Register volume

Purpose

Use Register Volume to align 3D image stacks, obtained from intact brain specimens, to a reference-brain atlas. You can also use register Volume to refine the registration of brain images that were registered using Atlas-constrained, section registration, then mapped to Intermediate Space. 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 volume for data obtained using MRI and microCT.

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

See also Registration windows and tools reference for an overview of the "registration mode" software interface and shared tools.

Before you start

1. Mouse brain atlases are installed when NeuroInfo software is installed. If you are working with images from animals other than mice, our download center may have an atlas for your species. Alternatively, brain atlases can be configured for use in NeuroInfo.

   Click here for instructions on installing brain atlases from our download center and configuring atlases for use in NeuroInfo.

2. Open a 3D image stack obtained from intact brain specimen of a 3D brain reconstruction using any of the following methods.

   You can also open a brain image that was registered using Atlas-constrained, section registration, then mapped to Intermediate Space.

   • 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 > Image Stack.

   If your brain image is not visible or is almost offscreen, go to the Image ribbon and click Fit to Screen.

   The image file format we recommend for use in NeuroInfo is .jpx, however, in many cases, proprietary microscope-system files can be used (e.g., .nd2, .lif, .czi, .vsi).

   If you encounter any problems with using your image files in NeuroInfo, we recommend that you convert the files to .jpx using our free MicroFile+ software designed to help researchers manage large quantities and types of microscopy image data.

3. 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 white, yellow, or pale orange to display the selected color channel(s).

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

   Note that these display adjustments do not impact registration results, but they can help you to visually evaluate and manually adjust registration. Registration utilizes raw image data at its acquired intensity bit-depth for more accurate results.

Procedure

A. Enter volume registration mode and navigate to an area near the center of the brain

1. 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.

2. Navigate to an area in the experimental brain with good contrast and distinguishable features, such as the hippocampus and fiber tracts, using the 3D Slice Scroll slider or the Orthoview tool. If you're working with a whole brain, a location near the center is typically ideal. If you're working with a single brain lobe or a partial brain, go to a location near the center of one of the brain lobes. is best when working with a partial brain or brain images from

B. Activate, create, or confirm your Current calibration

Context

Calibration provides NeuroInfo with enough information about how your brain image(s) 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

• approximate optical-sectioning angle

There is a setting in Registration preferences, Use the global calibration file that determines whether NeuroInfo software shares calibration files among all users (box is checked) or only displays/edits the calibration files associated with the current User profile (box is unchecked). This setting can help multiple users share calibration settings for experimental specimens produced using the same methodology or can help prevent inadvertent changes to calibration settings configured by another researcher.

To set the calibration:

1. Expand the Current calibration: section at the top of the Register volume to atlas panel.

   Click any calibration in the table to display information about its associated atlas.

2. Then do one of the following:

   • Verify that the appropriate calibration is activated; this is indicated by a green check mark in the Active column of the table.

     Information about the atlas associated with the active calibration is displayed below the table.

   • Activate a calibration that was created previously.

     1. Click the calibration in the table that you want to use; information about the atlas associated with the calibration is displayed below the table; you may want to review it.

     2. Click Activate to use the selected calibration for registration.

        Confirm that there is a green check mark for the calibration in the Active column.

     You can open a data file that was registered to a brain atlas previously and activate a calibration associated with a different atlas to update the transforms for that registration to the active calibration. In other words, you can use this function to change the atlas associated with you previously registered image. This may be useful for comparing results with online atlas databases or with results from other laboratories using the same reference atlas.

   • Create a new calibration by clicking Create New (see instructions below).

     You will need to create a new calibration in the following circumstances:

     • The first time you use NeuroInfo

     • For images of brains sourced, processed and/or imaged differently than those represented in the list of calibrations

     • To modify the transforms from a previous registration to use an updated or different reference-brain atlas.

       For example, if you registered an experimental sample using the AllenCCF Default calibration that is based on the Mouse Allen/MBF CCFv3 25um atlas (formerly called Allen2017Coronal_25um), you may want to change the reference brain atlas to the unmodified, sagittal orientation Mouse Allen CCFv3 25um (or 10um) atlas to use the data and tools available through the Allen Institute or to compare with data that was generated by registration with the Allen Mouse Brain CCFv3.

   • Edit an existing calibration by selecting it and clicking Edit (see instructions below). You may want to edit a calibration for the following reasons:

     • To refine the calibration to include the transforms created by registering the experimental image to the atlas. In other words, you can update the calibration after completing the registration so that it may be more useful for registering images of other brains that were sourced, processed, and imaged similarly.

     • To reflect different processing or imaging protocols, or to test alternate calibration settings.

C. Register (align) your experimental-brain image to the atlas

It's a good idea to double-check that the calibration you want to use is activated; look for the green check mark in the Calibrations table and for the name of the calibration displayed in the Current Calibration: dropdown heading.

The registration can be repeated multiple times if desired; this will update and potentially further improve the registration result.

1. If you haven't already done so, navigate to an area in the experimental brain image that exhibits good contrast and distinguishable features.

2. If you are registering a complete brain image, you can skip this step if desired.

   If you are registering a partial-brain image, manually line it up with the atlas and adjust the scaling as follows:

   Click Adjust and do the following:

   • Use the Scale (size) sliders to make the atlas size more closely match the size of the experimental brain

   • Adjust the Shift sliders to align the atlas to the experimental brain; this is particularly important when working with a single brain lobe or other partial-brain image.

3. Click Setup to establish the atlas location, tissue-mounting angle, and scale relative to the atlas.

   Search Select Use automatic search in atlas and click Search to launch the registration.

   • NeuroInfo searches the entire atlas to find the best registration of the current optical section with 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 that of the experimental section. You can use the manual registration tools available when you click Adjust to modify the alignment if desired.

4. Click Register and refine the 3D brain volume registration by running Linear and/or Nonlinear transformation as follows:

   • Run Linear or Run Nonlinear Click Run Linear or Run Nonlinear to register the brain image to the atlas.

   • Clear Transforms Click Clear Transforms to clear the previous registration results.

   • We recommend that you run Linear transformation first, evaluate the precision of the alignment throughout the brain volume, then repeat the Linear transformation or run Nonlinear transformation and re-evaluate the alignment.

   Linear Transformation options

   • Lock sectioning angle: Check to hold the sectioning angle constant

     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 angles identified in parts of the brain with less contrast and more homogeneous anatomies.

   • Lock relative scale: Check to use the relative scale (compared to the atlas) identified for the reference section and keep the registration scale parameter constant while running a linear registration.

   Nonlinear Transformation options

   Nonlinear transformation optimizes a transform that is defined by a grid of parameters that are spatially aligned to the experimental image.

   You can run nonlinear registration multiple times with different settings, and the method will start from the previous result. So, for example, you could do your own multiple resolution registration by running one level at a series of decreasing target spacings.

   • Method

     • SyN: We recommend using the symmetric normalization method SyN in most cases. Its grid is typically finer and the parameters directly describe the displacement field. The SyN method has the major advantages of building smooth, invertible, topology-preserving displacement fields.

     • BSpline: With the BSpline method, the transform is described by displacement of control points with smooth interpolation between control points provided by BSpline basis function interpolation. This typically results in a relatively coarse grid.

   • Target spacing: Controls the resolution of the final level of the registration. Finer resolutions could potentially improve accuracy at the cost of increased memory, storage, and computation time. For a 25 µm voxel spacing image, 100 µm target spacing is typically a good balance of good results and computer resources.

   • Levels: Controls how many levels of resolution to use during registration. More levels can be helpful to handle larger scale distortion; fewer levels can be used to refine already accurate registrations. We typically recommend using 3 levels the first time that you run Nonlinear registration, then 1 or 2 levels for subsequent runs.

   • Iterations: Controls the number of optimization iterations to run at each level of resolution; we recommend 128 iterations as a starting point in many cases. Like the other settings, higher values take more time and resources than lower settings.

D. 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 set by clicking Registration transforms on the Registration ribbon, then Save transform set from the drop-down menu. The transform set includes atlas-transformation information for each section that was registered.

E. Identifying anatomical structures

See also 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.

 

Volume Registration tools reference

Following are descriptions of the tools available for registering an image volume to a brain atlas.

These tools are in the Registration menu in the Register Volume to Atlas panel.

They are organized in the tables below as they appear in NeuroInfo, which represents the generally recommended order for use. Note, however, that these tools are flexible and can be used in different sequences, depending on the experiment and user preference/experience.

Volume Tools: Tools for registering images of intact (unsectioned) brain to the reference-brain atlas.

Landmarks Not available yet, but coming soon: Tools for marking structures of interest (fiducial points) in your images to direct their registration to the atlas.