Intro

Your task is to identify specific muscle cells in an image of C. elegans in a process called annotation.  This includes naming cells, adding comments, and editing the segmentation of cells as needed.

 

Basic Information

C. elegans muscle cells

The muscle cells that will be annotated are body wall muscle (bwm) cells, colored green. These cells are located on the circumference of the worm, and are divided into four groups, corresponding to each quadrant.  Quadrants are Ventral Left (VL), Ventral Right (VR), Dorsal Left (DL), and Dorsal Right (DR).  The DL, DR, and VR quadrants all contain 24 bwm cells, while the VL quadrant only contains 23 bwm cells.

 

VANO

VANO is the program used to manually annotate worms.  To view a worm in VANO, double click the VANO icon, and click “No” on the pop-up window.  Next drag a .ano or a .new.ano file onto the following window.  When loading is complete, six views of the worm will appear.  The upper three of these are images of a worm taken through a microscope.  These three views are cross sections along three perpendicular planes.  Two of these views are long rectangles, of these, the upper one is the XY plane, and the lower is the XZ plane.  It is possible to scroll through each of these planes.  The third view, the square on the right, is a cross section of the YZ plane.  Scrolling in each of these views is done along the perpendicular axis.  For example, in the XY plane, scrolling is done along the Z axis. 

On the bottom right of the window, the color channels visible in the top three windows can be changed.  Looking at different channels separately is useful in evaluating segmentation accuracy.  Often the Blue and Blue (gray) channels can help show when multiple distinct cells in the segmentation are only a single cell in the worm and vice versa.

The bottom three views correspond to those on the top, and function similarly.  However, these views display the segmentation of the worm by the computer.  The colors of these cells are random, and can be changed by through the keyboard shortcut Shift+C.  Changing the colors of these cells can be useful in determining if cells have been fused, or if they need to be merged.  These cells can be edited to better match the pictures taken of the actual worm.

 

Annotation

When you first open the image...

The anterior and posterior (A/P) ends of the worm can be distinguished through a comparison of the two ends of the worm--the posterior end of the worm tapers, while the anterior end is rounded and thicker. The best view of this is seen in the XY plane.

To determine the dorsal/ventral orientation of the worm, look for the ventral nerve cord.  The ventral nerve cord consists of a line of small round blue nuclei that stretches the length of the worm. Look at the XY plane. The ventral nerve cord is either located on the top of the worm or the bottom of the worm in this view. Scroll up and down on the Z plane until you see a horizontal line of small round blue cells. This is usually best seen at the posterior half of the worm. When you think you have located the ventral nerve cord, right click one of the ventral nerve cord cells and view this worm on the XZ plane to make sure you see a line of small round blue cells just to double check.

Now that the A/P and D/V axes have been established, the next step is to deduce the left and right sides of the worm. Look at the YZ plane. The top and bottom are either ventral or dorsal. The left side of the XY plane (tail or head depending on the image) is always towards you and “projecting” out of the screen in the YZ plane and the right side of the XY plane (the other end of the image) is thought to “project“ into the screen further away from you in the YZ plane. Using this YZ plane, orient yourself mentally or physically as the worm and establish the worm’s left and right.  Using these axes, divide the YZ plane mentally into four quadrants (DL, DR, VL, VR).

 

Finding Cells

You are looking for green body wall muscle cells. Please note however, that the “green” color of these cells will often vary. Most of the body wall muscle cells are a strong glowing green color. However, some cells may look white or may even have a little pink in them. Use your discretion in picking out which are body wall muscle cells and which are not. Also, rarely, though there seems to be something green on the screen, this does not always necessarily mean there is a nucleus there. GFP may have gotten to the mitochondria so check to make sure a nucleus is present (if questionable) by switching to the Blue(gray) color format.

Scroll from the head towards the posterior in the YZ view, and look for green body wall muscle cells in the top three views.  These cells often come in left-right pairs, especially in the head.  When a cell appears, find its corresponding segmented cell in the lower three views.  Check in all three windows to ensure that the segmentation is correct and that no cells need to be merged or are fused.  If this is the case, label the cell according to its location in the worm, and comment it appropriately.  If, instead, the muscle cell either has no equivalent segmented cell, is segmented into multiple cells, or is fused with another cell, you will need to edit the segmentation.  Once this has been accomplished and comments have been added if necessary, continue on to the next cell in the quadrant.

In general, odd-numbered cells in the same quadrant are more central (closer to the top or  bottom of the YZ view), while even-numbered cells are more lateral (closer to the sides).  A notable exception to this pattern is the first two cells in each quadrant, where cell 1 is always more lateral than cell 2.  Here, even if the more lateral cell is more posterior, it should still be labeled as cell 1. Also, the pattern does not always continue in last 8 or so cells in each quadrant.  Also, on the dorsal side, often only one of the quadrants clearly follows the pattern.

Annotating the ventral side should be fairly straightforward until around cell 16.  The bwm cell can be difficult to distinguish form the muscles of the vulva.  Both of the ventral quadrants contain four vulva muscles arranged somewhat symmetrically in two pairs.  Two of the muscles are relatively large, while the other two are small.  Bwm cell 16 is usually either between the two pairs or past them.  When these four muscles have been identified, the next muscle cell will be bwm cell 16.  After finding cell 16, you should be able to continue all the way to bwmVL23 and bwmVR24, completing the ventral half of the worm.

In annotating the dorsal side, be aware that cells in the DR quadrant are often anterior to cells of the same number in the DL quadrant.  The DR cells often get 1-2 cells ahead of the DL cells.  This is due to the depressor (dep) muscle, which is on the dorsal side of the worm at the posterior end, past all the dorsal bwm cells. To distinguish the depressor muscle from bwmDR24, look at the sizes of the two last green cells on the DR side. The depressor muscle is usually smaller than DR24.

 

When Worm Cells are Missing...

Usually this problem results when a muscle cell fails to produce GFP.  If this happens, check for possible candidates in an appropriate position among the other cells as one of these may be the absent muscle cell.  It is rare, but not unheard of for a muscle cell not to form; this is most frequently observed in posterior dorsal muscles.

 

Naming Cells

The naming convention for body wall muscle cells consists of three parts.  First is “bwm”, which stands for body wall muscle.   Next, indicate the quadrant within the worm; the four options for these are VL (ventral left), VR (ventral right), DL (dorsal left), and DR (dorsal right).  Finally, cells are given a number which describes their rank within the quadrant in order from anterior to posterior.  For example, the 3rd green cell in the Dorsal Left quadrant should be labeled bwmDL3.  Similarly, the 21st body wall muscle cell in the Ventral Right quadrant should be labeled bwmVR21.

The depressor muscle is named simply “dep”.

 

Adding Comments

    When a cell is named, a comment must be added to indicate that the cell has been manually annotated.  Shorthand for comments includes the following:

Š   s -- Automatic annotation Satisfies

Š   m -- Automatic annotation Missed this cell

Š   r -- Cell needed to be Renamed

Š   f -- This nucleus is Fused with others

Note that the first three comments refer to the annotation, while the fourth refers to the segmentation.  If cells have been added, merged, or split, no comment beyond the automatic one is necessary.  Only one comment should be written, with segmentation comments taking precedence over annotation comments.

 

Editing Segmentation

When no cell is selected, the ‘e’ key allows the user to add a cell.  If a cell is selected, the ‘e’ key will allow the user to split the cell or merge it with others.

Adding a Cell

Adding a cell is necessary when the segmentation did not create a cell corresponding with a worm cell.  To add a cell, click on where the center of the cell should be and press the “e” key.  Yes, you want to add a cell.  Tweak the coordinates as needed and change the radius so that the new cell fits the worm cell as much as possible.

Merging Cells

When a worm cell was segmented incorrectly into multiple pieces, it is necessary to merge them.  To merge cells, click on one of the cells that require merging, and press the “e” key.  Select the “Merge cells” option, and change the x, y, and z coordinates until the Focus Cross Lines are on another cell that must be merged.  Click “OK”, and tell the program if you need to merge more cells.  Repeat until all incorrect cells have been merged.  

Splitting a Cell

Splitting a cell is only necessary if multiple muscle cells have been segmented into a single cell.  If it is only a muscle cell fused with another non-muscle cell (as is often the case with fused cells), just label the cell as if it were a normal muscle cell, but type “f” into the comments to indicate that cells were fused.  However, if multiple muscle cells are indeed fused, then click on the faulty segmentation, press “e”, and select the option “Split this cell”.  You will be asked to define coordinates and radii for the improperly segmented cells, repeating this until each of the worm cells corresponds to a distinct segmentation.  When the process is complete, the fused cell is automatically deleted.  Alternatively, if there are more than two cells that have been fused, it may be easier to split the original segmentation into just two cells, and then add other cells separately as needed.

 

Other Stuff

.ano vs. .new.ano files

The difference between files with the suffix .ano and those with is suffix .new.ano is that those ending with just .ano have not been automatically annotated.  Hence it is not necessary to comment on the accuracy of the automatic annotation when using .ano files.  Comments should still be added if cells are fused.

 

Tips for Beginners

Š   Increasing the brightness in the color of your display makes seeing the cells easier.

Š   The Stickies application provides an easy way to keep track of the ventral, dorsal, left and right sides of the worm in the YZ view. 

Š   Checklists can save time and assist you in remembering the number of the current cell.

 

Tips For Advanced Users

Š   It can be helpful to work on the left and right quadrants of each half of the worm simultaneously.

 

If all else fails, try contacting Xiao Liu at xiaoliu2@stanford.edu.