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MicroCT for comparative morphology: simple staining MicroCT for comparative morphology: simple staining methods allow highmethods allow high--contrast 3D imaging of diverse contrast 3D imaging of diverse nonnon--mineralized animal tissuesmineralized animal tissues
Arun Torris
Brian D MetscherBrian D MetscherDepartment of Theoretical Biology, University of Vienna, AustriaDepartment of Theoretical Biology, University of Vienna, Austria
BioMed Central PhysiologyBioMed Central Physiology, 9:11, 2009, 9:11, 2009
10 September 2009 Journal Club 2
Overview:
• Introduction
• Methods
� MCT imaging systems
� Sample preparation
� Illustrations
• Results & Discussion
� Vertebrates
� Embryos
� Insects
� Invertebrates
• Conclusion
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Introduction
Methods of 3D visualizations:
I. Serial sections
• Laborious process
• Specimen sectioning
• Destructive
II. Whole-volume imaging
• Non-destructive
• Imaging instrumentation
E.g.: Micro-MRI, OPT
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Computed TomographyPrinciple of Imaging:
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Reconstruction & Voxels
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Pixel & Voxel
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Micro-CT Vs Medical CT
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Micro-CT units
• Lab-based scanner
– Commercial x-ray source
– 120,000 to 400,000 Euro
• Synchrotron systems
– Much finer resolution
– Requires beamline
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Limitations in comparative morphology:
� Low x-ray contrast of non-mineralized
tissues
� Only few techniques for imaging soft tissues
� Organically-bound iodine
� Osmium staining
� Reduced-silver
� Contrast resin perfusion
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Major Contributions so far…..
• Imaging of mouse and rabbit – de Crespigny et al, 2008.
• Phenotyping mouse embroys – Johnson T J et al, 2006.
• Honeybee brains – Ribi W et al, 2008.
• Drosophila brains – Mizutani R et al, 2007.
• Arthropod vasculature – Wirkner et al, 2007.
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What it can offer ?
• Linear and volumetric size changes in development
• Comparison between control and genetically manipulated
specimens
• Quantitative data for modeling of developmental and
evolutionary changes
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MethodsMicro-CT imaging systems used in the study
Xradia MicroXCT System
5µm to 500nm
SkyScan 1174 scanner
30µm to 6µm
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Imaging Parameters
• No single optimal set of constants
• Requirements of investigation determines
• Lower voltage provides higher projection exposure
Specimens• Vertebrates
• Vertebrate embryo
• Insects
• Insect pupae
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4 μm3 hrs90 kV, 4WPTA IKIgluteraldehydeSquid Hatchlings
4.2 μm1 hr40 kV, 8 WPTABouin'sBryozoan Cristatella
3.2 μm1 hr60 kV, 8 WOsO4EM fix, resin blockFalcidens
7.7 μm2.75 hrs50 kV, 8 WPTAhot ethanolFly Pupa
0.9 μm8 hrs60 kV, 4 WPTABouin's, 70% ethanolInsect Tibia
2 μm16 hrs60 kV, 5 WI2EBouin's, 70% ethanolInsect Head
4.3 μm4 hrs60 kV, 5 WI2EBouin's, 70% ethanolInsect Thorax
8.2 μm----OsO4EM fix, resin blockMouse Embryo
9.6 μm3 hrs80 kV, 8 WPTAparaform-aldehydeMouse Embryo
9 μm2.6 hrs80 kV, 8 WIKIparaform-aldehydeMouse Embryo
2.1 μm4 hrs60 kV, 8 WPTAformalinXenopus Embryos
9.2 μm2 hrs40 kV, 8 WI2MDent's, methanolSturgeon Pectoral Fin
5.1 μm2.6 hrs80 kV, 8 WI2Mformalin, methanolPolyodon Head
15 μm4.2 hrs50 kV, 8 WI2Eformalin, 70% ethanolLamprey
4 μm4 hrs30 kV, 6 WIKIformalinPike Hatchling
9.6 μm3 hrs60 kV, 8 WPTAglyoxal, 70% ethanolAxolotl
2.1 μm12 hrs40 kV, 8 WPTAformalinGrayling Section
4.3 μm2.8 hrs80 kV, 8 WPTABouin's, 70% ethanolPolyodon Sections
5.6 μm2.2 hrs60 kV, 8 WPTABouin's, 70% ethanolPolyodon Head
VoxelsTimeVoltageStainFixation, StorageObject
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Contrast stains
• Inorganic iodine in alcohol
– Diffuses rapidly into fixed tissues
– Ability to stain in few hours
• PTA – Phosphotungstic acid
– Larger molecule
– Require overnight incubation
– Binds heavily to proteins and connective tissue
– Electron energy matches x-ray source emissions
• PMA – Phosphomolybdic acid
• Osmium Tetroxide
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Same as routine EM processing.
Osmium-stained samples can be scanned in resin blocks, with some loss of contrast.
standard EM post-fixation
Osmium tetroxide
Use at full concentration or dilute in absolute alcohol.
Take samples to 100% alcohol.
Stain overnight or longer. Wash in alcohol.
Stain does not need to be completely washed out before scanning.
1% iodine metal(I2) dissolved in 100% ethanol (I2E) or methanol (I2M)
I2E, I2M
Dilute to 10% in water just before use.
Rinse samples in water.
Stain overnight. Wash in water.
Can be scanned in water or dehydrated to alcohol.
1% iodine metal(I2) + 2% potassium iodide(KI) in water
IKI
Mix 30 ml 1% PTA solution + 70 ml absolute ethanol to make 0.3% PTA in 70% ethanol.
Keeps indefinitely. Take samples to 70% ethanol.
Stain overnight or longer.
Change to 70% ethanol. Staining is stable for months.
Scan samples in 70% – 100% ethanol
1% (w/v) phosphotungstic acid in water
PTA
Staining ProcedureStock SolutionStain
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Fixatives
• Neutral-buffered formalin
• Paraformaldehyde
• Gluteraldehyde
• Bouin's fluid
• Alcoholic Bouin's
• Glyoxal
• Dent's fixative
• Hot alcohol
10 September 2009 Journal Club 18Samples are dropped into 70% ethanol at about 60°C. Mainly used for fixing soft-bodied animals, such as insect larvae and pupae.
hot alcohol
80% methanol, 20% DMSO. Rapid dehydrating fixative. Expect some tissue shrinkage. Often used for immunostaining.
Dent's fixative
A cross-linking dialdehyde prepared in acidic buffers and marketed as formalin substitute. Much less volatile and toxic than formaldehyde.
Very good tissue preservation; especially good for immunostaining.
Glyoxal
Refers to either a mixture of Bouin's fluid and ethanol (1:1), or to the fixative also known as Bouin-Duboscq-Brasil. The alcoholic solutions penetrate more readily and are sometimes favored for arthropods.
alcoholic Bouin's
75 parts (v/v) saturated aqueous picric acid, 25 parts formalin (37% formaldehyde), 5 parts glacial acetic acid.
A standard and excellent histological fixative
Bouin's fluid
4% (or 3.7%) formaldehyde + 1% gluteraldehyde in phosphate buffer.
Common fixative for electron microscopy.
4F1G
Strong cross-linking fixative, often prepared in cacodylate buffer or a less toxic alternative such as HEPES. Common fixative for electron microscopy.
gluteraldehyde
Polymerized formaldehyde, usually dissolved in buffer (e.g. PBS) at 4% w/v when a chemically-controlled fixative is required. Similar to 10% NBF.
paraformaldehyde
Formalin = 37% formaldehyde solution (aq.). in phosphate buffer at pH 7.0. The most common, but rarely the best fixative.
neutral-buffered formalin (10% NBF)
NotesFixative
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Sample mounting
• Scanned in liquid media
• Polypropylene tubes
– Low x-ray absorption
– Conical shape
• Absolute alcohol
– Fewer bubbles
– Better tissue contrast
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Preparation of illustrations
• 3D viewing and imaging software's
• Transparency function
– to show both internal and external features
• Arranged with Photoshop CS3
• False color was added to the volume renderings
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Specimens…..
Paddle Fish – Polydon spathula
European grayling –Thymallus thymallus
Axolotl – Ambystoma mexicanum
Pike - Esox lucius
Green sturgeon -Acipenser medirostris
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Specimens…..
Lamprey – Lampetra
Xenopus embryos
genus Sysira
Diptera –
Calliphora vicinia
Bryozoan Cristatella mucedoSquid - Ideosepius pygmeus
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Anatomy – Plane of view
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Results & Discussion
• Vertebrates – Paddle Fish (Polyodon spathula)
• Lateral line receptors
• Nasal capsules and
muscles
Multiple views from a single scan of a 7-day post hatching paddlefish
Fixed in Bouin's, stored in 70% ethanol, stained with PTA.
5.6 μm voxels
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Paddle Fish
4 days post-hatching
Paddle Fish
27mm length
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Virtual Sections
Optic nerves,Layers in retina,Jaw adductor muscles.
Neurocranial cartilage,Otic chambers.
External naris,Olfactory epithelium,Cranial cartilage.
4.3 μm voxels
2.1 μm voxels
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Axolotl (Ambystoma mexicanum)
External views from the dorsal
Glyoxal-fixed, stored in 70% ethanol, PTA stained.
o Muscles and
nervous tissues.
o Sensory organs.
o Nasal capsules.
o Neuromasts.
9.6 μm voxels
Scale = 500 μm
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Pike (Esox lucius)
Volume renderings and virtual sections - fish’s head
Fixed in formalin and stained with IKI.
o Layers in the brain.
o Jaw adductor
muscles.
o Gill-arch cartilages.
o Retinal layers.
o Connections with optic nerves, lenses.
4.0 μm voxels
Scale = 500 μm
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Volume Reconstruction
Pike (Esox lucius)
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Juvenile lamprey (Lampetra)
Fixed in formalin and stained with I2E after storage
in alcohol (10 cm)
Top: Ventral View; Central: External View; Bottom:
Section
o The effects of previous
dehydration are evident
15 μm voxels
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Xenopus embryos, stage ca. 27
Fixed in formalin and stained with PTA (left) and IKI (right).
Fixed in formalin and stained with IKI.
o Pharyngeal pouches
o Optic vesicles
o Ciliated epidermal cells.
o Neural tube.
2.1 μm voxels
Scale = 100 μm
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Mouse embryos
Left: Paraformaldehyde-fixed and IKI-stained
Center: PTA-stained; Right: Osmium-stained
9.0 μm voxels
9.6 μm voxels
8.2 μm voxels
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2D sections of Mouse embryo
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Volume Reconstruction
Mouse embryo Theiler stage 21
Mouse embryo Theiler stage 22
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A neuropteran insect (genus Sisyra)
Fixed in Bouin's fluid and stained with I2E
o Musclature.
o Chitinous and soft tissues
4.3 μm voxels
2 μm voxels
Scale = 100 μm
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Tibia of a mantophasmid insect
Stereo pair for convergent (cross-eyed) viewing.
Shows the vibration-sensitive scolopidial organ
o Scolopidial organ
o Sensory cells and fiber
o Muscle fibers
o Single blood cells
0.9 μm voxels
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Pupa of the flesh fly Calliphora vicinia (Diptera)
Fixed in hot ethanol and stained with PTA.
Pupae must be perforated for PTA to penetrate
o Metamorphosis
o Near-adult morphology
7.7 μm voxels
Scale = 1 mm
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A caudofoveate mollusc (Falcidens sp).
Stained with osmium tetroxide and embedded
in Spurr's resin, scanned in resin block
Left: a low-resolution scan (1.4mm);
Center: High resolution, Right: Section
3.2 μm voxels
1.6 μm voxels
Scale = 1 mm
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Bryozoan Cristatella mucedo
Fixed in Bouin's and stained with PTA.
Scanned in alcohol (2 mm)
o Extraction of soft tissue
characters important for study
of the diversification of life
[systematics]
4.2 μm voxels
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Squid hatchlings, Ideosepius pygmeus, ca. 2 mm long
Fixed in gluteraldehyde, stored in cacodylate buffer,
and stained with PTA (left) and IKI (right).
o Emphasizing
the importance
of testing
different stains
on each new
kind of sample
4 μm voxels
4.4 μm voxels
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Discussion
• Examples are intended to illustrate some possibilities for
microCT investigations of diverse problems that require or
will benefit from 3D morphological data
• Each new type of sample must be tested with different
fixations and stains to find the best treatment for the
imaging required
� Accurately calibrated 3D images of musculoskeletal systems
can be also used to quantify
� Muscle fiber numbers and cross sectional areas,
� Muscle attachment areas,
� Bone or cartilage sizes and shapes, and
� Facilitate functional modeling
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� PTA Vs Iodine stain
� The PTA and iodine stains were found to impart strong tissue
contrast to fish and amphibian samples
� Especially PTA staining of Bouin's or glyoxal-fixed material
with IKI staining after formalin-fixation
� PTA is known to bind to collagen, proteins and musculature
� Cartilage does not stain strongly with PTA, but appears as gaps in
volume renderings
� It is worth noting that iodine did not stain effectively in 70%
alcohol, and so samples had to be transferred to 100% alcohol
before staining
� Nervous tissues are also demonstrated well with PTA & IKI, and
different layers of the brain can be distinguished easily.
Discussion
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� Osmium staining
� Most common contrast stain,
� Has electron binding energies favorable for strong x-ray absorption
� Bind to cell membranes and other lipid-rich structures
including nerves
� Very toxic
� Penetration is slow
� Expensive to dispose of
� Does not stain well if samples have been in alcohol
Discussion
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� PTA staining
� Penetrates tissues slowly
� Far less toxic
� Much simpler to use
� Effectively stain alcohol-stored samples
� PTA did not readily penetrate the cuticle
� Inorganic iodine readily penetrates all soft tissues tested so far, and it has proven to be versatile and robust contrast stain.
Discussion
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Questions
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Evolution….
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Future Directions
Nanotomography
Synchrotron Radiation Facility
Resolution : <150nmObject Size : 11mm max. diameter
ESRF, Grenoble, FranceHasylab, GermanySLS, SwitzerlandNSLS & APS in USA
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Micro CT - Principle
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