Olympus Spinning Disk - 3: Overviews + multi-positions + xyzt images

When acquiring high resolution images, it can make sense to acquire an overview image of your sample at a lower magnification. This allows you to identify and navigate between your positions/regions of interest.

Choose the 4x objective (or another low magnification objective).

Choose the imaging setting you want to use for the overview (e.g BF, 640) and adjust it accordingly (laser intensity, exposure time) in the Live mode.

You can define the overview area on the Stage Navigator window (bottom left).

Click "Define Overview Area".

Move to the top left and bottom right as indicated in the pop up messages.

Click here to initiate the overview.

It is also possible to acquire a multichannel overview using the dropdown menu.

It won't be possible to add positions outside the overview.

You have the option tosave your overview here.

Here you can load the "Default Overview Area" or other previously saved overviews.

Select your objective of choice and define your imaging settings.

If you need further information on how to set up your imaging please refer to the appropriate guides:

Spinning disk mode: Multichannel + Z-stack acquisition

Widefield mode

Add the hardware autofocus through a Z-Drift compensation command before your acquisition sequence.

Add a Stage loop to define the positions you would like to image.

While using RTC Fast for z-stacks please make sure it is in the appropriate position (middle=150um) before starting to define positions.

Additionally you can also add a time lapse command if you wish to loop through all positions.

Here you can define the time interval

Or choose the minimal possible time interval by checking the "As fast as possible" box.

Activate ZDC offset measurement when adding a new position.

Go to your first position.

You can either add the current stage position here,

or add a select position directly in the overview.

To create a stitched image of a larger area than your field of view you can acquire and generate a MIA (Multiple Image Alignment).

After adding a new position, focus your specimen.

In a MIA the central position will be used to assess the ZDC offset.

Click OK once you are satisfied with your focus.

You will hear a beep if the offset is successfully determined. If you hear 2 beeps, the measurement failed. Check for any air bubbles in immersion oil and repeat the measurement.

You can check the coordinates of your positions in the Position List.

Here you can also redefine xy or z coordinates as well as re define the ZDC offset.

"Use current Z-position"

When selected: all Z-positions that you have set during the definition of the XY-positions and MIA scan areas are ignored. The images are acquired at the current microscope Z-position.

Recommended when using stage loop including ZDC. Make sure you are at the start position and in focus when starting the experiment.

When cleared: your software saves the current Z-position at each defined XY-position and at each defined MIA scan area. The saved Z-positions are used when acquiring the image.

Once you've added all your positions you can group them (e.g. according to the imaging protocol you'd like to run on each group).

Use the arrow tool to select your positions (for multiple positions keep the shift key pressed while selecting the different positions)

Selected positions will be formatted in red.

Hover over the selected position and right click. Select Group positions.

Name your new group.

Click on the stage loop box.

You can now select (bottom right) if you want to image all your defined positions or just a restricted group .

Under the Experiment Manager window you can redefine how you'd like to name your images.

Open Acquisition settings.

Chose "Document naming".

Choose Process/Experiment.

Here you can see how the naming is currently defined.

Here you can redefine the document naming.

Here you can check what is the predicted total duration of your experiment.

You can now start your experiment!