Tutorial for Joining Serial Section Tomograms with eTomo
(IMOD Version 3.11)

Cindi L. Schwartz

Boulder Laboratory for 3D Electron Microscopy of Cells


I. Initial steps

This tutorial provides a step-by-step guide to joining together tomograms from serial sections, using a small sample data set containing three volumes to be joined.  Before you try to join your own tomograms, please refer to the more comprehensive Guide to Joining Tomograms for additional detail about the decisions and options at each step. 

To get started, download the sample data set from our web site.  Unpack the data by typing:

        tar -xzf joinTutorialData.tar.gz

This will create a directory, joinTutorialData, containing the three serial volumes, named basal-1.rec, basal-2.rec, and basal-3.rec.

II.  eTomo Setup

To start eTomo, at the command line type:     etomo

This will load the eTomo's Front Page. To get to the join setup page press the New Join button.



This will load the Join Interface shown below. The interface is divided into three panels: Setup, Align, Join, Model, and Rejoin. The Setup panel allows you to identify which serial tomograms you would like to join, define the surfaces at which they should be joined, flip or rotate the volumes relative to each other, and extract sample slices from each serial tomogram so that you can visualize the boundaries between the serial tomograms and align them. You will notice that you cannot use the Align or Join tabs until you have finished filling in the Setup information and calculated your sample file.



To get started, select your Working directory and Root name for output file. In this example, we used a directory called join and used unicross for the root name. You can enter the Working directory by clicking on the yellow file selection button associated with the Working directory field, or by typing in the directory path and file name directly in the field.  Since you already have all of the individual reconstructions in the joinTutorialData directory, your can make that be your working directory.



Next, you need to select the serial tomograms you want to join by pressing the Add Section button. It will take you to your Working directory and allow you to select a file for joining. (Note that you are not required to put your serial tomograms in your working directory. eTomo will keep track of where your files are located). You must add each serial tomogram individually. The initial order is not important because you can change the order later.



Once you have input all three datasets, click on the arrow in the Order subsection to highlight unicross1.rec. Now click on the Open in 3dmod button. This will open unicross1.rec using 3dmod. These tutorial datasets are small in size; however, for future data, you may need to use the binning option to view all of your serial tomograms at once. Now, open the other two serial tomograms with the Open in 3dmod button. Once all three tomograms are loaded, you will typically movie through them to figure out the order of the serial tomograms and what slices you would like to use to create the samples that are used in the aligning process. It is important to note that Bottom and Top are used in the join software to denote that the 'top' of the section is the part of the tomogram that matches up with the 'bottom' of the next section. 'Top' and 'bottom' DO NOT refer to the high Z and low Z portions of the tomogram. So, the top of a section can be at either high or low Z. The join programs will take care of any inversions in Z, both in extracting sample slices and in assembling the final volume. The 'Bottom' entry in the first row and the 'Top' entry in the last row are not necessary because they don't match up to another tomogram. To determine the Sample Slices you need, see the entries we have used below. The goal here is to find a small subsection of the ends of each serial tomogram that you can use to align. Note that the Bottom of unicross2.rec starts at Z=5. This is because the gold on the surface does not give you any information to align with, so you need to go deeper into the tomogram. You can also select the Final Start and Final End of each tomogram now, but you will get a chance to change this later on in the Join tab.  These numbers determine which slices will be placed into the final joined volume.



Once you are satisfied with your decisions, press the Make Samples button. This creates unicross.sample which will be used in conjunction with Midas to visually align the serial tomograms together. After unicross.sample is created, you can now access the Align and Join tabs. You will notice that you can always come back and use Change Setup to pick new Sample Slices and start over.



III.  Aligning the Sample Slices

The Align tab allows you to align the serial tomograms before joining them together. It is a good idea to Open Sample Averages in 3dmod. By toggling through Z, you can see whether you have chosen the correct Sample Slices. For instance, toggle between Z=1 and Z=2. You can see these are a close match by looking at the cluster of vesicles in the lower right corner, but they are not aligned to each other. Now you can choose to try auto alignment or manual alignment of the serial tomograms. These tutorial tomograms lend themselves well to auto alignment. Click on Initial Auto Alignment and wait for done to appear in the process bar. Now, click on Midas. This will load the program midas which allows you to see how well the auto alignment worked.



The first thing you will see in Midas is an overlay view showing contrasting magenta and green colors that help you align images. What is first displayed is the 'top' of unicross1.rec aligned with the 'bottom' of unicross2.rec. Midas considers each tomogram as a single 'chunk' of slices, so it is actually showing the alignment of the bottom of 'chunk 2' to the top of 'chunk 1'.  You can use the Toggle Ref/Cur button to see the alignment better by toggling between the images, and the PageUp and PageDown keys can also be used to see one image at a time. If the sample slices being displayed do not have a clear image of the structures at the surface of the volume, then you can step deeper into either section by reducing the Reference Sec. number or increasing the Current Sec. number.  You can play around with Midas and try to align things manually using the program. Midas lets you shift, rotate, or stretch the current image with the left, middle, and right mouse buttons, respectively.  Otherwise, make the Current Chunk 3 to align the next pair of  tomograms.



Now you see the 'top' of unicross2.rec aligned with the 'bottom' of unicross3.rec. You can also try and manually improve this alignment. If you make changes you want to keep, be sure to save them under File-Save Transforms.



If you make changes and realize that they aren't as good as the auto alignment, you can always use the Revert to No Transforms button and start over.



IV.  Joining the Serial Tomograms

Once you are happy with the alignments, you can now move to the Join tab. First, you can pick the starting and ending slices of each serial tomogram you would like to keep for the final volume. Again, you are given the option to view each tomogram by using the Open in/Raise 3dmod button. These serial tomograms provide an example where using the Get Max Size and Shift button can be useful. The program will calculate the Size and Shift in X and Y required to keep all original data in the final volume and automatically put the numbers in the proper fields. Now you can use the Trial Join button to get a very quick idea of what your final volume will look like. This is very useful with larger datasets because the join process can take a long time. Once the Trial Join is finished, Open Trial in 3dmod to view it. The Get Subarea Size And Shift button is used if you wish to crop the final volume to a particular area with the rubberband feature in 3dmod. If you are happy with your Trial Join, click Finish Join to create your final volume. This could take a long time depending on the size of your original datasets. Once the program has finished, use Open in 3dmod to see your final volume called basal.join. Again, there is no need to use the binning feature with these data, but it will likely be necessary for real datasets.