Non invasive in vivo investigation of hepatobiliary structure and function in STII medaka (Oryzias latipes): methodology and applications

Additional file 1:

STII medaka, 12 dpf, left lateral view. Lacking dermal and visceral pigmentation, internal organs are readily visible through the body wall, and amenable to in vivo observation/imaging. Note peristalsis in the gut on a temporal scale. Liver (L), Gut (Gt), Otic Vesicle (Ov), Spleen (Sp), Air/Swim Bladder (AB), Heart (H).

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Additional file 2:

In vivo confocal imaging of hepatobiliary system, STII medaka, 9 dpf. Example of a confocal stack acquired in vivo. Parenchyma elucidated here with β-Bodipy C5 ceramide. Hepatocyte nuclei (HN) appear dark (non fluorescent), cytosol is distinct. Red blood cells can be seen in circulation through sinusoids (S/r). Note differential fluorescence between sinusoid lumen vs. cytosol. Endothelial cells lining sinusoids are also distinct. Stack size [x : y : z = 192 × 192 × 62 μm], Scaling [0.37 × 0.37 × 0.7 μm].

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Additional file 3:

Three-dimensional reconstruction of canaliculi and sinusoids from in vivo confocal image stack: relationship of sinusoids to intrahepatic biliary passageways, STII medaka, 30 dpf. Shown is an isolated section of the parenchyma from a 3D reconstruction. Canaliculi (C, green), which average 1.3 μm in diameter, are green, sinusoids (S, red). Examples of metrics acquired from 3D reconstructions are given for illustrative purposes. The example movie shown here, extracted from an Amira 3D reconstruction, is limited to rotation in a single plane. Actual 3D reconstructions can be rotated in any plane, at virtually any magnification, allowing detailed study of hepatobiliary structure/function relationships.

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Additional file 4:

Example of 3D reconstruction of hepatic parenchyma from in vivo confocal image stack: bile preductules and preductular epithelial cells, STII medaka, 24 dpf. Shown is an isolated section of the parenchyma showing the 3D characteristics of bile preductular epithelia (BPDEC) and bile preductules (BPD), the latter a unique morphological feature created by junctional complexes between hepatocytes and BPDECs. Hepatocytes, which occupy the negative/empty space, are not rendered for visual clarity. A canaliculus (C, green) is shown joining a bile preductule (BPD, green). The background grayscale image is a single optical section from a confocal image stack. Red blood cells can be seen in circulation through sinusoids of the liver (S/r) in confocal image. To our knowledge this was the first rendering of this bile preductule junctional complex in 3D, the evaluation of which provided novel insights into parenchymal organization. The movie given here, extracted from an Amira 3D reconstruction, is limited to rotation in a single plane. Actual 3D reconstructions can be rotated in any plane, at virtually any magnification, allowing detailed study of hepatobiliary structure/function relationships.

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Additional file 5:

Example of 3D reconstruction of parenchyma from in vivo confocal image stacks: relationship of sinusoids to intrahepatic biliary passageways, STII medaka, 30 dpf. Sinusoids (S) are red, canaliculi (C) in green. All space between sinusoids and surrounding canaliculi (empty) is hepatocellular space, not rendered for visual clarity. Morphometric and volumetric analyses of 3D reconstructions assisted elucidation of parenchymal architecture, and relationship of canaliculi to sinusoids. These types of investigations revealed medaka hepatic parenchyma to be more akin to a muralium like structure (as opposed to tubular architecture). Grayscale confocal image can be seen in the background. The movie given here, extracted from an Amira 3D reconstruction, is limited to rotation in a single plane. Actual 3D reconstructions can be rotated in any plane, at virtually any magnification, allowing detailed study of hepatobiliary structure/function relationships.

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Additional file 6:

Example of 3D reconstruction of hepatobiliary architecture from in vivo confocal image stack: sinusoids and parenchymal architecture, STII medaka, 30 dpf. Sinusoids (S) are red. All space between sinusoids (empty) is hepatocellular space, not rendered for visual clarity. Morphometric and volumetric analyses of 3D reconstructions allowed elucidation of parenchymal architecture, and revealed medaka parenchyma more akin to a muralium like structure (as opposed to tubular architecture). Background grayscale image is a single frame from a confocal image stack from which the 3D model was generated. The movie given here, extracted from an Amira 3D reconstruction, is limited to rotation in a single plane. Actual 3D reconstructions can be rotated in any plane, at virtually any magnification, allowing detailed study of hepatobiliary structure/function relationships. STII medaka, 30 dpf.

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Additional file 7:

Example of 3D reconstruction of the hepatobiliary system from in vivo confocal image stacks: relationship of sinusoids to intrahepatic biliary passageways, STII medaka, 12 dpf. Sinusoids (S) are denoted in red, intrahepatic biliary passageways (IHBPs) in green/gold. All space between sinusoids and surrounding IHBPs is hepatocellular space, not rendered for visual clarity. These types of reconstructions permitted 3D morphometric and volumetric analyses, which assisted in elucidation of hepatobiliary architecture. Grayscale confocal image can be seen in the background. The movie given here, an image capture from an Amira 3D reconstruction, is limited to rotation in a single plane. Actual 3D reconstructions can be rotated in any plane, at virtually any magnification, allowing detailed study of hepatobiliary structure/function relationships.

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Additional file 8:

Example of 3D projection of liver and gall bladder from in vivo confocal image stacks: STII medaka, 12 dpf. 3D projections of confocal image stacks, in conjunction with 3D reconstructions, aided in evaluation of 3D architecture of the hepatobiliary system. Intrahepatic biliary passageways (IHBPs) of the liver (L), and gall bladder (GB) were elucidated with fluorescein isothiocyanate.

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Additional file 9:

Example of 3D projection of parenchyma from in vivo confocal image stacks: STII medaka, 12 dpf. 3D projections of confocal image stacks, in conjunction with 3D reconstructions, aided in evaluation of 3D hepatobiliary architecture. Intrahepatic biliary passageways (IHBPs) of the liver, denoted by increased fluorescence, elucidated with β-Bodipy C5 Phosphocholine (HPC).

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