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 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:

   • 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.

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.

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.
		Setup: Tool for initializing registration by searching the entire atlas using the NeuroInfo machine-learning based automated search. When to use Setup: To conduct an initial registration when working with whole-brain images or when working with partial brain images, after Shifting the atlas to align with the partial brain and adjusting its Scale if needed How to use Setup Use automatic search in atlas: Check the box to direct NeuroInfo to search the atlas to establish the optical-sectioning angle, and scale relative to the atlas. Click Search to start the search and create a volume-registration transform that registers (or maps) the experimental image volume to the atlas. A progress window opens while the search runs and closes when it is complete.
		Register: Tools for refining the registration of the experimental image volume. Linear transformation: Applies a uniform set of transformation parameters to the entire 3D volume. We recommend trying linear transformation first and evaluating the results. Nonlinear transformation: Applies a transform that is non-uniform across the 3D volume. Nonlinear transformation can improve the registration in some cases; we recommend trying it if the results of linear transformation do not meet your expectations.
		Adjust: Tools for interactively (manually) registering or refining the registration by moving, scaling and deforming the atlas slice image so that it aligns with the displayed experimental image slice. Use the sliders to Shift, and adjust Rotation and Scale to transform the atlas slice so that it matches 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 Clear transform: Click to clear the atlas transform Note that one of the Shift sliders (which one depends on the specimen orientation) changes the brain-atlas plane (slice) displayed. Use it to display the location of the atlas brain that most closely matches the current experiment slice 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. Moving any of the sliders creates a transform.

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.

Procedure

A. Getting started

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

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

Note: There is a setting in Registration preferences that determines whether NeuroInfo software shares calibration files among users or only displays/edits the calibration files associated with the current User profile. 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 the thumbnail for 10 sec video showing how to expand the Current Calibration controls.)

2. Then do one of the following:

   • 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 in the following circumstances:

     • the first time you use NeuroInfo

     • for images of brains processed and/or imaged using a different protocol than those represented in the list of calibrations.

     • to work with images from other species

   • Change an existing calibration to reflect different processing or imaging protocols, or to test alternate calibration settings.

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

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.

   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:

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

   • Click Clear Transforms to clear the 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.

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.