MicroCT For Comparative Morphology

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


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


Computed TomographyPrinciple of Imaging:


Reconstruction & Voxels


Pixel & Voxel


Micro-CT Vs Medical CT


Micro-CT units

• Lab-based scanner

– Commercial x-ray source

– 120,000 to 400,000 Euro

• Synchrotron systems

– Much finer resolution

– Requires beamline


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


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.


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


MethodsMicro-CT imaging systems used in the study

Xradia MicroXCT System

5µm to 500nm

SkyScan 1174 scanner

30µm to 6µm


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


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


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


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


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


Sample mounting

• Scanned in liquid media

• Polypropylene tubes

– Low x-ray absorption

– Conical shape

• Absolute alcohol

– Fewer bubbles

– Better tissue contrast


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


Specimens…..

Paddle Fish – Polydon spathula

European grayling –Thymallus thymallus

Axolotl – Ambystoma mexicanum

Pike - Esox lucius

Green sturgeon -Acipenser medirostris


Specimens…..

Lamprey – Lampetra

Xenopus embryos

genus Sysira

Diptera –

Calliphora vicinia

Bryozoan Cristatella mucedoSquid - Ideosepius pygmeus


Anatomy – Plane of view


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


Paddle Fish

4 days post-hatching

Paddle Fish

27mm length


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


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


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


Volume Reconstruction

Pike (Esox lucius)


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


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


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


2D sections of Mouse embryo


Volume Reconstruction

Mouse embryo Theiler stage 21

Mouse embryo Theiler stage 22


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


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


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


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


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


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


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


� 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


� 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


� 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


Questions


Evolution….


Future Directions

Nanotomography

Synchrotron Radiation Facility

Resolution : <150nmObject Size : 11mm max. diameter

ESRF, Grenoble, FranceHasylab, GermanySLS, SwitzerlandNSLS & APS in USA


Micro CT - Principle


Technology

MicroCT For Comparative Morphology