Image scaling dialog

Purpose

When you open an image or image stack and BrightSLICE software does not find or cannot interpret image-scaling information, the Image Scaling dialog is displayed where you can enter scaling information. Once the scaling information has been entered, click OK to apply it to the file you're opening.

Procedure

Entering scaling information

Image stacks (3D images): The Image Scaling dialog will display sections for both Z–scaling, and X and Y–scaling information when you open an image stack.

2D (single plane) images: Only the X and Y–scaling information section of the dialog is displayed for two-dimensional images.

Z Scaling Applied to Images

Distance between images: Enter the distance between images (Z-step).

Distance Type: Click the radio button to select Focal or Physical Distance to indicate which category of distance you entered above.

  • Focal Distance: Image stacks acquired using spacing that describes the focal plane separation.

  • Physical Distance: The physical movement of the microscope stage as images are acquired.

    If you select Physical Distance, choose the medium used between the lens and the specimen and BrightSLICE will autofill the appropriate correction factor. If you select Other, enter the refractive index of the medium; it will be used as the correction factor.

Create sections for this stack: Check the box to auto-populate the Serial Section Manager with a section for each plane in the image stack, using the Distance between images you specified as the Z-spacing.

X and Y Scaling Applied to Images

X: and Y: µm/pixel or voxel: if available, values for the current X and Y scaling are displayed. If scaling information is not present/interpretable in the image file, enter the correct scaling information:

Source of X and Y Scaling: radio buttons

  • File: X/Y scaling information embedded in the image file. If this information is incorrect, you can update it in the User Specified section and save it with the image file by clicking Update Image File.

  • Current lens: X/Y scaling information is from the lens currently selected in BrightSLICE software. Setting up a lens with X/Y scaling information may save time if you are working with many images that do not have embedded X/Y scaling information. See instructions for Creating a New Lens.

  • User Specified: You can enter the X/Y scaling information manually. Enable this by clicking the checkbox, Override X and Y scaling.

Override X and Y scaling: Click the box, then select User Specified (under Source of X and Y Scaling). You can then type in X and Y scaling information.

Expansion microscopy correction

Expansion microscopy correction enables you to adjust scaling after tissue expansion. This ensures that any measurement analyses provide data based on an object's biological size, not their expanded physical size.

Apply expansion factor: Check the box to enable expansion microscopy correction. Enter the expansion factor into the text box. For example, if the tissue has been expanded 3 times its biological size, enter 3. The scaling will be isotropically reduced to 1/3 of the original image scaling.

Adjusted scaling (µm): The adjusted scaling for the X, Y, and Z axes is displayed.

Applying scaling information

  • Click OK to apply the scaling information to the file you're opening.

    The scaling information will not affect the image/image stack file unless you choose to update the image file (see next bullet), but will be saved with your data file (if you save a data file).

  • Click Update Image File to save the image/image stack file with the scaling information you entered.

    1. The Update Image File Scaling window will open :

      • Check the box to Allow scaling to be written to the image file, confirming your choice to update the image file.

      • (optional) Check the box to Persist this setting for this session... for files that do not contain scaling information that BrightSLICE software can interpret. This can be a real time saver if you create a new lens with scaling information and you are working with many images that do not have embedded X/Y scaling information.

      • Click OK.

    2. In the Save As window that opens, navigate to the file storage location and choose a file type from the drop-down menu. We recommend that you save as a .jpx file or use the original file format.

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You can also drag and drop files into the BrightSLICE software window. The Image Opener dialog box will open. Choose options to specify how you want BrightSLICE software to handle your files.