MD ImageXpress HCS.ai - 2. targeted acquisition

Leave all toggles unchecked for the overview acquisition.

Go to your Plate library

Select the plate configuration you are using

Select the fields you want to include in the overview scan

For overview acquisition, Widefield mode is recommended over spinning disk. It allows faster acquisition through lower exposure times and is generally sufficient for detecting targets of interest.

The 10× Plan Apo Lambda D objective offers the best trade-off between image resolution, speed, and data size.

Set the Magnification Changer to 1×.

Set Binning to 1×1.

Only one channel can be used for target selection during the analysis. Additional channels can be set up if desired, but they will not be used further in the workflow.

Adjust the laser power and/or exposure time until your signal falls within the optimal range for your sample.

Set up the sites you want to scan.

Give the protocol and acquisition informative names.

Save your data to your data folder on the Z: drive for easy access from the ZMB MetaExpress VM.

Be mindful of the data size.

Browse to the acquisition data in your data folder on the Z: drive.

Select your data

Select the position and field of view (FOV) for target selection.

Select the FlexiProtocol 2D

Press New to create a new detection protocol.

If you have already created a protocol, you can select it from the list to rename, modify, or run it.

Select Cells as the target type.

The SINAP segmentation mode works well for most use cases.

Give your targets a descriptive name.

Select the channel to be used for segmentation.

Select the color for the segmentation masks.

Set the compartment to Everywhere and target linking to Non-linked.

Configuring the segmentation settings properly, under Analysis Settings, has the biggest effect on your target detection.

Several segmentation options are available. We recommend Cell.d, which generally gives good results.

Cell.d also lets you provide a rough estimate of the object size you are selecting.

Make sure the correct channel is selected for segmentation.

Press Apply to test the settings.

Click Masks to adjust mask settings.

Switch to Random color to better distinguish individual segmented objects.

Change the mask type from Solid to Outline for a clearer view of underlying cells.

Under Filter in the Analysis Settings, the segmented objects need to be further defined.

Refine the segmentation by filtering cells by size and intensity

Make sure the correct channel is selected.

Suppress overexposed signals and cells touching the image border.

Under Advanced, define the minimum number of pixels required between the border and segmented objects.

Only a few segmented objects should remain. These will be used as your targets.

Press Run Protocol

Save your protocol with a descriptive name and an informative description

Select the well you want to run the analysis on.

Start the analysis.

The analysis is finished, and the results are shown here. You can now move on to target acquisition.

Select Overview Acquisition, Use Regions, Quick ID Targeted Acquisition, and Z-Series.

The Targeting button will appear in the sidebar.

For target acquisition, switch to a higher resolution.

Select the 60 µm Pinhole spinning disk.

Select the 60× Plan Apo VC XA Water objective.

Set the Magnification Changer to 1.5×

Leave binning at 1×1.

Setup the channels of interest

Set up the Z-series Before you move to Targeting

Select the maximum number of objects per well you want to load.

Load the objects using the Update Wells and Regions button.

Navigate to the experiment folder in your data folder on the Z: drive.

Select the analysis that has been done with IN Carta

The targets will load into the Navigation window, where you can also delete overlapping areas.

Give your protocol and data appropriate names

Run your protocol.