TITLE: Supporting protocol for use-case 2: Multi-modal 3D image reconstruction in "M2aia - Interactive, fast and memory efficient analysis of 2D and 3D multi-modal mass spectrometry imaging data"
AUTHORS: Jonas Cordes

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We provide 10 consecutive brain slices of an APP NL-G-F mouse model [1], imaging both lipid and peptide features. The objective of use-case described in the following is to demonstrate the applicability of M²aia v2021.01.01 [2,3] for mono- and multi-modal 3D image reconstructions by showing how to embed the peptide information into the lipid structural context in three dimensions using M²aia.</div><div class = "text-block">To demonstrate mono-modal 3D reconstruction, all 10 slices of the lipid dataset are loaded into M²aia and used for slice-wise reconstruction of 3D image stack. For multi-modal 3D-reconstruction, the peptide dataset will be pair-wise registered with the respective lipid slices of the previously reconstructed 3D lipid image stack.</div><div class = "text-block"><span>[1] </span><a href="http://dx.doi.org/10.5524/100909" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Cordes J; Enzlein T; Marsching C; Hinze M; Engelhardt S; Hopf C; Wolf I (2021): Supporting data for "M²aia - Interactive, fast and memory efficient analysis of 2D and 3D multi-modal mass spectrometry imaging data" GigaScience Database. http://dx.doi.org/10.5524/100909</span></a><span style = ":;"></span></div><div class = "text-block"><span>[2] </span><a href="http://github.com/jtfcordes/m2aia" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">http://github.com/jtfcordes/m2aia</span></a></div><div class = "text-block"><span>[3] </span><a href="https://github.com/jtfcordes/m2aia/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">M²aia (RRID:SCR_019324): MSI applications for interactive analysis in MITK</span></a><span style = ":;"></span></div></div>

[STEPS]
?. [3D reconstruction]
Open the Preferences of M²aia, e.g. from the menu: Window > Preferences  or Ctrl+P.Select M²aia's preference page and set the paths to the elastix and transformix executables, respectively.
?. [3D reconstruction]
Open the Data view, e.g. from the menu: Window > Show View > DataOpen the Imaging tab in the Data view.enable TIC normalizationdisable smoothingdisable baseline correction
?. [3D reconstruction]
Load the dataset into M²aia: File > Open File  or Ctrl + O.Open all lipid files at once:Ensure, that all *.nrrd and *.ibd files are available at the same directory level.
?. [3D reconstruction]
Load the dataset into M²aia: File > Open File  or Ctrl + O.Open all peptide files at once:Ensure, that all *.nrrd and *.ibd files are available at the same directory level.
?. [3D reconstruction]
Setup Standard Display for 2D visualization (optional).When the export utility saves the original world coordinate origin (with respect to the MSI acquisition device), the image origin is available in world coordinates. If several such datasets were loaded at the same time, they may appear stacked or may be offset.Open the Options tab in the Data view. Set Rows to 4 in the Tile images box and hit Apply.Maximize the top view display by pressing the blue square at the top right of the display menu.
?. [3D reconstruction]
Open the Data view, e.g. from the menu: Window > Show View > Data or click on the Data view tab.Navigate to the Ion imaging box and create ion images at m/z 2250 +/- 50 Da. and m/z 865 +/- 50 ppm.
?. [3D reconstruction]
Pre-align images (optional). This can increase the registration accuracy.Open the Position view: Window > Show View > Position.Open the Data Manager view: Window > Show View > Data Manager.Select image nodes in the Data Manager and use the Position view to align the images.
?. [3D reconstruction]
Open the Reconstruction 3D view, e.g. from the menu: Window > Show View > Reconstruction 3DSelect the Image selection tab.Drag&Drop all peptide images to the right table.Select a reference slice in the left table (with lipid images) and click "Start stacking".Progress can be traced in the console window.
Two new nodes are listed in Data Manager (Stack_0 -> lipids, Stack_1 -> peptides).
?. [3D reconstruction]
Minimize the top view again to see all render windows by pressing the blue square at the top right of the display menu.
?. [3D reconstruction]
Select one or both nodes of the  image stacks in the Data Manager and perform a Reinit and "Show only selected nodes" by right-clicking on the node(s).
?. [3D reconstruction]
In the Data view, select the Initialization  tab and enable Only visible.Now perform 3D ion imaging by using the Spectrum view or the Ion imaging utilities (see step 6.).