Fluigent Aria 3: Combine microfluidics with ZEISS microscopes

Turn ON the "Main Switch" and also switch the laser key to "ON".

Switch ON the "Components" on the table.

Switch ON the fluorescence lamp.

Once turned on, the lamp should remain on for a minimum of 30 min.

Turn ON the computer.

Sign-in with your ZMB core credentials.

Start the "ZEN 3.x (blue edition)" software.

Choose "ZEN system".

Make sure there is no sample on the microscope stage and calibrate the stage by clicking "Calibrate Now".

Open the "Aquisition" tab and press "Smart Setup".

Select "Widefield", press "Dyes and Contrasts" double-click"Coherence Contrast"

Make sure the 2.5x objective is selected.

Check "Tiles"

Open the "Tiles" tab and click on "Show viewer"

Press "Live"

Navigate the crosshair to the top left corner of your sample , choose the "Marker-position mode" and press "+"

Repeat this step for the lower right corner of your sample

Press "Start Experiment"

Drag and drop the image from the "Images and Documents" Tab onto the "Tile Viewer"

You can now use the Tile Viewer to navigate within your sample

Select the "40x" and press "Load"

Remove the entire sample holder and set it down within the microscope without moving the sample in the sample holder or disconecting the tubing

Not moving the sample within the sample holder avoids large discrepancies between the 2.5x overview and the later acquired 40x images.

Apply a generous amount of oil on the objective

Carefully place the sample holder back

Press "Work" in the Focus tab

Check "Z-Stack" and choose "Full Z-Stack per Track".

Open "Smart Setup".

Press "LSM" , press "Dyes and Contrast" and select your dyes.

Select "Smartest (Line)" and press "OK".

Alternatively select "Best Signal"

Go to the "Z-Stack" tab and select "Center".

Press "Live" and focus by either using the focus wheel or by pressing Ctrl and using the mouse wheel.

Once you have focused, press "Center".

Choose the size of your Z-Stack and of the steps.

Press "Optimal" for optimal step size

Go to the "Focus Strategy" Tab and select "Combine Software Autofocus and Definite Focus".

Press "Expert", check "Tile Regions/Positions" and check "Repeat Every 1 Region/position".

Go to the "Software Autofocus" tab, uncheck "Automatic Range" and set "Range" to 40 micro meters.

Go to the "Auto Save" tab and choose a naming strategy.

Press the Gear icon, select "Save As".

Choose a name for your Zen Protocol and press Enter on the keyboard.

Open the Aria Software and load a sequence using this guide.

For ALL "Start Acquisition" steps make sure to select the correct Zen Protocol

Press the Start Button in the lower right corner.

Check if you have enough volume in all reservoirs

To start the sequence press "Start injection delivery"