(A) Whole octopus histotomography enabled by custom wide-field detector. Centimeter-wide rotational projections of X-rays passing through the rotating formalin-fixed, metal-stained O. bimaculoides (pink) onto a scintillator (tan disc, (B)) were magnified by the custom lens and mirror onto a medium format sCMOS chip camera (B). (C) 3D Dragonfly of render the reconstructed hatchling octopus, with a spherical section removed virtually to reveal the long-range pathway of the brachial nerves extending from the arms in the periphery, through the nerve ring in the mantle, and into the central brain. 2D slices of tomographic reconstructions were used to segment and pseudocolor microanatomical structures (shown together, top left).Bottom panels (D) include the digestive, circulatory, whole nervous system, and its arm branches. (E) Snapshot taken from the peripheral nervous system, highlighting segmentations of the nerve ring (AAUT shown here), brachial nerve (BN), oblique connectives (OCs), and oral intramuscular nerve cords (oINCs).

3D mapping of the long-range connections from sucker to brain in Octopus

(A) 3D render of hatchling reconstruction with spherical cutout illustrating the brachial nerves entering the body from the arms and projecting into the central brain. Each brachial nerve contributes to the portion of the cerebrobrachial connective that enters the inferior frontal lobe (shown here from the right 4 arms only for visual simplicity). Dotted line indicates contiguous brain regions (frontal lobes and subvertical lobe). (B) 2D sagittal cross section of the hatchling octopus from arm (left side) to brain (right side). Schematic lines illustrate continuous textures labeled connecting the oral nerves to the intermediate longitudinal tracts (see also Fig. 3) to the brachial nerves, which project to the cerebrobrachial connectives. The CBCs then project to the buccal lobe and the inferior frontal lobe (IFL). Dotted lines indicate connections between brain regions that exist but are not visible at the chosen 2D cross section.

Intermediate longitudinal tracts (iLT) illustrate distinct pathway linking oral nerves from the suckers to the proximal brachial nerve

(A) Sagittal cross-section of proximal end of arm L4 within the hatchling octopus reconstruction, illustrating the texture we labeled as the iLT descending separately from the brachial nerve into the neuropil of the arm. (B) Transverse cross-section of the excised adult arm reconstruction illustrating the relative position of the iLTs within the neuropil and their intersection with the oral nerves. (C) Coronal cross-section of the excised adult arm reconstruction illustrating the paired iLT structures running parallel within the neuropil. (D) 3D rendering of the adult arm reconstruction with a cutout revealing the oral nerves, their intersection with the iLT, and the spatial positioning of the iLT and brachial nerve within the axial nerve cord (ANC). (E) Alternative angle illustrating the proximity and contact of oral nerves with the sucker ganglion. Segmented oral nerves are rendered in color, and additional oral nerve traces are rendered in situ.

Subdivisions of the octopus nerve ring

A. Axial cross-section of the reconstructed octopus histotomogram at 0.7-micrometer resolution with nerve ring segmentation rendered in 3D. The green subdivision delineates what we refer to as the Arm-to-Arm U-Tract (AAUT), connecting the brachial nerves (BN) of neighboring arms via nerve fascicles that flow through each interbrachial commissure. The blue subdivision represents the nerve ring, which flows continuously across each arm-to-arm junction while also linking the neuropil of neighboring arms. The AAUT and inner nerve ring subdivisions were labeled to provide context to the segmentation shown in (A). B.) Higher-power examination of an arm-to-arm junction reveals microanatomical features of the octopus at the cellular level.

Hatchling Octopus bimaculoides are fully formed miniature octopuses

There is no larval or metamorphic stage; they become a typical benthic octopus immediately after hatching out of the egg. Mantle length is ca. 6-8mm.

Contextualization of the 10 mm detector systems in relation to other imaging modalities

(A) Comparison of objective lenses by numerical aperture (NA) and FOV. Our custom lens is labeled in blue according to its diagonal FOV and NA. (B) 2 histotomograms of our hatchling octopus sample stitched in the projection domain with a grid overlay corresponding to equivalent area captured by a conventional 2k x 2k detector. The grid shows how many tomograms (48) would need to be computationally stitched at 15% overlap to cover the same FOV as our custom detector system. (C) X-ray sensitivity specifications for the sCMOS sensor employed within our custom system.

FOV and resolution characterized using a QRM phantom

A. 3D render of the phantom wafer embedded within its sample tube labelled for size. B. QRM phantom as seen in the projection do-main prior to the initiation of a histotomographic scan. The full 10 x 7.5 mm field encompasses all surface area of the 5mm-wide sample tube as well as the full incident X-ray wavefront. The area that would be covered by a 2k x 2k pixel detector array of equivalent pixel size is represented by the solid white box within the phantom wafer (dotted white box). B’. Ten-slice average of the reconstructed QRM phantom showcasing the resolving power of our detector system. C-D. Higher-powered visualizations of the phan-tom line pairs and dot patterns in the reconstruction domain. E. Modulation transfer function (MTF) cal-culated from the L feature of the reconstructed QRM phantom. The MTF was taken as the discrete Fou-rier transform of the derivative of the edge-spread function recorded from a 10-slice average of the L visi-ble in B’.

Whole-organism phenotyping allows for identifications of complex structures at the cellular level across multiple organ systems within a single scan

A: The Octopus visual system focuses light through the lens (1) and into a retina formed from long, single-type photore-ceptor cells (2). Signals pass into the large, paired optic lobes, divided into the outer deep retina (3) and inner medulla (4). B: The octopus siphon (5) is used for respiration and locomotion. Glandular epithelium (6) lines a portion of the inner surface. C: The Stellate ganglia (7) control mantle musculature (8) through a series of radial stellar nerves. D: During inspiration, water trav-els through an opening in the collar (9) and proceeds across the gills (10), driven by muscle movements controlled by the stellate ganglions. The branchial hearts (11) perfuse the gills, sepa-rate to the systemic heart (not pictured), while blood departs through the efferent branchial vessel (12). E: A virtual slice shows parts of the digestive system, including the edge of the large diges-tive gland (13), esophagus (14), crop (15), and portions of the posterior salivary (venom) glands including glandular cells (16) and striated ducts (17). A major artery carrying blood from the systemic heart to the brain is also visible (18).

Axial cross-section of the interbrachial commissure as observed via histotomog-raphy (left) and confocal microscopy (right)

Segmentations were hidden from the histotomog-raphy image for an unbiased visualization of the regional microanatomy. The boundary delineat-ing the AAUT subdivision of the nerve ring appears as a white stripe in both imaging modalities. The histotomography image (A) was taken from a hatchling octopus, while the confocal image (B) was taken from a 3-month-old octopus. The axon bundles of the U-ring are labeled with or-ange bars in each image. (C.) Representative hematoxylin and eosin-stained section taken from the Interbrachial commissure of a separate hatchling octopus. (D.) Whole-animal imaging al-lowed us to create high-power 2D snap shots from each of the 8 interbrachial commissures in-cluding annotation of the corresponding segmented AAUT (teal green) and the inner nerve ring (blue). Each snapshot is oriented such that the top of the image is lateral to the octopus and the bottom of the image is medial.

Wide-field histotomography enables continuous volumetric segmentation of the oral intramuscular nerve cords (oINCs) and oblique connectives (OCs)

(A.) Transverse dig-ital cross section of the reconstructed octopus arm with 3D segmentations of the oINCs rendered in situ. (B.) Sagittal view of octopus arm L4 with rendered segmentations of ipsilateral oral and aboral INCs and their oblique connectives (OCs) that form an arcade-like motif. (C.) 3D render-ing with cut out illustrating the anatomical context of the L4 OC within the broader volume of the animal. (C.) Full 3D segmentations of the oral INCs illustrating their overlap and tortuosity. R1 = Right arm 1, R2 = Right arm 2.

Euclidean distance between oblique connective insertions at the oral INCs and spacing of sucker ganglia reveal a similar pattern

A.) 3D point tracing of each visible sucker ganglion (white) rendered alongside point tracings of the oblique connectives (maroon) as they intersect with the oral INCs. B.) Mean and standard deviation of the Euclidean distance between the insertions of oblique connectives (OC) at each oral INC. Distances are plotted for each set of OCs, yielding 2 sets per arm (L1 OC O1, L1 OC O2, etc). C.) Histogram of all measured dis-tances between the OC insertions not stratified by arm. The overall mean is indicated with a dashed line.

Quick start guide to navigate the image and segmentation using our customized Neuroglancer web interface

A) Transverse planar view of the octopus. Scrolling up parses dis-tally while scrolling down parses proximally. B) Coronal planar view of the octopus. C.) The control panel on the left of the web page allows the user to select which elements of the dataset are visible, allowing them to hide segmentations, ontological labels*, 3D rendering, and the mi-cro-CT reconstruction itself. D.) Sagittal planar view of the octopus. E) Panel dedicated to 3D rendering of the reconstructed dataset and segmentations. F) Source, Rendering, and annotation control panel. The user can use this panel to not only alter the appearance of the image, but also to record coordinates of regions of interest and add their own custom point annotations and measurements by selecting “Annotations” at the top right and control-clicking on a voxel within panels A, B, or E. G) Example ontological label as seen within the web viewer. See Video S9 for basic demonstration.

Major integrative connections to and from the subvertical lobe

A) Multiplanar visualization allowed validating alternative views of connections between the subvertical lobe and other brain regions that may play an important role in sensorimotor integration involved in learning and memory. A) Transverse 2D cross-sectional slices at 0.7-micrometer resolution used to recognize and color-label major connections in 2D, and example of which is shown in (B). Collective 2D segmentations validated by multiplanar inspection were used to create 3D repre-sentations shown in (C) that were then incorporated into the digital octopus. The subvertical lobes have extensive connections to other brain regions, the largest of which are the optic to lat-eral subvertical lobe tracts that link them to the larges brain lobes, the optic lobes after passing through the lateral subvertical lobes. C) The optic lobes also have direct connections to each other, primarily the large dorsal and ventral optic commissures, with some smaller paths such as the tract passing through the precommissural lobe. The eyes are lateral to the optic lobes.