High-contrast 3D imaging of diverse non-mineralized tissues

MicroCT for comparative morphology: simple staining
methods allow high-contrast 3D imaging of diverse
non-mineralized animal tissues

Brian D Metscher
Department of Theoretical Biology, University of Vienna, Austria

BioMed Central Physiology, 9:11, 2009

Arun Torris

Overview:
• Introduction
• Methods
MCT imaging systems
Sample preparation
Illustrations
• Results & Discussion
Vertebrates
Embryos
Insects
Invertebrates
• Conclusion
10 September 2009 Journal Club 2

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 Tomography
Principle 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


Methods
Micro-CT imaging systems used in the study

SkyScan 1174 scanner
Xradia MicroXCT System 30µm to 6µm
5µm to 500nm


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


Object Fixation, Storage Stain Voltage Time Voxels
Polyodon Head Bouin's, 70% ethanol PTA 60 kV, 8 W 2.2 hrs 5.6 μm
Polyodon Sections Bouin's, 70% ethanol PTA 80 kV, 8 W 2.8 hrs 4.3 μm
Grayling Section formalin PTA 40 kV, 8 W 12 hrs 2.1 μm
Axolotl glyoxal, 70% ethanol PTA 60 kV, 8 W 3 hrs 9.6 μm
Pike Hatchling formalin IKI 30 kV, 6 W 4 hrs 4 μm
Lamprey formalin, 70% ethanol I2E 50 kV, 8 W 4.2 hrs 15 μm
Polyodon Head formalin, methanol I2M 80 kV, 8 W 2.6 hrs 5.1 μm
Sturgeon Pectoral Fin Dent's, methanol I2M 40 kV, 8 W 2 hrs 9.2 μm
Xenopus Embryos formalin PTA 60 kV, 8 W 4 hrs 2.1 μm
Mouse Embryo paraform-aldehyde IKI 80 kV, 8 W 2.6 hrs 9 μm
Mouse Embryo paraform-aldehyde PTA 80 kV, 8 W 3 hrs 9.6 μm
Mouse Embryo EM fix, resin block OsO4 -- -- 8.2 μm
Insect Thorax Bouin's, 70% ethanol I2E 60 kV, 5 W 4 hrs 4.3 μm
Insect Head Bouin's, 70% ethanol I2E 60 kV, 5 W 16 hrs 2 μm
Insect Tibia Bouin's, 70% ethanol PTA 60 kV, 4 W 8 hrs 0.9 μm
Fly Pupa hot ethanol PTA 50 kV, 8 W 2.75 hrs 7.7 μm
Falcidens EM fix, resin block OsO4 60 kV, 8 W 1 hr 3.2 μm
Bryozoan Cristatella Bouin's PTA 40 kV, 8 W 1 hr 4.2 μm
Squid Hatchlings gluteraldehyde PTA IKI 90 kV, 4W 3 hrs 4 μm

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

Stain Stock Solution Staining Procedure
Mix 30 ml 1% PTA solution + 70 ml absolute ethanol to
make 0.3% PTA in 70% ethanol.
1% (w/v) Keeps indefinitely. Take samples to 70% ethanol.
PTA phosphotungstic Stain overnight or longer.
acid in water
Change to 70% ethanol. Staining is stable for months.
Scan samples in 70% – 100% ethanol

1% iodine metal Dilute to 10% in water just before use.
(I2) + 2% Rinse samples in water.
IKI
potassium iodide Stain overnight. Wash in water.
(KI) in water Can be scanned in water or dehydrated to alcohol.
Use at full concentration or dilute in absolute alcohol.
1% iodine metal Take samples to 100% alcohol.
(I2) dissolved in
I2E, I2M Stain overnight or longer. Wash in alcohol.
100% ethanol (I2E)
or methanol (I2M) Stain does not need to be completely washed out before
scanning.
Same as routine EM processing.
Osmium standard EM post- Osmium-stained samples can be scanned in resin
tetroxide fixation blocks, with some loss of contrast. 16
10 September 2009 Journal Club

Fixatives
• Neutral-buffered formalin

• Paraformaldehyde

• Gluteraldehyde

• Bouin's fluid

• Alcoholic Bouin's

• Glyoxal

• Dent's fixative

• Hot alcohol

Fixative Notes
neutral-buffered Formalin = 37% formaldehyde solution (aq.). in phosphate buffer at pH
formalin (10% NBF) 7.0. The most common, but rarely the best fixative.
paraformaldehyde Polymerized formaldehyde, usually dissolved in buffer (e.g. PBS) at 4%
w/v when a chemically-controlled fixative is required. Similar to 10% NBF.
gluteraldehyde Strong cross-linking fixative, often prepared in cacodylate buffer or a less
toxic alternative such as HEPES. Common fixative for electron microscopy.
4F1G 4% (or 3.7%) formaldehyde + 1% gluteraldehyde in phosphate buffer.
Common fixative for electron microscopy.
Bouin's fluid 75 parts (v/v) saturated aqueous picric acid, 25 parts formalin (37%
formaldehyde), 5 parts glacial acetic acid.
A standard and excellent histological fixative
alcoholic Bouin's 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.
Glyoxal 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.
Dent's fixative 80% methanol, 20% DMSO. Rapid dehydrating fixative. Expect some tissue
shrinkage. Often used for immunostaining.
hot alcohol 2009
10 September
Samples are dropped into 70% ethanol at about 60°C. Mainly used for
Journal Club 18
fixing soft-bodied animals, such as insect larvae and pupae.

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…..

European grayling –
Thymallus thymallus
Paddle Fish – Polydon spathula

Pike - Esox lucius

Axolotl – Ambystoma mexicanum
Green sturgeon -
Acipenser medirostris

Specimens…..
Diptera –
Calliphora vicinia

Lamprey – Lampetra

genus Sysira

Xenopus embryos

Squid - Ideosepius pygmeus Bryozoan Cristatella mucedo

Anatomy – Plane of view


Results & Discussion
• Vertebrates – Paddle Fish (Polyodon spathula)

• Lateral line receptors

• Nasal capsules and
muscles

5.6 μm voxels

Multiple views from a single scan of a 7-day post hatching paddlefish
Fixed in Bouin's, stored in 70% ethanol, stained with PTA.

Paddle Fish Paddle Fish
4 days post-hatching 27mm length


Virtual Sections
Neurocranial cartilage,
Otic chambers.

External naris,
Olfactory epithelium,
Cranial cartilage.

2.1 μm voxels

4.3 μm voxels

Optic nerves,
Layers in retina,
Jaw adductor muscles.

Axolotl (Ambystoma mexicanum)
o Muscles and
nervous tissues.
o Sensory organs.
o Nasal capsules.
o Neuromasts.

9.6 μm voxels

Scale = 500 μm
External views from the dorsal
Glyoxal-fixed, stored in 70% ethanol, PTA stained.

Pike (Esox lucius) 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 renderings and virtual sections - fish’s head
Fixed in formalin and stained with IKI.

Volume Reconstruction

Pike (Esox lucius)

Juvenile lamprey (Lampetra)

o The effects of previous
dehydration are evident

Fixed in formalin and stained with I2E after storage μm voxels
15
in alcohol (10 cm)
Top: Ventral View; Central: Club
10 September 2009 Journal
External View; Bottom: 30
Section

Xenopus embryos, stage ca. 27
o Pharyngeal pouches
o Optic vesicles
o Ciliated epidermal
cells.
o Neural tube.

2.1 μm voxels

Scale = 100 μm
Fixed in formalin and stained with PTA (left) and IKI (right).
Fixed in formalin and stained with IKI.

Mouse embryos

Left: Paraformaldehyde-fixed and IKI-stained 9.0 μm voxels

Center: PTA-stained; Right: Osmium-stained 9.6 μm voxels
8.2 μm voxels

2D sections of Mouse embryo


Volume Reconstruction

Mouse embryo Mouse embryo
Theiler stage 21 Theiler stage 22

A neuropteran insect (genus Sisyra)

o Musclature.
o Chitinous and soft
tissues

4.3 μm voxels

2 μm voxels
Fixed in Bouin's fluid and stained with I2E Scale = 100 μm


Tibia of a mantophasmid insect

o Scolopidial organ
o Sensory cells and
fiber
o Muscle fibers
o Single blood cells

0.9 μm voxels

Stereo pair for convergent (cross-eyed) viewing.
Shows the vibration-sensitive scolopidial organ


Pupa of the flesh fly Calliphora vicinia (Diptera)

o Metamorphosis
o Near-adult
morphology

7.7 μm voxels

Scale = 1 mm

Fixed in hot ethanol and stained with PTA.

Pupae must be perforated for PTA to penetrate


A caudofoveate mollusc (Falcidens sp).

Stained with osmium tetroxide and embedded
3.2 μm voxels
in Spurr's resin, scanned in resin block
Left: a low-resolution scan (1.4mm); 1.6 μm voxels
Center: High resolution, Right: Section Scale = 1 mm

Bryozoan Cristatella mucedo

o Extraction of soft tissue
characters important for study
of the diversification of life
[systematics]

4.2 μm voxels

Fixed in Bouin's and stained with PTA.
Scanned in alcohol (2 mm)

Squid hatchlings, Ideosepius pygmeus, ca. 2 mm long

o Emphasizing
the importance
of testing
different stains
on each new
kind of sample

4 μm voxels
4.4 μm voxels
Fixed in gluteraldehyde, stored in cacodylate buffer,
and stained with PTA (left) and IKI (right).

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

Discussion
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.


Questions


Evolution….


Future Directions

Nanotomography

Resolution : <150nm
Object Size : 11mm max. diameter

Synchrotron Radiation Facility
ESRF, Grenoble, France
Hasylab, Germany
SLS, Switzerland
NSLS & APS in USA


Micro CT - Principle


High-contrast 3D imaging of diverse non-mineralized tissues

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