TITLE: Labeling of Zebrafish Organs and Substructures in Light Data in Amira 3D Classic Segmentation Workroom
AUTHORS: Alyson Petruncio, Aparna Dev, Virginia Ruetten, Gudrun Ihrke, Aubrey Weigel, Project Technical Resources, CellMap Project Team

[DESCRIPTION]
Intended for use on light microscopy datasets of larval zebrafish ubiquitously expressing soma- and membrane-based fluorescent proteins. Methods are subject to change based on ultimate segmentation goals and expansion into variable light microscopy datasets.

Segmentation Summary
Amira Project and Segmentation Setup
Mask Segmentation
Overview of Class Types
Glossary and Segmentation Techniques

See protocol in References for general Amira segmentation tips.

[STEPS]
SECTION: Amira Project and Segmentation Setup
1. 
After opening a new Amira project, select File > Open Data, or click+drag the raw data tiff to the empty space under the Project tab. Confirm coordinates and voxel size in the Image Read Parameters window. 

For higher label resolution: Select the arrow of the raw data object in the Project tab. In the window search bar, type ‘resample’. Select the Resample object. With the Resample object selected, adjust Voxel Size: under Properties to half of the original resolution value. Select Apply, and select the newly generated raw data object as the new working raw data object.

Select the Segmentation tab to automatically generate a label field. Confirm Image: dropdown under Segmentation Editor is the raw data. Under Materials, ensure the Exterior (Not Assigned) material remains unlocked. The existing Inside material may be renamed and used as the first cell segmentation material. 
SECTION: Mask Segmentation
2. Before segmenting organ classes, creating a mask of the zebrafish sample will aid in determining organ segmentation boundaries and later mesh visualization. 
SECTION: Overview of Class Types
3. All substructure and organ classes fall under a class type. Amira label fields may be organized by class type to track classes with similar patterns in the data.

Muscle 

Trunk Muscles
The fish contains two main truck muscles, the epaxial and hypaxial muscles separated by the transvese septum. 

Pectoral Girdle Muscles
Three muscles take their origin at the ventral tip of the posterior cleithrum, the first bony structure to emerge. This is just lateral to the caudal edge of the heart.
Abdominal muscle
The abdominal muscle (in the literature called posterior hypaxial muscle) sends long muscle fibers caudo-dorsally to join the ventral boundary of the hypaxial muscle where there is a fascial thickening. It attaches to the ventral side of the epaxial myotomes at the level of myotomes 4, 5, 6.
Sternohyoid muscle
The sternohyoid muscle courses anteriorly and is formed of two consecutive myotomes reaching the urohyal cartilage.
Sternobranchial muscle
Sternobranchial muscle (my name). The third muscle of the group courses dorsally and attaches to the base of the skull at the level of the base of 5th ceratobranchial arch. 

Branchial Muscles
Branchial Levator 5.
This muscle attaches at the caudal rim of the ear bone and project ventro-medially to attach to the fifth branchial arch. This is one of the muscles which keep together the cranium and the jaw. 

 

Cartilage

Neurocranium
The cranium is formed of different cartilaginous structures. The base of the brain can be divided into the prechordal elements and the para and postchordal elements. The ethmoid plate connects to the basal plate (also parachordal cartilage) via trabeculae. This basal plate abuts the notochord. The basicapsule emanates from the basal plate encircling the base of the developing ear. It comprises the anterior, lateral and posterior basicapsular commissure. This is also sometimes referred to as the auditory capsule. At the anterior portion of the basal plate a perforated fossa is present formed by lateral commissure through which vessels run. The basal plate extends caudal to merge with the occipital plate which extends two dorsal elements, the occipital arches. 
The cleithrum, a prominent vertical structure, acts as a central element of the zebrafish skeletal system. 
 
Notochord. 
A structural and signaling element located directly under the spinal cord containing large vacuolated cells surrounded by notochord sheath cells.

The pharyngeal teeth pad is situated at the upper region of the pharynx.

Gills are protected by the operculum, a covering that shields the delicate structures underneath. The gills themselves are individually labeled.
 

The pectoral fins are anterior limbs situated on each side of the fish.
 

Sensory organs include the eyes, with distinct labels for structures such as the iris and exterior, and the ears, which encompass the entire organ along with its internal compartments. Hair cells line the base of the ear in uniform lines.
 

Organs include:
Gastrointestinal tract: starts at the mouth, extending along the body cavity.
Heart: includes the ventricle, a large, heavily lined cavity, the atrium, the more dorsal/caudal, thin cavity, and ductus arteriosus, the muscular outlet. 
Kidney: looping structure located in the midline and extending along the body cavity.
Swim Bladder: characterized by its large size and dark interior pixels.
Gallbladder: notable for its contrast between the muted interior and bright surrounding membrane.
Pancreas: divided into exocrine regions, featuring wiry tubules near the swim bladder, and endocrine clusters of cells adjacent to the exocrine area.
Liver: identified by its dark border and unique pattern, distinct from the surrounding muscle.
 

Nervous system includes:
Spinal Cord: Extends out from the brain, surrounded by epaxial muscle and dorsal to the notochord.
Pineal gland: Located dorsally in the midline of the brain.
Habenulae: A paired structure situated lateral to the pineal gland.
Brain: Includes internal features while excluding surrounding muscle and nearby ears.
Posterior lateral line ganglion: A dorsal ganglion directly behind the ear.
Sympathetic Nervous System/Adrenals: Bright clusters of cells located near blood vessels.
 

 

SECTION: Glossary and Segmentation Techniques
4. For all classes, carefully annotate intermittent slices of objects down the XY plane, then interpolate (ctrl+i) and add selection to the appropriate class material. Check the object segmentation in XZ and YZ planes to ensure segmentation does not overlap with any other data patterns relating to a different class. 

Note: Smoothing may cause excessive material shrinking in datasets where the Z dimension is relatively short. 

In many instances, multiple class patterns will have signal overlap in the same channel (Figure 12). Visible muscle fibers and cartilage should always be segmented in instances of overlap with other classes.
 

SECTION: Mask Segmentation
2.1. Under the Segmentation tab, adjust the 2D data contrast range to the data histogram by selecting the 'Data Histogram' option in the Display Control window (Figure 1). This will present the general shape of the sample, which can be roughly segmented in XY, interpolated through the Z plane, and globally smoothed (Figure 2). The final fish mask should include all signal in the data (Figure 3).

 

 

 

SECTION: Mask Segmentation
2.2. Create a new label field for each class type (see Overview of Organ Class Types) to organize each class and avoid overwriting segmentations. 

Note: Since class types have a range of signal across multiple light channels, multiple channel datasets were used across class types. The best channel dataset was chosen for each class type segmentation, and segmentations in different channels were overlayed after Amira export.
SECTION: Glossary and Segmentation Techniques
4.1. Muscle

Epaxial and hypaxial muscles

Clearly defined dorsal and ventral bundles (top and bottom) are segmented separately and avoid the spinal cord and notochord, respectively.
 

Abdominal muscle

More dispersed, streaking pattern extends across the ventral surface up towards the hypaxial muscles. Small, bright nerve signals can be included in the more sparse muscle patterns.
 


Pectoral girdle muscles and cranial muscles
Short, small muscle bundles that wrap around organs like the gut and surround the gills, heart, and mouth.
 

SECTION: Glossary and Segmentation Techniques
4.2. Cartilage

Fragments run throughout the body, primarly surrounding the ears, brain, and gills. Texture pattern is hollow, porous, and bright.
 

Cleithrum
Small, bright bars pass behind the ears and gills, extending towards pectoral fins. The cleithrum segments do not have a distinct cartilage signal pattern.
 

Pharyngeal teeth pad
Packed, bright, and webbed signal pattern towards the back of the pharynx, distinct from surrounding gut folds.
 

SECTION: Glossary and Segmentation Techniques
4.3. Gills

Two sets of four gills have a soft, gray dotted signal pattern, with gill 4 (yellow) being closest to the cleithrum and gill 1 (green) being closest to the eye.
 


SECTION: Glossary and Segmentation Techniques
4.4. Pectoral Fins
The pectoral fins have small muscle fiber and cartilage signal pattern. Muscle and cartilage may be segmented separately and combined to create the total pectoral fin segmentation.
 

SECTION: Glossary and Segmentation Techniques
4.5. Sensory Organs

Eye
The most anterior organ, the eye has a soft, radial signal pattern with a thin, bright membrane.
 

Ear and auditory hair cells
Ear signal pattern is primarily empty compartments with hair cells (blue) lining the base of the ear. 
 


SECTION: Glossary and Segmentation Techniques
4.6. Organs

Gastrointestinal tract

Extending from mouth to anus, the gut texture is hollow with irregular folds of soft, gray dots.
 

Heart

The ductus arteriosus (red) is the smallest compartment and closest to the eye with a signal pattern of soft gray dots throughout, the ventricle (blue) is most heavily lined with a visible valve separating the ventricle from the atrium (green), which is thinly lined with visible blood cells dispersing out of the compartment.
 


Kidney nephron

The nephron and glomerulus with a thick, soft gray signal pattern that bundle dorsal to the liver, caudal to the ears, and dorsal to the gallbladder. Ducts are segmented separately to generally track nephric structure.
 

Swim bladder

A mainly empty compartment with minimal signal, the swim bladder takes up a large portion of the body cavity and is directly above the gut.
 

Gallbladder

Surrounded by the liver, the gallbladder often has a dark, soft interior signal pattern, lined with brighter, sparse dotted signal. While generally spherical, some invagination may be present.
 

Swim-bladder duct and gall bladder duct

 



Liver

Wedged between the gut and heart, the unique liver signal texture appears as a gradient of intensity and surrounds the gallbladder. Adjust data contrast as needed in order to include all liver signal.
 

SECTION: Glossary and Segmentation Techniques
4.7. Nervous System and Notochord

Notochord

Surrounded by bright nerve signal, the notochord passes between epaxial and hypaxial muscles and into the main body cavity. Signal pattern is porous with thin bright strands passing vertically through a cross-section.
 


Spinal cord

Directly dorsal and parallel to the notochord, the spinal cord has a varied soft gray dotted signal pattern with some bright nerve signal dispersed throughout. 
 


Pineal gland

Smaller, granular signal pattern is surrounded by slightly large gray cells located dorsally, between the eyes. The gland is physically separate from other brain regions.
 

Habenula

Both lobes flank the pineal gland, with identical signal patterns to one another; dark soft interior is lined with slightly lighter gray cells.
 


Brain

Composed of multiple signal patterns, the brain includes most gray signal in the dorsal cephalic region of the dataset. The brain connects directly to the spinal cord, which shares a similar signal pattern, and includes the pineal gland and habenula.
 


Nodose Ganglion

Bundle of soft, gray dots clustered behind the ear, forming an upside down triangle shape. 
 


Autonomic Nervous System

Saturated signal is thresholded out using the Magic Wand tool (3) under the Segmentation tab. Once selection is added to a material, noise and excess signal is removed from the segmentation and significant nerve signals are refined.
 
Figure 31.