cochleogram 0.8.1

Naming conventions for LIF files

All Z-stacks for a single cochlea should be stored in a single LIF file. Files should be named with the identifier (e.g., animal ID and ear) followed by the label for each channel in the order the channels were imaged. For example:

B009-8L-GluR2-CtBP2-MyosinVIIa

Inside each file, the pieces must be numbered sequentially from base (hook) to apex. If more than one image is required for a piece, use letters for the suffixes (i.e., “piece_2a”, “piece_2b”, etc.). The order in which the images for a single piece are labeled does not matter since the program will automatically align them based on the stage coordinates stored in the file.

To exclude an image, add an underscore to the beginning of the stack name, e.g.:

_piece_2a_high_power

Naming conventions for CZI data

All Z-stacks for a single cochlea should be stored in a single folder. Folders should be named with the identifier (e.g., animal ID and ear) followed by the label for each channel in the order the channels were imaged. For example:

B009-8L-GluR2-CtBP2-MyosinVIIa

Inside teh folder, each Z-stack should be numbered sequentially from base (hook) to apex. If more than one image is required for a piece (e.g., because you are not using tile mode), use letters for the suffixes (i.e., “piece_2a”, “piece_2b”, etc.). The order in which the images for a single piece are labeled does not matter since the program will automatically align them based on the stage coordinates stored in the file. An example of the filenames that could be found in the folder:

BP1-FL_piece_1.czi
BP1-FL_piece_2.czi
BP1-FL_piece_3.czi
BP1-FL_piece_4a.czi
BP1-FL_piece_4b.czi
BP1-FL_piece_5.czi

The underscore before and after piece is important. The pieces should be numbered sequentially from hook (starting at 1) to apex. If the field of view is too small to capture the full piece and you are not using tiling, you can add a letter suffix after the piece number (e.g., “a”, “b”, etc.).

To exclude an image, add an underscore to the beginning of the filename, e.g.:

_BP1-FL_piece_5.czi

Missing pieces

If a piece is missing, you can copy the image stacks for a matching piece from another file (for LIF files, you can use LAS X Office). To indicate that the piece is a copy, it must have the suffix _copied_<note>. For example, if you copy piece_4a and piece_4b from B009-8L to the file/folder containing data for B021-3L, the copied images should be named “piece_4a_copy_B009-8L” and “piece_4b_copy_B009-8L”. The note will appear in the composite generated for the frequency map.

Mouse interaction

left click

Select tile

left click + drag

Pan image

mouse wheel

Zoom in/out

Keyboard shortcuts

t

Switch to tile mode

i

Switch to IHC mode

1

Switch to OHC1 mode

2

Switch to OHC2 mode

3

Switch to OHC4 mode

4

Switch to extra mode

s

Select spiral tool

e

Select exclude tool

c

Select cell tool

n

Select next tile (tile mode only)

p

Select previous tile (tile mode only)

arrow keys

The behavior of the arrow keys will depend on whether tile mode is selected. If tile mode is selected, then the arrow keys will move the tile. If any other mode is selected, the arrow keys will pan the image (this can be useful when in spiral or cell mode to move through the cochlea when zoomed in). To move the tile (or pan the image) in smaller steps, hold down shift at the same time.

Analysis

Analysis requires the following steps:

• Aligning the tiles so that they overlap as accurately as possible.

• Tracing a spiral through each row of hair cells.

• Marking individual hair cells.

• Marking regions containing uninterpretable data.

Tools are provided to facilitate each step. Be sure that you are satisfied with the result of the current step before moving to the next step. Although you can go back and edit a previous step, it may affect your analysis (e.g., if you need to move a tile after marking hair cells, you may have to manually edit a large number of hair cells).

Tile mode

Start by selecting “tiles” from the edit buttons, then left-clicking to select the tile that is misaligned. Using the arrow keys, you can move the tile until it is properly aligned with the other tiles. If you need to move the tile in smaller steps, hold down the shift key at the same time as the arrow keys. It may be helpful to toggle “highlight selected” so that you get a transparent overlay. When in “highlight selected” mode, the currently selected tile will be shown with a red border.

left click

Select tile

mouse wheel

Zoom in/out

arrow keys

Move currently selected tile (large steps)

shift + arrow keys

Move currently selected tile (small steps)

n

Select next tile

p

Select previous tile

An “align tiles” tool is provided to facilitate this step. It uses an automated algorithm that attempts to align the tiles based on the correlation between the images (using the MyosinVIIa channel).

Spiral mode

Once you are satisfied with the alignment of the tiles, select “IHC” from the edit buttons and be sure the spiral tool to the right of the edit buttons are selected. The very first point you mark should be on the end of the row of hair cells facing the most basal region of the cochlea. This point will be highlighted with a red circle. If you realize you made a mistake, you can select a different point as the start of the spiral by control + right-clicking that point when in spiral mode.

You must select a minimum of four points to create the spiral. You can add points in between existing points and the spiral will be rerouted through those points. The algorithm assumes that the “next” point in the path is the one closest to it (i.e., the order in which you add the points does not matter).

Repeat the process for OHC1, OHC2, and OHC3. Be sure that the spiral bisects the nuclei (IHCs) or cuticular plate (OHCs) as that will facilitate the semi-automated algorithms implemented by the program to help mark hair cells.

right click

Add point

shift + right click

Remove point

control + right click

Set point as origin for spiral

Cell mode

After marking the spiral, run the algorithm to automatically detect cells. You can play with the settings (each time you run, it will delete the existing cells and create new ones). You will likely have to manually edit the automatically-detected cells. Select the cell tool and then use right click to add cells and shift + left click to delete cells.

right click

Add cell

shift + right click

Remove cell

ctrl + right click (only for IHCs)

Labels cell as supernumerary (See Rask-Andersen et al. 2017; Supernumerary human hair cells - signs of regeneration or impaired development? A field emission scanning electron microscopy study).

From time to time there will be a fourth row of OHCs. These should manually be identified by selecting “extra” for the cell you would like to edit and then adding the cells using the cell tool. Since the fourth row tends to be very short in length, you cannot mark a spiral or mark the region as excluded.

Exclude mode

Finally, go back through each row of hair cells. If there was a region you felt you could not intepret properly, select the exclude tool. Right-click the spiral at one end of the region then right-click again at the other end of the region you wish to exclude.

right click

Start region. Click again to end region.

shift + right click

Remove region under mouse cursor.

escape

Cancel current region.

Some additional tools are made available to facilitate this process:

• You can merge all excluded regions across the OHC spirals into a single set of excluded regions that apply to all OHC spirals (Combine OHC exclusions button).

• You can simplify a set of excluded regions for a particular spiral if they are overlapping (this will combine overlapping exclusion regions into a single exclusion region) using the Simplify exclusions button.