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In this second video of the Well Plate Navigator series,

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I’ll show you how to acquire multi-channel Z-stack images
from multiple wells.

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If you haven’t already, make sure you watch the first video in this series

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where I discuss the necessary preparation steps.


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Let’s get started.

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First, make sure the Well Navigation layout tab is open.

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Click Load Overview Area to open the well plate data
that you registered.

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If you haven’t registered your plate, please refer
to the first video in this series.

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Select the objective lens you’re using for the acquisition.

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Then click, Open a dialog box to define the current pattern.

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In this dialog box, you can define the XY acquisition
coordinate pattern in each well.

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A variety of patterns are available.

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In this example, select Full well in and then click OK.

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Click a well to assign the pattern to it.

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The whole area inside the well is registered as an acquisition area.

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If you want to delete the pattern, click the delete button
and then click on the well.

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The pattern will disappear.

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For more information about other patterns,
use our online help tool.

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Here, we’ll use Custom rectangle.

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In this mode, you can place any number of
rectangle-shaped frames in each well.

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In this example, the number of rows and columns are set to three.

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You can also define the space between each frame.

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If you click Tile, there will be no space,

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and if you click Spread, the space will be set to maximum.

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Click Spread and then OK.

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To copy this pattern to multiple wells,
left click your mouse and move it across each well.

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The pattern is clearly visible in the preview window

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3 columns, 3 rows, with spread spacing.

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Click the Position List to check the registered positions.

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The XY position, Well ID, and Well Position ID
are registered at each point.

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We’ll define the Z position later.

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Click OK to close the Position List.

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Now we’ll enter the acquisition settings.

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Open the Process Manager tool window.

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Click the Multi Channel, Z-Stack, and XY-Positions/MIA icons
to enable them.

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Time-lapse is also available, but we’ll skip it here to save time.

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Assign DAPI and RFP channels.

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Click on the DAPI channel.

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The microscope icon indicates that your system is
ready to acquire DAPI.

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Start Live and adjust the focus and exposure time.

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Click Read Setting to register the current exposure time.

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Repeat this for the RFP channel.

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Then click Read Settings.

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Stop Live, and then we’ll adjust the Z-stack settings.

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Select Range to acquire a Z-stack centered on the current Z-position.

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The optimal step size can be calculated using
the theoretical optical z resolution.

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Click to apply the recommended step size.

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Lock the step size, and then decide the number of Z-slices.

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Let’s set it to 5.

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Checking Z before channel can shorten the acquisition time

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by reducing the number of filter turret rotations.

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Now that the Z-stack range is registered,
let’s register the center of the Z-stack.

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You should choose the best focus position,
which differs depending on the XY position.

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Because it’s difficult to register the focus for all positions,

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we recommend using a TruFocus Z-drift compensator.

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The TruFocus hardware-based autofocus system
keeps the sample in sharp focus faster

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and with less damage to your cells than software-based autofocus.

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It detects the vessel bottom by emitting
an infrared laser and detecting its reflection.

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It moves the objective lens to compensate for any Z-drift,

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keeping your sample in focus.

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We can register the same Z-drift offset at several positions

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since we expect the cells to be captured
at almost the same distance from the vessel bottom.

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Registering the Z-drift compensation offset is simple.

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Open the Position List, and select the positions
that have the same offset value.

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In this example, we’ll select them all.

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Click Read Offset.

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Live starts automatically so that you can find the focus position.

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Once you’ve done that, check Apply to all and then click OK.

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Make sure that the Z-drift compensation offset was registered.

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In this example, we registered the same offset
for all positions to save time.

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You can also register different offsets
for different wells for more accurate focus.

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Click the AF icon and check Z-drift compensation
to activate TruFocus during the experiment.

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Before starting the experiment, let's check the save location and name.

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Click Acquisition Settings.

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Then click Process/Experiment under Document Name in the tree view.

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Click All Options, and you’ll be able to add and remove properties.

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Select Experiment Name, Process type, Date,
Counter, Well Position ID, and Separator.

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You can change the order by clicking the Up and Down buttons.

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Check the text in the preview to confirm that it’s correct,
and then click OK.

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Check Reset automatically so that the counter sets to 1 automatically.

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Then, click Process/Experiment under Saving in the tree view.

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Select File System as the save destination.

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Check Create Subdirectory and click Customize
to see the subdirectory’s name.

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Click Folder and Modify.

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Give the file any name that you want, then click OK.

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Check Close after save since the Navigator acquires a lot of images.

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Click OK to close the Acquisition Settings.

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We’re ready to start the acquisition.

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Click Start.

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Two-channel Z-stack images are acquired at each point.

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The number of frames and time remaining
are displayed on the lower left of your screen.

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The focus is good thanks to the TruFocus Z-drift compensator.

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The acquired images are not opened,
but instead are saved per our acquisition settings.

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OK, the experiment is done.
Let’s check the save location.

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Make sure the folder is available according to the acquisition settings.

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90 images are available.

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You can quickly overview the images.

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Make sure the file name shows Experiment Name,
Process type, Date,

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and Well Position ID as we specified in the Acquisition Settings.

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We can drag and drop images to open them
for more detailed observation.

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Overview images are available in the Gallery Tool window,
making it easy to browse images.

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Thanks for watching!

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In the next video, I'll explain how to group wells
and set individual acquisition settings.

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For more information, visit olympus-lifescience.com.