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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 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 E100 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. 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.
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.
Rotate the correct phase ring into position. Check the list of objective lenses to see which phase ring 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 pE300 and 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 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.
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 to get a live camera image. Click the pE300 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. 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 phase contrast
Select the brightfield 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. Tick ‘Close active shutter during Z movement’ to reduce photobleaching.
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