Register volume

Purpose

Use Register Volume to align 3D image stacks, obtained from intact brain specimens, to a reference-brain atlas.

The Register Volume tools are designed for 3D images in which the image planes in the file were acquired from tissue that was intact, i.e., not sectioned or sliced prior to imaging. You can work with 3D images obtained from cleared tissue, blockface imaging, or any tissue that was imaged whole.

Use Register sections (rather than Register Volume) for working with 3D reconstructions, created from images of serial sections using Serial Section Assembler.

See also Registration windows and tools reference.

Procedure

A. Getting started

  1. If you are working with specimens from animals other than mice, download a brain atlas for the species from the NeuroInfo section of the MBF Bioscience download center.

    When you install the brain atlas for your species, it will be available in the Atlas: dropdown menu.

  2. Open your 3D brain image stack, using any of the following methods:

    • Drag and drop the file onto the NeuroInfo software window.

    • Click Open an image file button in the Quick Access bar or type Ctrl+O.

    • Go to File > Open and click Image Stack.

  3. Click Register Volume in the NeuroInfo tools section of the Registration ribbon.

    • The Register Volume to Atlas window opens on the right; this is where you'll do most of the work.

    • The Registration ribbon displays functions relevant for registering a 3D volume to the atlas. Other tools are not displayed until you click Leave Registration.

  4. Navigate to approximately the center of the brain using the 3D Slice Scroll slider or the Orthoview tool. Find an area that exhibits good contrast and distinguishable features, such as the hippocampus and fiber tracts.

  5. Using the Image Adjustment tool (available on the Workspace ribbon), we recommend making the following adjustments:

    • Display only the color channel(s) with the anatomic information that you want to use for alignment. Deselect the other color channels so that they are not displayed.

    • Choose a light color, such as yellow, pale orange or pale grey to display the selected color channel(s).

    • Adjust the histogram so that structures in the image are clearly visible.

B. Select or create an atlas calibration [Current calibration section]

Context

Calibration provides NeuroInfo with enough information about how your 3D reconstruction relates to the reference-brain atlas to enable the effective use of automatic registration (alignment) tools. It establishes:

  • the brain atlas you are using as the reference

  • the anatomic orientation of the experimental image data

  • approximate relative scaling of the brain specimen(s) to the atlas

Do one of the following to set the calibration:

  • Select an appropriate calibration from the list. You can typically use the same calibration for registering brain specimens from the same species that were processed and imaged using equivalent protocols.

  • Create a new calibration:

    • the first time you use NeuroInfo

    • for specimens processed and/or imaged using a different protocol

    • to work with specimens from other species

  • Change an existing calibration: Select a calibration in the list and click Edit. Note that you will overwrite the existing calibration when you save the edited version.

  1. To create a new calibration, click Create New in the Calibrations section, under Current Calibrations.

    • A dialog box opens that prompts you to Enter Calibration Name. Type in a name that reflects the specimen and its processing method, then click OK.

    • You'll see the name for the new calibration at the top of the registration panel, Current Calibration: Your New Calibration.

    To edit an existing calibration, select a calibration and click Edit. [Note that if you Edit a calibration, you will overwrite the existing calibration when you save the edited version.]

    Information to guide you through the atlas calibration process is displayed. Follow the steps outlined onscreen (and below)

  2. Choose (or verify) the reference-brain atlas for the registration using the Atlas: dropdown menu

    • Allen2017Coronal_25um: Recommended for most registrations.

      This atlas was created in the coronal orientation, with 25 µm sampling, by the Allen Institute for Brain Science in 2017.

    • GerfenNissl2017: You may want to choose this atlas if your specimen is labeled with a fluorescent dye that targets Nissl bodies (such as NeuroTrace dyes from ThermoFisher Scientific), and you are not satisfied with the precision of registration results using the Allen2017Coronal_25um atlas.

  3. Check the Anatomic Orientation and click Change Orientation if the orientation of your 3D reconstruction is not accurately represented.

    The Orientation Selector dialog opens and prompts you to enter specimen-orientation information.

  4. Expand the Registration Info section and review/update the Image Info:

    • Modality: select the microscopy mode used to process and image the experimental specimen.

    • Channel: Select the color channel with the cytoarchitectural information that will be used to align/register the experimental image volume to the atlas.

  5. Use the sliders in the Manual Registration Adjustment section to visually align the experimental and atlas slice images:

    1. Click to expand the Manual Registration Adjustment and Visualization Options tools.

      [If you haven't already done so, navigate to a section in the experimental brain with good contrast and distinguishable features, such as the hippocampus and fiber tracts. Typically a section near the center of the brain is ideal.]

    2. Move, scale and deform the atlas slice image so that it aligns with your experimental slice image:

      • Use the sliders in the Manual Registration Adjustment panel to Shift, adjust Rotation, and Scale the atlas slice to match the experimental slice image.

        The sliders are labeled as follows:

        • D and V: dorsal and ventral

        • R and C: rostral and caudal

        • L and R: left and right

      • Note that one of the Shift sliders (which one depends on the specimen orientation) changes the plane of the atlas displayed. Use it to display the plane of the atlas brain that most closely matches the experimental plane displayed.

      • It's helpful to turn the atlas overlay on and off as you align the images. Do this by toggling the checkbox and/or adjusting the slider to Show atlas over experiment slice in the Visualization Options section of the panel.

  6. Press Run linear in the Registration section of the panel to refine the alignment between atlas and experimental slice images.

    If needed, you can repeat steps 5 and 6 to further refine the alignment.

  7. Save the calibration when you are satisfied with this initial alignment/registration; click Save in the Calibrations section at the top of the registration tools panel.

    • Once saved, the calibration will be selected by default the next time that you open NeuroInfo; it can be used with other specimens from the same species that were prepared using the same tissue-processing procedures.

C. Register (align) your experimental images to the atlas

The order of the steps is flexible and the registration can be repeated multiple times if desired. Running the registration algorithms multiple times will update and potentially further improve the registration result.

  1. Use the Local search function to establish the atlas location, tissue-mounting angle, and scale relative to the atlas for one to a few sections near the center of the brain as follows:

    1. Navigate to a section that exhibits good contrast and distinguishable features, Click Local Search to register (align) the current section to the atlas.

      • NeuroInfo searches the entire atlas to find the current section in the atlas, then refines the alignment by determining the angle that provides the best match between the current optical and atlas sections.

      • When the search is complete, the atlas slice image should closely match the experimental section. You can use the controls in the Manual Registration Adjustment section to modify the alignment if desired.

    2. You may want to register 2-4 sections across the center of the brain using Local Search.

  2. Refine the 3D brain volume registration to the atlas using the Refinement tools. The tools are described below; use them in the order you prefer, and repeat the registration multiple times to further refine registration.

    Refinement Controls:

    These check boxes constrain Linear registration refinement only:

    • Lock sectioning angle: Check to hold the specimen angle constant while running a linear registration.

      We recommend using this constraint in most cases; the sectioning angle identified near the center of the brain, where there is good contrast and multiple distinguishable features is typically more accurate than sectioning angle identified in parts of the brain with less contrast and more homogeneous anatomies.

    • Lock relative scale: Check to keep the registration scale parameter constant while running a linear registration.

    Run linear/Clear: Use linear registration, with the selected constraints, to refine the alignment of the experimental brain volume to the atlas. / Clear the linear registration results

    Run nonlinear/Remove: Use nonlinear registration to refine the alignment of the experimental brain volume to the atlas. / Clear the nonlinear registration results.

  3. Save your work.

    • Save the data file by clicking Save in the quick access menu, typing Ctrl+S, or by going to File > Save > Data file.

    • > Save your transform by clicking Manage transforms on the Registration ribbon, then Save transform from the dropdown menu. The transform includes atlas-alignment information for the volume registration; saving the transform enables you to compare results between different transforms.

C. Identifying anatomical structures

Once you have registered (aligned) your experimental image volume to a reference atlas, you can identify atlas structures in your experimental image as follows:

  • See the name of the anatomical structure at the cursor location: Hover your mouse over the Experimental or Atlas Slice images (top and bottom images on the right) to display the name of the brain structure in the lower left corner of the image panel.

  • Display the outline of the anatomical feature:

    • Ctrl + click the location with your mouse

    • Click Select Anatomy in the Registration ribbon and click the location with your mouse.

      Click Deselect Anatomy to turn off display of anatomical structures.

    • Click the check box for the structure name in the list displayed on the Atlas Ontology tab to display its outline. Uncheck the box to stop its display.