Creating a new lens or calibrating an existing lens from an image

Purpose

Use this procedure to create software lenses from images with known scaling or that contain an accurate scale bar.

Software lenses can be used to assign scaling information to image files that:

  • Do not contain scaling information.
  • Contain inaccurate (wrong) scaling information.
  • Contain scaling information that Neurolucida 360 cannot recognize/interpret.

Materials needed

An image with known scaling, for example one of the following:

  • Image file that includes scaling information, for example, such as .jp2 or .jpx files captured using an MBF Bioscience imaging system

  • Image of the MBF Bioscience calibration-grid slide or a graticule slide with known scaling:

    Acquire an image of the calibration-grid slide using the same objective that you plan to use in your study.

    If you are using multiple objectives, acquire an image of the calibration grid slide with each objective.

    • 10x and lower: Acquire an image of the large grid (250 µm).
    • 20x and above: Acquire an image of the small grid (25 µm). Immersion media (oil, water, etc.) are not required for this step.

  • Image that includes an accurate scale bar

Procedure

Using an image file that includes scaling information to create a new lens

  1. Open the image with known scaling in Neurolucida 360 and continue to the next step unless the Image Scaling dialog box opens.

    The Image Scaling dialog box opens if Neurolucida 360 cannot interpret the scaling information in the file or if your Images > Openpreferences are set to Show image scaling dialog on opening image.

    In the Image scaling dialog dialog, check the Source Of Scaling selected in the X and Y Scaling Applied to Images section of the dialog:

    • If File is selected, verify that the X and Y values are accurate, modify them if needed, and Click OK to apply the scaling information and continue to the next step.

      If you changed the scaling information, you may want to click Update Image File to save the image/image stack file with the scaling information you entered.

    • If a different choice is selected (i.e., anything other than File), do the following:

      1. Check the box to Override X and Y scaling.

      2. Select User Specified as the Source Of Scaling.

      3. Enter the correct values for X and Y scaling in the boxes.

      4. Click OK.

  2. If you have more than one image open, use the Image Organizer to set the relevant image to be visible and hide any other images.

    For example, if you are calibrating a 5x objective, the eye icon in the Image Organizer should only appear next to the large grid (250 µm) image.

  3. Go to File > Calibration and click New lens.

    If the image is not displayed at its original magnification, it is automatically adjusted to display at 100% resolution.

  4. Follow the instructions for Creating a New Lens:

    • Enter the lens name, units, category and Z correction factor in the Create New Lens window.

    • Select Use Scaling From Image for the Calibration Type to populate the Scale Factor and Aspect Ratio fields with scaling information from the active image.

  5. Verify that the calibration is correct using the Measure Line tool (Trace ribbon>Measure line drop-down).

Using an image of a calibration-grid slide or an image that includes an accurate scale bar to create a new lens

  1. Follow steps 1-2 of the procedure above (for using an image file that includes scaling information to create a new lens).
  2. Go to File > Calibration and click New lens.
    • If the image is not displayed at its original magnification, it is automatically adjusted to display at 100% resolution.

    • Follow the instructions for Creating a New Lens:

      • Choose Manual Calibration for the Calibration Type

      • Enter the size of the grid that is displayed onscreen under Calibration Box Setup.
      • If you are calibrating from an image with a scale bar instead of from a calibration-grid slide, check the Force Square checkbox.
      • Click OK to launch manual calibration. See Manually calibrating lenses for instructions.
  3. If you need to define multiple lenses from calibration grid images, show the image appropriate for the lens power and hide other calibration grid images by clicking to add or remove the eye icon in the Image Organizer and repeat this process for the other lenses.
  4. Verify that the calibration is correct using the Measure Line tool (Trace ribbon>Measure line drop-down).