Basic procedure for setting up a timelapse experiment using GFP4

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 wall switch labeled "microscope devices".  This turns on the following devices: microscope stand, motorised stage, light sources and camera. Login using your agendo username and password. Double-click the NIS-Elements icon on the desktop and enter your user credentials.  When the driver selection window pops up, ensure the 'first driver' is set to 'Phasics', and the 'second driver' is set to 'ANDOR Neo/Zyla'.  'Enable Multi Camera' has to be ticked.  Then click on 'OK'.

View DIC down the eyepieces

Mount your specimen on the stage. Direct light to the microscope eyepieces by pressing the EYE button on the front operation panel. Press the FL1 button on the Ti-RCP remote control panel and select the empty filter position (-----). Turn on the pE100 and set the brightness to a suitable level. Coarsely adjust the focus of the condenser so the two pieces of purple tape line up. If you are using the CO2 chamber be careful not to break the lid with the condenser lens. Use a low power lens like the 10x to focus on your specimen and then adjust the condenser for Köhler illumination. Once the microscope is in Köhler rotate the correct DIC prism into position. Check the list of objective lenses to see which DIC prism to use.

View fluorescence down the eyepieces

Turn the pE100 off. Press the FL1 button on the remote control pad and select a filter position. Check the list of filters for the one you need. Turn on the pE-4000and adjust the brightness accordingly.

Using the Perfect Focus System

The Perfect Focus System (PFS) keeps the microscope in focus by constantly correcting the distance between the specimen and objective lens. All the objective lenses on GFP4 are compatible with the PFS, although the higher magnification immersion lenses must be used with a coverslip-thickness-glass-bottomed imaging vessel containing aqueous medium. Make sure the specimen is in focus. Check that the amber DM IN/OUT light on the PFS Offset Controller is on. Check that the ‘Focus’ LED on the front operation panel is on. Press the ON button next to the LED. It should emit two tones and go solid green. Readjust the focus if necessary using the wheel on the PFS Offset Controller.

If the ON button flashes green and emits several short tones the PFS is not working for some reason. Check that you aren’t using the 4x lens, that the specimen is in focus, the dichroic mirror is in position and the ‘Focus’ LED is on. If they are all correct then for some reason the specimen is not compatible.

Optical configurations

The bottom left corner of the Nikon Elements software is set up with different optical configurations. Select 'Zyla' to set up routine imaging.  Select 'Advanced' for more control over imaging parameters, and 'Sid4Bio' to image with the Phasics camera

Imaging fluorescence

Select the 'Zyla' tab in the bottom left of the screen. 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 to get a live camera image. Click the pE-4000 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. In 'Zyla 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.

Imaging DIC

Select the Transmitted Light optical configuration and start the live preview and adjust, exposure, gain and binning as for fluorescence. Set the pE100 brightness using the keypad.

Timelapse Imaging

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. Tick the box at the bottom of the window that says ‘Close active Shutter during Filter Change’ if you want to reduce photobleaching.

Setting multiple points

Click the XY tab and click the Add button to add points as you move the stage around. Make sure the ‘Close active Shutter during Stage Movement’ at the bottom is ticked to reduce photobleaching and that the ‘Include Z’ box is ticked so the Z position is included along with the XY position. If the PFS is switched on the offset will be saved in the PFS column along with the X, Y and Z value at each position. It may be necessary to adjust the PFS offset at different positions, especially when marking points that are very distant to one another. If you mark a position with the PFS off and the focus changes when you turn the PFS on then the offset is different at that position. You can adjust it using the wheel on the PFS Offset Controller and then update the value in the software by clicking the arrow that appears next to the PFS column. X, Y and Z positions can be updated in the same way. Finally, marked positions can be reordered using the up and down arrows in the toolbar and pressing the Optimize button will re-order the points so that the stage moves the shortest distance.

Setting up Z series

Click the Z tab to set the range and step size for Z series. Click on the ‘Defined by top bottom’ button 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 time loop to test the settings or Run Now to begin the timelapse.