Acquiring Data

This guide introduce the basic steps for acquiring data with navigate. It assumes that you have already completed:

• Software Installation

• Configuring Navigate

If your microscope hardware is not fully configured yet, you can still complete all steps below in synthetic hardware mode using Virtual Devices.

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Launch the Software Package

1. Activate the same environment you used during installation.

2. Launch navigate:

# Use configured hardware
navigate

# Or use Virtual Devices (synthetic hardware mode)
navigate -sh

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Configure the Channel Settings

• Select the Channels tab, which is located on the upper left of the main window.

• Under the Channel Settings section, select the number of channels needed for imaging. For each channel selected, you will need to configure the acquisition settings:

  

  • Select the appropriate Laser from the dropdown menu.

  • Select the appropriate Power for the laser.

  • Select the appropriate emission Filter from the dropdown menu.

  

  • Specify the camera Exp. Time (ms). A good default value is 100 or 200 ms.

  • Specify the Interval to be 1.0. While this feature is not currently implemented, future releases will allow users to image different channels at different time intervals.

  • Specify the Defocus to be 0. This feature allows you to adjust for chromatic aberrations that result in focal shifts between each imaging channel.

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Configure the Camera Settings

• Select the Camera Settings tab.

• For standard imaging applications, select Normal in the Sensor Modes dropdown menu within the Camera Modes section.

• If you are using the rolling shutter, select Light-Sheet and specify its Readout Direction and Number of Pixels.

Note

For more information on how to configure the rolling shutter for ASLM operation, please refer to ASLM.

• Choose the size of your camera’s field of view.

  • Specify the Region of Interest Settings by entering the appropriate Number of Pixels for both the Width and Height values. Alternatively, one can select from one of several default values in the Default FOVs section.

  Note

  The FOV Dimensions (microns) is automatically calculated based on the Number of Pixels and the pixel_size as specified in the zoom section of your configuration.yaml file.

  zoom:
    pixel_size:
      20x: 0.325 # magnification, and pixel size in microns
  

  

Note

If multiple channels are selected, each channel will be acquired with the same:

• Sensor Mode

• Readout Direction

• Region of Interest Settings.

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Acquire in a Continuous Scan Mode

• Select “Continuous Scan” in the dropdown next to the Acquire button in the acquisition bar.

  

• Press Acquire. This will launch a live acquisition mode.

  Note

  If multiple channels are selected, each channel will be imaged sequentially. The order of imaging is determined by the order of the channels in the Channel Settings section of the Channels tab, and will proceed from the top to the bottom of this channel list.

  

• Move the stage to identify the location of the sample.

  • Select the Stage Control tab, and use the graphical user interface to move the stage. This includes buttons for moving the stage in X, Y, Z, F, and Theta directions.

    • The step size for each axis can be adjusted with the spinbox next to each button.

    • For stage axes configured as Virtual Devices, buttons will be disabled.

    • Absolute positions can be entered in the text boxes next to each button.

    • Check Advanced Configuration settings for more information.

  • Alternatively, if available, use the manufacturer-provided joystick to position the sample.

  Note

  The axes for a light-sheet microscope vary in the literature. Here, we define the Y axis as the direction of the light-sheet propagation, the Z axis as the direction of the detection objective, and the X axis as the direction perpendicular to the light-sheet and detection objective axes. The F axis typically controls the position of the detection objective along the detection axis. The Theta axis typically controls the rotation of the sample. This is discussed in more detail in the coordinate system section.

  Warning

  One should always be careful when moving the stage. If the stage is moved too quickly, the sample and/or microscope may be damaged. We strongly recommend that you implement stage limits in your configuration file. Please refer to configuration settings for more information, or open Stage Control ‣ Advanced Stage Parameters.

  

• Press the Stop button in the acquisition bar to stop acquisition.

  

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Acquiring a Single Image

• Check the Save Data box in the Timepoint Settings section under the Channels tab to save the acquired images. Check this box before acquiring data.

  

• Select Single Acquisition from the dropdown next to the Acquire button.

  

• Press Acquire to open the File Saving Dialog interface. Enter the sample parameters, notes, location to save file, and filetype in the File Saving Dialog that pops up.

  

• Press Acquire Data to initiate acquisition. Acquisition will automatically stop once the image is acquired.

  Note

  Each acquisition will be saved in a separate folder (e.g., Cell01, Cell02, …) within the directory specified in the File Saving Dialog interface. If you prefer a different prefix for the folder names, you can specify this in the Prefix field. Data will not be overwritten between acquisitions.

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Acquiring a Z-Stack

• Using the Stage Control, go to the desired z-position in the sample. Make sure that the sample is in focus. To use the autofocus feature, please refer to the Autofocus Settings.

  

• Under the Channels tab, in Stack Acquisition Settings (μm) press Set Start Pos/Foc.

  

• Using the Stage Control, go to a different z-position within the sample. Again, make sure that the sample is in focus.

  

• Under the Channels tab, in Stack Acquisition Settings (μm) press Set End Pos/Foc.

  

  Note

  If there is a shift in F between the start and stop positions, the F axis will be ramped synchronously with Z to maintain focus. Check configuration settings for more information to determine if focus is enabled in hardware. Refer to Imaging on a mesoSPIM BT section for an example of how to acquire a z-stack with a focus ramp.

• Type the desired step size in microns in the Step Size dialog box in Stack Acquisition Settings (μm).

  Note

  The minimum step size, and increment between steps, are graphical user interface defaults that are specified in the configuration.yaml file. More information can configuration settings.

  gui:
    stack_acquisition:
      step_size:
        min: 0.100
        max: 1000
        step: 0.1
  

  

• If using multiple channels for imaging, select either Per Z or Per Stack under Laser Cycling Settings in the Stack Acquisition Settings (μm) section under the Channels tab.

  • Per Z acquires all channels before moving the stage to a new position.

  • Per Stack acquires all images in a stack acquisition for a single channel before moving the stage back to the start position and restarting acquisition for the subsequent channel until all channels are imaged.

  

• Select Z-Stack from the dropdown next to the Acquire button. Press Acquire.

  

• Enter the sample parameters, notes, location to save file, and filetype in the File Saving Dialog that pops up.

• Press Acquire Data to initiate acquisition. Acquisition will automatically stop once the image series is acquired.