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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Press the L100 button to redirect the light to the camera. Click the fluorescence optical configuration you need in the OC Panel. Click the live preview button in the toolbar toolbar (red arrow B below) to get a live camera image (red arrow B below). Click the pE-300 Pad tab and adjust the brightness of the illumination if necessary. A red exclamation mark appears in the OC Panel if there are any changes. Click the arrow next to the optical configuration to save the new brightness (red arrow A below). In DS-Qi2 Settings change the exposure, binning and gain as necessary. Changes to the exposure are automatically written to the optical configuration so you don’t need to click the arrow.
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In the ND Acquisition window tick the Save to File box and define a filename and path for the timelapse (see image below).
Setting up fluorescence and transmitted light channels
Click the lambda tab and select the channels you want to image by ticking the box at the start of each row and selecting from the drop-down list of optical configurations (red arrow below). Tick the box at the bottom of the window that says ‘Close active Shutter during Filter Change’ if you want to reduce photobleaching.
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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.