Volume registration

This tutorial will show how to generate a project from multimodal volume images and register them.

To download the Electron microscopy (EM) dataset, head to https://www.ebi.ac.uk/empiar/EMPIAR-11537/, make sure to de-select all data except #4 “Downscaled (20nm) aligned FIB SEM stack resliced to match the Airyscan dataset” and click the individual download button (choose HTTP).
The Fluorescence microscopy (FM) dataset is stored at https://www.ebi.ac.uk/biostudies/bioimages/studies/S-BSST1075. You can download the raw data file directly from here.

open the LM data in Fiji using the BioFormats Importer plugin. (Plugins > Bio-Formats > Bio-Formats Importer)
you should get a Hyperstack image with 4 channels. (MitoTracker, Agglutinin, GFP-TGN46, and Hoechst)
adjust the lookup table for each channel to fit the fluorophore. (Image > Lookup Tables)
Currently, the MoBIE project creator only supports single channels, so we have to split the fluorescence stack into separate channels using Image > Color > Split Channels.

create a new MoBIE project: type “mobie” in the search bar or choose Plugins > MoBIE > Create > Create New MoBIE project...

name the project as you like and create it in a local directory.
add a new dataset, name it as you like. Do not tick “2D”.
select the first channel image in Fiji
add a new source current displayed image, call it appropriately (channels are sorted inverse), make sure the Image type is “Image” and do not tick make view exclusive.
choose the selection group name as “fluorescence” or “FM”
open the MoBIE project to see how the scaling and LUT are transferred

continue the same way with the other channels, adding them to the same dataset and selection group.
open the other channel views using the MoBIE UI and explore the multi-channel volume
right click into the multi-channel viewer and Save current view
Save as new view, save to projcect
Call the view something like “all channels” and make it part of the “FM” group
close MoBIE

open the EM data in Fiji
add the volume to MoBIE under the selection group “EM” (make new selection group) to the same dataset. The format conversion can take a couple of minutes.
open the MoBIE project and view your multichannel FM and EM together
make all fluorescence channels but the Hoechst invisible by clicking on S
change contrast and transparency settings for the relevant sources (B)

right click the image “registration manual”, select the EM image as we want to keep the multi-channels where they are.
Click Start manual transform
use the right mouse button to drag the image around.
Press the z key to make sure rotations are around the viewing axis and use the right and left arrow keys to rotate.
You can also use the mouse wheel to translate in z, and the up and down arrows to scale (dangerous).
If necessary adjust brightness or opacity of the images using MoBIE’s main window (B).
click cancel manual Transform to undo the translation and bring the image back to its original position.
click Accept manual Transform to make it permanent and store the view into the project. This will only save the transformed EM source. Create a new selection group “registered”.
clear the viewer
open the registered view an all FM channels. Check their registration by scrolling through the volume.

push CTRL + y to view the volume from the side. Make sure te mouse pointer is in the center of the feature, as it determines the viewer’s rotation axis.
repeat the manual registration of the EM volume until your registration matches in all axes.
You could also use the mitochondria or another channel for refining it.

Note that when clicking Accept manual transform the single transformed source will be saved as a new view. In case you like to transform more.

To save a complete view with all sources (channels + EM), do right click > save current view and save your view as desired.