DATA ACQUISITION & ANALYSIS

SPECIMEN PREPARATION

Phoronis muelleri, actinotrocha larvae:

Actinotrocha larvae of Phoronis muelleri were fixed with 2% glutaraldehyde in 0.1M sodium cacodylate buffer at a pH value of 7.4. Subsequently, specimens were post-fixed using 1%OsO4. For microCT scanning, a specimen was stained with I2KI (0.1% (w/v) elemental iodine (I2) and 0.2% (w/v) potassium iodide (KI) in distilled water) and embedded in 1.5% low melt Agarose in a plastic pipette tip. For light microscopic investigation, a specimen was embedded in Agar low viscosity resin (Agar Scientific, Stansted, UK) and serially sectioned using a Jumbo diamond knife (Diatome AG, Biel, Switzerland) on a Reichert UltraCut-S microtome with a section thickness of 0.5µm.

Oikopleura sp:

Specimens of Oikopleura sp. were fixed with 2% glutaraldehyde in 0.1M sodium cacodylate buffer at a pH value of 7.4. Subsequently, specimens were post-fixed using 1%OsO4. For microCT scanning, a specimen was stained with I2KI (0.1% (w/v) elemental iodine (I2) and 0.2% (w/v) potassium iodide (KI) in distilled water) and embedded in 1.5% low melt Agarose in a plastic pipette tip. For light microscopic investigation, a specimen was embedded in Agar low viscosity resin (Agar Scientific, Stansted, UK) and serially sectioned using a Jumbo diamond knife (Diatome AG, Biel, Switzerland) on a Reichert UltraCut-S microtome with a section thickness of 0.5µm.

Noctiluca scintillans:
Specimens of Noctiluca scintillans were fixed with 4% buffered formalin. For microCT scanning, a specimen was stained with I2KI (0.1% (w/v) elemental iodine (I2) and 0.2% (w/v) potassium iodide (KI) in distilled water) and mounted in distilled water in a plastic pipette tip. For light microscopic investigation, a specimen was post-fixed using 1%OsO4 and embedded in Agar low viscosity resin (Agar Scientific, Stansted, UK) and serially sectioned using a Jumbo diamond knife (Diatome AG, Biel, Switzerland) on a Reichert UltraCut-S microtome with a section thickness of 0.5µm.

Tomopteris helgolandica:

Specimens of Tomopteris helgolandica were fixed with 4% buffered formalin. For microCT scanning, a specimen was stained with I2KI (0.1% (w/v) elemental iodine (I2) and 0.2% (w/v) potassium iodide (KI) in distilled water) and mounted in distilled water in a plastic pipette tip.

MICROCT SCANNING
For microCT scanning of the different planktonic species, the plastic pipette tips containing the samples were vertically mounted in a microCT sample holder. MicroCT scans were acquired with an Xradia microXCT-400 (Carl Zeiss X-ray Microscopy, Pleasanton, CA). For the Tomopteris helgolandica specimen the 4x optical magnification lens was used for scanning, while all other samples (Phoronis muelleri actinotrocha larvae, Oikopleura sp., Noctiluca scintillans) were scanned using the 20x optical magnification lens. Samples were imaged at 40kVp source peak voltage and 200µA intensity. Depending on sample contrast, projections were recorded with 30-120s exposure time and an angular increment of 0.2°. Depending on sample size, isotropic voxel resolution of reconstructed volumes varied between 0.53µm and 1.98µm.

LIGHT MICROSCOPY
Serial light microscopical sections were imaged using an Aperio ScanScope slide scanner with a 20x objective. Single sections were cropped from whole slide images in Adobe Photoshop CS5 (Adobe, San José, CA). These light microscopic images and videos were used as motion and color references.

IMAGE SEGMENTATION
The image volumes generated by microCT and from serial light microscopical sections were used for segmentation of the whole animal outline and specific features of each animal, such as the alimentary tract. Image segmentation was performed with the 3D software package Amira 6.1. (FEI Visualization Sciences Group, Mérignac Cédex, France) by combining intensity-based and manual segmentation tools. Based on the image segmentation, polygon surface meshes were triangulated. Subsequently the surfaces were smoothed and re-meshed in order to obtain isotropic surfaces triangles.

3D-MODEL PREPARATION AND  COMPUTER ANIMATION

The obtained polygon surface mesh was then imported into Blender 2.78 (Blender Foundation, Amsterdam, Netherlands), were editing was necessary to reach the final goal of various rendered animations. The laborious processes of preparing the 3D-geometry varies for every organism due to different complexity and deformations of the sample while preparation. Therefore, we combine all the worksteps into one linear workflow.