Basic procedure for setting up a
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timelapse experiment using GFP3
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Make sure the Oko-lab environmental chamber is set to the correct conditions for your experiment. In no particular order turn on the computer and the red button on the expansion strip labelled 'GFP3 Devices' behind the microscope. The red button turns on the following devices: microscope stand, motorised stage, light sources and camera. Login to the User account; there’s no password. Double-click the NIS-Elements icon on the desktop and enter your user credentials.
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Click the Z tab to set the range and step size for Z series. Click on the ‘Defined by top bottom’ button (see red arrow below) to set a top and a bottom for the Z series. Set the Step size. Clicking the button next to the Step field will set the step to the optimum for the objective lens. For multi-position acquisitions the Z series must be Relative to compensate for the different Z positions of the marked points. Click the ‘Symmetric mode defined by range’ button next to 'Defined by top bottom' and click Relative to make a symmetric z series around every point whose range is defined by the top and bottom you set above. The third button for ‘Asymmetric mode defined by range’ is more difficult to set up since you need to be able to type in the range above and below the Relative home position. Use the Order of Experiment button to define the order of acquisition for channels and Z slices.
Setting up the length of the timelapse and frequency of acquisition
Click the Timing… button to check how long a single loop will take; this will determine the minimum Interval between time points. Click the Time tab and select at least one tick box at the start of a row. Specify the desired Interval and a Duration or number of Loops. Ticking a box in the row below will create another time Phase, so for instance you can have a fast acquisition for a short period followed by a slow acquisition over a longer period. Click the ‘Close Active Shutter when Idle’ box to reduce photobleaching.
Click ‘1 1 time loop’ to loop to test the settings or ‘Run Now’ Run Now to begin the timelapse.