=========================== Acquire an Image (Beginner) =========================== This guide will describe how to acquire a single image and a z-stack using the **navigate** software package. Launching the Software Package ============================== Open Anaconda Prompt -------------------- To start, you need to open the Anaconda Prompt. Follow these steps: 1. On Windows, click on the Start menu. 2. Type ``Anaconda Prompt`` into the search bar. 3. Click on the Anaconda Prompt application to open it. .. note:: Ensure that Anaconda and **navigate** are already installed on your system. If not, please refer to our :ref:`Quick_Start_Guide` for more information. Activate Conda Environment -------------------------- Once the Anaconda Prompt is open, activate the desired conda environment. By default, the command prompt will open the base environment (as shown in parentheses). To activate **navigate** environment, type the following command into the Anaconda command window and press :kbd:`Enter` .. code-block:: console (base) conda activate navigate Launch the Software Package --------------------------- After activating the environment, **navigate** should now be shown in parentheses. After you have already :doc:`configured ` **navigate**, you can launch it by typing the following command into the Anaconda command window: .. code-block:: console (navigate) navigate The **navigate** software package will launch and the main window will appear. .. image:: images/beginner/open-navigate.png :alt: Opening **navigate**. ------------------------- Configure the Channel Settings ============================== * Select the :guilabel:`Channels` tab, which is located on the upper left of the main window. * Under the :guilabel:`Channel Settings` section, select the number of channels needed for imaging. For each channel selected, you will need to configure the acquisition settings: .. image:: images/beginner/channel-selector.png :alt: Channel settings in the **navigate** software package. * Select the appropriate :guilabel:`Laser` from the dropdown menu. * Select the appropriate :guilabel:`Power` for the laser. * Select the appropriate emission :guilabel:`Filter` from the dropdown menu. .. image:: images/beginner/channel-selector-filter.png :alt: Changing the emission filter in **navigate**. * Specify the camera :guilabel:`Exp. Time (ms)`. A good default value is ``100`` or ``200`` ms. * Specify the :guilabel:`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 :guilabel:`Defocus` to be ``0``. This feature allows you to adjust for chromatic aberrations that result in focal shifts between each imaging channel. ------------------------- Configure the Camera Settings ============================= * Select the :guilabel:`Camera Settings` tab. * For standard imaging applications, select :guilabel:`Normal` in the :guilabel:`Sensor Modes` dropdown menu within the :guilabel:`Camera Modes` section. * If you are using the rolling shutter, select :guilabel:`Light-Sheet` and specify its :guilabel:`Readout Direction` and :guilabel:`Number of Pixels`. .. note:: For more information on how to configure the rolling shutter for ASLM operation, please refer to :doc:`ASLM `. .. image:: images/beginner/sensor-mode.png :alt: Changing the camera sensor mode in **navigate**. * Choose the size of your camera's field of view. * Specify the :guilabel:`Region of Interest Settings` by entering the appropriate :guilabel:`Number of Pixels` for both the :guilabel:`Width` and :guilabel:`Height` values. Alternatively, one can select from one of several default values in the :guilabel:`Default FOVs` section. .. note:: The :guilabel:`FOV Dimensions (microns)` is automatically calculated based on the :guilabel:`Number of Pixels` and the `pixel_size` as specified in the `zoom` section of your your ``configuration.yaml`` file. .. code-block:: yaml zoom: pixel_size: 20x: 0.325 # magnification, and pixel size in microns .. image:: images/beginner/ROI-definition.png :alt: Changing the camera region of interest in **navigate**. .. note:: If multiple channels are selected, each channel will be acquired with the same camera :guilabel:`Sensor Mode`, :guilabel:`Readout Direction`, and :guilabel:`Region of Interest Settings`. ------------------------- Acquire in a Continuous Scan Mode ================================= * Select "Continuous Scan" in the dropdown next to the :guilabel:`Acquire` button in the :ref:`acquire bar `. .. image:: images/beginner/continuous-scan-dropdown.png :alt: Selecting the continuous scan mode in **navigate**. * Press :guilabel:`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 :guilabel:`Channel Settings` section of the :guilabel:`Channels` tab, and will proceed from the top to the bottom of this channel list. .. image:: images/beginner/continuous-scan-acquire.png :alt: Launching the continuous scan mode in **navigate**. * Move the stage to identify the location of the sample. * Select the :guilabel:`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 stages loaded in a synthetic mode, buttons will be disabled. * Absolute positions can be entered in the text boxes next to each button. * Check :doc:`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. .. 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 the :doc:`configuration settings ` for more information. .. image:: images/beginner/stage-movement-panel.png :alt: Moving the stage in **navigate**. * Press the :guilabel:`Stop` button in the acquisition bar to stop acquisition. .. image:: images/beginner/stop-acquisition.png :alt: Stopping the continuous scan mode in **navigate**. ------------------------- Acquiring a Single Image ========================= * Check the :guilabel:`Save Data` box in the :guilabel:`Timepoint Settings` section under the :guilabel:`Channels` tab to save the acquired images. Check this box before acquiring data. .. image:: images/beginner/save-data.png :alt: Saving data in **navigate**. * Select :guilabel:`Single Acquisition` from the dropdown next to the :guilabel:`Acquire` button. .. image:: images/beginner/single-acquisition-dropdown.png :alt: Selecting the single acquisition mode in **navigate**. * Press :guilabel:`Acquire` to open the :guilabel:`File Saving Dialog` interface. Enter the sample parameters, notes, location to save file, and filetype in the :guilabel:`File Saving Dialog` that pops up. .. image:: images/beginner/save-dialog-box.png :alt: Saving data in **navigate**. * Press :guilabel:`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 :guilabel:`File Saving Dialog` interface. Data will not be overwritten between acquisitions. .. image:: images/beginner/save-dialog-box-acquire.png :alt: Saving data in **navigate**. ------------------------- Acquiring a Z-Stack =================== * Using the :guilabel:`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 :ref:`Autofocus Settings `. .. image:: images/beginner/stage-control-start-pos-zstack.png :alt: Adjusting the stage position in **navigate**. * Under the :guilabel:`Channels` tab, in :guilabel:`Stack Acquisition Settings (μm)` press :guilabel:`Set Start Pos`. .. image:: images/beginner/press-start-pos.png :alt: Adjusting the stage position in **navigate**. * Using the :guilabel:`Stage Control`, go to a different z-position within the sample. Again, make sure that the sample is in focus. .. image:: images/beginner/stage-control-end-pos-zstack.png :alt: Adjusting the stage position in **navigate**. * Under the :guilabel:`Channels` tab, in :guilabel:`Stack Acquisition Settings (μm)` press :guilabel:`Set End Pos`. .. image:: images/beginner/press-end-pos.png :alt: Adjusting the stage position in **navigate**. .. 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 :doc:`configuration settings ` for more information to determine if focus is enabled in hardware. Refer to :doc:`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 :guilabel:`Step Size` dialog box in :guilabel:`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 :doc:`configuration settings ` .. code-block:: yaml gui: stack_acquisition: step_size: min: 0.100 max: 1000 step: 0.1 .. image:: images/beginner/define-step-size.png * If using multiple channels for imaging, select either :guilabel:`Per Z` or :guilabel:`Per Stack` under :guilabel:`Laser Cycling Settings` in the :guilabel:`Stack Acquisition Settings (μm)` section under the :guilabel:`Channels` tab. * :guilabel:`Per Z` acquires all channels before moving the stage to a new position. * :guilabel:`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. .. image:: images/beginner/laser-cycling-settings.png * Select :guilabel:`Z-Stack` from the dropdown next to the :guilabel:`Acquire` button. Press :guilabel:`Acquire`. .. image:: images/beginner/z-stack-acquisition.png * Enter the sample parameters, notes, location to save file, and filetype in the :guilabel:`File Saving Dialog` that pops up. * Press :guilabel:`Acquire Data` to initiate acquisition. Acquisition will automatically stop once the image series is acquired.