Olympus ScanR HCS (Irchel) - Part2: Image Acquisition

Make sure the 4x objective is selected and in the lowest position. Otherwise:

Choose the 4x objective using the microscope touch panel.

Move the objective to the lowest position.

Start ScanR Acquisition software

Choose and place the appropriate holder.

Several holders are available in the different drawers.

Mount your sample.

In an inverted microscope the coverslip should face the objective.

Select "Edit" to define your plate/slide

On the next pop up window select "Edit plate types".

As a starting point you can use an existing template and adapt it according to your needs.

Chose a profile from the list that you would like to adapt, eg "z.mb_oneslide".

Create and rename your own profile by pressing "New".

Here you can define the Pattern / Spacing and Geometry of your Wells.

Press "Calibrate Z" to go to Live View.

Here you can change the "Defined Color Channels" (eg DAPI, etc).

Focus your sample using the microscope wheels and press "Set".

Press "Set" once you are satisfied.

Calibrate positions ("Plate type settings" window) using:

"3-point A1 measurement" - triangulation method

"A1 center position" - only necessary to define the center of the coverslip/well

Press calibrate A1

On the "Pattern Position measurement" window select which slide you want to measure.

Click "Measure" for Live view .

Move the stage so that the red cross is either at the center or at the edge of the slide, according to the chosen calibration strategy. Click "Add position".

Repeat for the 2 other borders of A1 (if 3-point A1 measurement was chosen).

Confirm with "OK" ("A1 measurement" window).

Once you are satisfied with your plate settings confirm with "OK".

Back in the "Edit Scan" window go to the "SW-Autofocus" tab.

Click "Test system settings".

If the focus is not ideal adjust the step size and/or range accordingly.

SW-Autofocus settings can be optimized with the help of the autofocus quality graph.

Confirm your settings by clicking on "OK".

Back in the "Edit Scan" window go to the "Acquisition" tab.

Here you can chose your channels for acquisition. (eg DAPI SEM, FITC SEM).

You can also create or remove channels accordingly.

When using an existing or defining a new channel please confirm:

(Re)name here.

Objective of choice.

Correct ilumination type and LED excitation (eg Fluorescence and Cy5).

Correct emission filters (eg. Cy5 705/72).

For transmission

In "Illumination" chose "Transmission".

In "Contrast insert" choose "Empty".

In "Emission" choose "Empty" Emission filter.

Decreasing the "Intensity" to 20 should avoid saturating the image.

Press"Live settings" to check settings on Live view.

Press "Focus". The AF settings will now be applied.

SW-autofocus settings can be optimized with the help of the autofocus quality graph.

Move the Live settings window to the side so you can change between the different channels and adjust them accordingly.

Adjust exposure time for each channel.

Make sure your image is not saturated - clipping should be 0%.

Check that "Focus offset" for each channel is on "0" .

Here you can define a time lapse.

Here you can define z-stacks.

Here you can define the magnification changer.

Define your destination folder here.

Make sure that the magnification changer on the right side of the microscope is correctly set. It should match the software settings.

Otherwise you will receive a message asking you to move it into the correct position.

Choose your imaging pattern here.

Finally choose the autofocus positions/pattern you would like to apply to each well.

Right click over defined positions.

To optimize and speed up acquisition we recommend that you include one position per well with the 3 options "AF Coarse", "AF Fine" and "AF Hardware".

For the remaining positions "AF Fine" might be sufficient, but this needs to be adjusted depending on the sample.

Increase the number of positions with "AF Coarse" and "AF Hardware" if necessary.

Start your scan.