Basic procedure for setting up a timelapse experiment using GFP3

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.

View phase contrast 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 orange tape line up (see red arrow below). 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 phase ring into position. Check the list of objective lenses to see which phase ring to use.

GFP3 front panel, Ti-RCP, pE100 and condenser

Aligning the microscope for Köhler illumination

Fully close the field diaphragm and adjust the condenser height so the image of the iris is in sharp focus. Centre the image of the iris using the two centring screws. Open the field diaphragm until it is just outside the field of view. Remove an eyepiece and look into the empty opening. Adjust the iris aperture until it is about 70% open and then replace the eyepiece. The microscope is now adjusted for Köhler illumination.

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 pE300 and adjust the brightness accordingly.

pE300 LED light source

The excitation wavelength is determined by the microscope filter set used but it is also possible to change the balance of the UV, blue and green/yellow/red brightness of the pE300 using buttons on the keypad and in the software. If you change the balance make sure that you use the same LED settings for any specimens that you want to compare.

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 GFP3 except the 4x are compatible with the PFS, although the 60x oil lens must be used with a 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.

Perfect Focus System controls

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.

Imaging fluorescence

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 (red arrow B below) to get a live camera image. 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.

GFP3 NIS Elements controls

Imaging phase contrast

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 (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.

ND Acquisition window with lambda tab

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 (see red arrows below). 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.

XY tab in ND Acquisition

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 (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.

Z tab in ND Acquisition

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.

Time tab in ND Acquisition

Click 1 time loop to test the settings or Run Now to begin the timelapse.

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