MicroCT For Comparative Morphology

4,818 views

Published on

MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues.
BioMed Central Physiology, 9:11, 2009
Brian D Metscher
Department of Theoretical Biology, University of Vienna, Austria

Published in: Technology
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
4,818
On SlideShare
0
From Embeds
0
Number of Embeds
19
Actions
Shares
0
Downloads
105
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

MicroCT For Comparative Morphology

  1. 1. 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
  2. 2. Overview: • Introduction • Methods MCT imaging systems Sample preparation Illustrations • Results & Discussion Vertebrates Embryos Insects Invertebrates • Conclusion 10 September 2009 Journal Club 2
  3. 3. 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 10 September 2009 Journal Club 3
  4. 4. Computed Tomography Principle of Imaging: 10 September 2009 Journal Club 4
  5. 5. Reconstruction & Voxels 10 September 2009 Journal Club 5
  6. 6. Pixel & Voxel 10 September 2009 Journal Club 6
  7. 7. Micro-CT Vs Medical CT 10 September 2009 Journal Club 7
  8. 8. Micro-CT units • Lab-based scanner – Commercial x-ray source – 120,000 to 400,000 Euro • Synchrotron systems – Much finer resolution – Requires beamline 10 September 2009 Journal Club 8
  9. 9. 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 10 September 2009 Journal Club 9
  10. 10. 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. 10 September 2009 Journal Club 10
  11. 11. 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 10 September 2009 Journal Club 11
  12. 12. Methods Micro-CT imaging systems used in the study SkyScan 1174 scanner Xradia MicroXCT System 30µm to 6µm 5µm to 500nm 10 September 2009 Journal Club 12
  13. 13. 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 10 September 2009 Journal Club 13
  14. 14. 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 10 September 2009 Journal Club 14 Squid Hatchlings gluteraldehyde PTA IKI 90 kV, 4W 3 hrs 4 μm
  15. 15. 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 10 September 2009 Journal Club 15
  16. 16. 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
  17. 17. Fixatives • Neutral-buffered formalin • Paraformaldehyde • Gluteraldehyde • Bouin's fluid • Alcoholic Bouin's • Glyoxal • Dent's fixative • Hot alcohol 10 September 2009 Journal Club 17
  18. 18. 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.
  19. 19. Sample mounting • Scanned in liquid media • Polypropylene tubes – Low x-ray absorption – Conical shape • Absolute alcohol – Fewer bubbles – Better tissue contrast 10 September 2009 Journal Club 19
  20. 20. 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 10 September 2009 Journal Club 20
  21. 21. Specimens….. European grayling – Thymallus thymallus Paddle Fish – Polydon spathula Pike - Esox lucius Axolotl – Ambystoma mexicanum Green sturgeon - Acipenser medirostris 10 September 2009 Journal Club 21
  22. 22. Specimens….. Diptera – Calliphora vicinia Lamprey – Lampetra genus Sysira Xenopus embryos Squid - Ideosepius pygmeus Bryozoan Cristatella mucedo 10 September 2009 Journal Club 22
  23. 23. Anatomy – Plane of view 10 September 2009 Journal Club 23
  24. 24. 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. 10 September 2009 Journal Club 24
  25. 25. Paddle Fish Paddle Fish 4 days post-hatching 27mm length 10 September 2009 Journal Club 25
  26. 26. 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. 10 September 2009 Journal Club 26
  27. 27. 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. 10 September 2009 Journal Club 27
  28. 28. 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. 10 September 2009 Journal Club 28
  29. 29. Volume Reconstruction Pike (Esox lucius) 10 September 2009 Journal Club 29
  30. 30. 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
  31. 31. 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. 10 September 2009 Journal Club 31
  32. 32. Mouse embryos Left: Paraformaldehyde-fixed and IKI-stained 9.0 μm voxels Center: PTA-stained; Right: Osmium-stained 9.6 μm voxels 10 September 2009 Journal Club 32 8.2 μm voxels
  33. 33. 2D sections of Mouse embryo 10 September 2009 Journal Club 33
  34. 34. Volume Reconstruction Mouse embryo Mouse embryo Theiler stage 21 Theiler stage 22 10 September 2009 Journal Club 34
  35. 35. 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 10 September 2009 Journal Club 35
  36. 36. 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 10 September 2009 Journal Club 36
  37. 37. 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 10 September 2009 Journal Club 37
  38. 38. 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 10 September 2009 Journal Club 38
  39. 39. 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) 10 September 2009 Journal Club 39
  40. 40. 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). 10 September 2009 Journal Club 40
  41. 41. 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 10 September 2009 Journal Club 41
  42. 42. 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. 10 September 2009 Journal Club 42
  43. 43. 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 10 September 2009 Journal Club 43
  44. 44. 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. 10 September 2009 Journal Club 44
  45. 45. Questions 10 September 2009 Journal Club 45
  46. 46. Evolution…. 10 September 2009 Journal Club 46
  47. 47. Future Directions Nanotomography Resolution : <150nm Object Size : 11mm max. diameter Synchrotron Radiation Facility ESRF, Grenoble, France Hasylab, Germany SLS, Switzerland NSLS & APS in USA 10 September 2009 Journal Club 47
  48. 48. Micro CT - Principle 10 September 2009 Journal Club 48
  49. 49. 10 September 2009 Journal Club 49

×