Neutron Tomography for Paleontological Studies Timothy D. Huang 黃⼤大⼀一 2016-10

New Paleontology ★

Think Outside the Box.

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Look Into the Bone.
 Timothy D. Huang, July 2010

What do we NOT see? Neutron Tomography is a new way of not only for exploring extant material, but more importantly for studying extinct organisms, ie., paleontological material (fossils). For the extant material such as plants or animals, they can be reproduced and experiments can be repeated in the lab. However, paleontology is not a repeatable science in the lab. Whatever happen happened. Whatever fossil we have is what the nature provides. We can not go back into the long fossilization time and environment, nor to meaningfully simulate such processes in the lab. What we can find from the field are what we can go by. Furthermore, the availability of fossil specimens is very different. Some are like trash ‘everywhere’. However, rare or even just a single specimen found in the whole world is a very real situation paleontologists have to face. Thus, for paleontological studies, non-destructive method shall be preferable to any semi-destructive or destructive ways. In our ongoing pursuit of “Dinosaur Embryology” by “Look Into the Bone” new paleontological approach, we are exploring several new avenues, such as but not limited to synchrotron radiation TXM (Transmission X-ray Microscopy) and synchrotron radiation FTIR (Fourier Transformation Infrared Spectroscopy, both are semi-destructive), high resolution (10µm) and very high resolution (<1µm) µ-CT, Multiple Harmonic Generation Microscopy (MHGM), Raman-CT, and Neutron Tomography. These various methodologies provide us many very valuable information never explored before in conventional paleontology. However, there are many important issues that we have to face when we are charting into the brand new unexplored water. One of which is “what are we actually seeing under such and such light/beam source?” In other words, what do we NOT see? This is more important and serious question we have to keep in mind. For example, sr-TXM can provide very high resolution (~50 nm) morphological information of fossil material, however, despite of it’s semi-destructive nature (~15 µm thin slice), can we see all composition besides the preserved heavier elemental morphology of that fossil? µ-CT suffers the same limitation as srTXM due to the imaging nature of X-ray light source. Again, for sr-FTIR, it’s semi-destructive nature (~15 µm thin slice as srTXM), we can obtain information about the chemical functional groups of the fossil composition. sr-XRF (synchrotron radiation Xray Fluorescence) provides chemical elemental mapping of the fossil specimen, mainly on inorganic composition. We published a Nature cover paper (April 11, 2013) on the preservation of organic remains (collagen type I) in Lufengosaurus embryonic bones by using sr-FTIR at BL-14A of NSRRC. Unlike the X-ray, neutron scan can provide much better images on lighter elements. So, in our pursuit of “Look Into the Bone” trying to “see” organic remains preserved in the fossils or matrix, it’s the more suitable methodology. Neutron scan is an excellent complimentary to µ-CT. The main compositional elements for organic material are light elements mainly hydrogen, carbon, nitrogen, and oxygen. These elements can not be seen by X-ray, but show very well by neutron scattering. Thus, it provides what X-ray can NOT see. As now, we already have some initial exciting results from our two previous neutron scans at ANSTO, and the results are reconfirmed by Raman-CT done at Sydney University. We are planning to explore much much more neutron tomography for our paleontological studies on various high caliber evolutionary important fossils from Northeastern and other places in China, including such study on dinosaur brain and tooth growth series and comparative morphology of two different but related new dinosaurs. Neutron tomography will be our main tool to accomplish these goals.

Extant vs Extinct

from Wiki

Here and now

~240 mya

Look Into the Bone Idea Situation DaWa Think, if we can do all of these, Embryonic Limb how powerful that will be? Bone This is a transversal cut of an embryonic limb bone from DaWa. It’s around the middle of the bone longitudinal.

Overlapping Images of: Optical, sr-µ-CT MHGM, Neutron Scan, Raman-CT, (non-destructive) & sr-FTIR on the same spot of the same specimen

THG SHG 60μm

sr-TXM and SEM 15µm

Seeing the cell-like morphological structures is not unique to the MHGM. It was first observed by sr-TXM. This structure is further confirmed by these MHGM scans. The size is around 10~30 µm. Furthermore, bacterial like structures were observed by SEM, and now in MHGM, attached on the inner wall of Ediacaran tubular creatures.

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20110819_HYC_m3-2Cells?.tiff

Multiple Harmonic Generation Microscopy

Emb003_05_redo2014_mip.mov

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This QTVR 3D models were reworks with color assignment of TPH/Yellow, SGH/Green, and TGH/ Magenta.

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There are some White areas in the 3D model, meaning both SGH and THG responses (green + magenta = white).

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The depth of higher resolution (0.47µm/px) scans of these were only 81 µm, far less than the average length of the primarily tubular cavity. So, only a section of each PTC can be seen. The average diameter of the PTC is about 50 100 µm, in which the ‘wall’ thickness is about 15 µm. Intensity adjustments of laser caused some irregularities.

sr-FTIR

Neutron Scan Facility •

ANSTO/DINGO

Image Station Neutron Beam

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27 µm Resolution

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180/360 Rotation

Specimen Aluminum Can

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~24 Hours per Scan

Rotation Stage

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Scan Volume 5x5x5 cm3

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Neutron Cross Section

Main elements of fossils Rare Earth Elements

Aluminum Can

Neutron vs µ-CT Scan •

µ-CT is based on X-ray density, i.e., heavier atoms will have bigger cross sections.

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Neutron scattering is more sensitive to lighter elements, such as organic remains in fossil.

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What can’t be seen in µ-CT, such as H, C, O, and N, can be seen clearly in Neutron, and vise versa.

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These two are very good complimentary to each other.

µ-CT vs Neutron 7 µm µ-CT scan

27 µm Neutron scan

Case of Massospondylus

27 µm Neutron scan

The top photo was taken under normal light. Three tiny tiny Massospondylus embryonic ribs can be seen clearly. When this was neutron scanned, these three ribs are not shown well. But, some amazing hazy areas (next slide) showed up very well. Wondering if these hazy areas are the organic remains of the decomposed nesting plant material (leafs).

Case of Massospondylus

(video)

Case of Lufengosaurus Embryonic Bones

7 µm Synchrotron-CT scan

The left photos (single slice) were taken by synchrotron µ-CT at 7 µm resolution on Lufengosaurus embryonic vertebrae. The video below was reconstructed from neutron scan at 27 µm resolution. (Further image processing needed.)

The white areas from µ-CT are apatites, while the white areas from neutron scan means preservation of organic remains. 27 µm Neutron scan

Confirmation of Organic Remains from Raman-CT of Lufengosaurus Embryonic Bones

This is a Raman CT of Lufengosaurus embryonic bone 3D distribution of Amide III peak, which is one of very representative functional group of proteins, peptides, and amino acids.

(Univ. of Sydney)

Combining neutron scan and RamanCT is a good way of studying Organic Remains in fossil.

Case of Lufengosaurus Embryonic Bones This Primary Tubular Cavities was obtained from a 2 µm very high resolution µ-CT scan on a Lufengosaurus embryonic femur. During the very fast growing embryo development stage, about 50% of the cortex bone spaces are voids and called PTC, which contains various liquid organic material. (Apatite bone areas in black, PTC in red)

This corresponds very well from what neutron tomography showed.

Interesting and Important Evolutionary Topics

Lufengosaurus in Action

Holy Shit (Coprolite) As can be seen from this neutron scan, there are ‘something’, very likely organic remains inside this “Holy Shit (dinosaur poop, coprolite, ~1x0.5cm)”. What did this dinosaur eat?

Watch outs • •

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The higher the resolution, the bigger the image size. Thus, requires more powerful computing systems with PLENTY of storage rooms. And may have to pay an arm or leg for commercial image processing software. Highway robberies exist. However, there are many 3D rendering freebies, such as ImageJ, Drishti, etc. available. (Medical) Image processing skills required. Paleo-anatomy knowledge required. Have a lot of patience, go drink a lot of coffee. Radioactive “hot” after neutron scan.

Thank you!