Olympus Spinning Disk - Widefield mode

To use the microscope on the Widefield mode different observation methods are available:

You can find them under My functions - "Widefield" for easier access during Live mode.

DAPI WF

GFP WF

CY3 WF

Cy5 WF

DAPI/Cy3 (using the dual camera mode)

Be aware that the Cy5 WF observation method might also show signals is the red range as a broad light source is used (no excitation filter is present).

Start a new Experiment on the "Experiment manager" tab.

For a 4 color sequential image you can use the template - Favorite Templates - "Widefield DAPI GFP Cy3 Cy5".

You can also design your experiment using the available widefield observation methods from the menu.

For multichannel images do not forget to add the respective option.

Double click on the observation method box to activate it.

Check the "Synchronize Shutter" box option if you would like to minimize exposure of your sample to light.

Press Live.

Adjust exposure time accordingly.

You can also change LED intensity under the "Microscope Control tab":

Scroll down until you find pE-300

the appropriate LED will be active and can be changed for each channel.

Make sure your image is not saturated with the help of the Adjust Display pannel.

Here you can toggle the camera bit-depth between 16 and 8-bit .

Using the camera at 16-bit (recommended), Max intensity should be less than 65 536. At 8-bit, the Max intensity should stay bellow 255.

For more details refer to the different camera modes here: Olympus Spinning Disk - 2: Multichannel + Z-stack acquisition.

Once you are satisfied with your settings right click on the observation method and select "Get settings".

Repeat the procedure for the remaining observation methods.

Name your experiment.

Press Start.

Visualize your image