A. Several things influence image speed acquisition:

1- Number of Channels: to achieve the fastest acquisition, image with one channel.

2- Exposure time: Make sure that you are using the minimum exposure time. If the signal is bad, try to use higher intensity and more sensitive camera (axiocam 702).

Exposure Time Ranges:

Axiocam506: 250 µs to 60 s.

Axiocam702: 0.1ms − 60 s.

3-Number of steps of a Z-stack: the less is better.

4- ROI: the smaller the ROI in Y-axis -the faster you will be able to image.

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5- Camera: CD7 has two cameras, Axiocam702 is faster however Axiocam506 has a smaller exposure time.

• CMOS Axiocam702: Up to 128 fps at full resolution and up to 1000 fps @ 1024 × 128 pixels.

• CCD Axiocam506: Binning Pixel: 1x1, Count (H x V): 2752 x 2208, Mode Mono, 20 FPS @ 1 ms.

6- Pupil component: If you want to do past imaging in the transmitted light channel (Oblique), make sure that the system doesn't waste time taking in and out the “pupil” component; it should be in.

In the “Imaging Setup” tab, go to advanced, go to “After oblique:…” and unclick the box “include in this setting”-red arrow.

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B. How to set up a fast acquisition:

Without tiles setup/ single position:

Once you set everything up (channels, position, focus, etc..)

Go to Time series, set the interval to 0ms, and click on “use camera streaming…” and “use burst mode…”.

**Once “Use Camera Streaming If Possible” is activated, the software tries to use the active camera's free-running/streaming mode.

**Once “Use Burst Mode if Possible“ is activated, the system buffers acquisition data in the main memory. Burst Mode enables the recording of the fastest framerates, which the used camera hardware can achieve. This mode supports only single-channel time-lapse acquisition.

 Start the experiament.

With tiles setup / Multiple positions:

Once you set everything up (channels, position, focus, etc..)

Go to the “Time Series” tab.

Set the interval to 0ms and check the box “Use camera streaming if possible”.

**Once “Use Camera Streaming If Possible” is activated, the software tries to use the active camera's free-running/streaming mode.

The final step is to go to the “Acquisition Mode” tab → Fast Acquisition.

Then click on “Triggered”.

After clicking “Triggered”, the button below “Validate Experiment” should be enabled for clicking.

(If the bottom “Validate Experiment” appear grey and unclickable, then it means the fast acquisition failed- and part of the issues that appear below in the “validation results” should be fixed- errors about “Motorized Stage” and “Motorized Focus” can be ignored).

Click on “Validate Experiment”.

After clicking “Validate Experiment”, the “Compromise” mode will appear green.

Click “Start Experiment” immediately.

Note that once you change something in the experiment setting, you will need to do this process again and validate the experiment again.