routine pauses the protocol for a short period (~8-15 seconds) and re-checks the position of the bottom of the coverslip at regular intervals; this shall occur, at most, once within each time interval specified on the Focus Stabilisation panel, and shall never interrupt a channel repeat, z-scan or acquisition of a montage.
The time interval can be specified by the user between 1 minute to 30 minutes.
If the coverslip position is unchanged, no action is taken and the protocol resumes. Conversely, if the coverslip position has altered due to Z-motor thermal drift, the objective lens focus is automatically adjusted by moving the Z-motor to match the coverslip-focus lock offset distance once again. Note: Focus Stabilization is only active during imaging protocols and applied to the current Z-position or the Z-positions associated with the Multi-Position Settings option of the protocol manager (if activated); The focus is not locked during a live preview of the specimen.
Inside the Focus Stabilisation panel the option “Always Execute at New Position“ will cause Focus Stabilisation to run when moving to a new position, or at the start of a montage acquisition.
This shall never interrupt the acquisition of a Montage, but shall override the Time Interval property if appropriate. Note: This option may significantly increase acquisition time.
A video guide can be seen here: Focus Seek and Lock
Re-Calibrating Focus Seek and Lock Module
On the rare occurrence that Find Coverslip was working, but now it keeps saying it is unable to perform, you will need to re-calibrate your focus seek and lock module. This can happen if the module has been recently replaced, your pc was replaced or your configuration files have been corrupted. This is not needed otherwise.
To begin, Navigate to the Preferences – And select Hardware tab
Turn Perform LED Light intensity calibration to ON
Ensure there is a slide in relative focus on the system and you are on an objective that can perform Find Coverslip (Any air would do but 40x is best if available). Before starting ensure coverslip is facing the objective.
Click on the Return to imaging button.
Create a Time Series protocol interval 45s and at least 2 repeats. Focus stabilisation needs to be selected.
Click on Acquire.
Once finished, Navigate to the Preferences – And select Hardware tab. Please check the Preference: Perform LED Light intensity calibration is now OFF.
If it is not, please repeat the protocol ensuring the 45s interval.
Once again, click on the Return to imaging button. Please move focus away from your coverslip and try to run Find coverslip.
If the issue persists, please reach out to your local Andor Support.