The corneal universe - mission possible by in vivo confocal microscopy

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My gratest honour for 2011 - "J Kersley" named lecture in Istanbul September 2011 at ECLSO congress

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The corneal universe - mission possible by in vivo confocal microscopy

  1. 1. The corneal universe - mission possible by in vivo confocal microscopy<br />Jonathan Kersley Lecture<br />Presented by <br />Christina Grupcheva<br />
  2. 2. Jonathan Kersley<br />(1939-2000)<br /><ul><li>Henry Jonathan Kersley was born in Bath
  3. 3. Educated at Cambridge University
  4. 4. Completed medical studies at St Bartholomew's Hospital
  5. 5. Member of the Royal College of Ophthalmology since founded in 1988
  6. 6. In 1974, he gave his first paper at the international meeting of the</li></ul>Contact Lens Society in Montreux<br /><ul><li>He was involved in clinical development of both Sauflon 70 and</li></ul>Sauflon85 by Contact Lenses Manufacturing Ltd<br /><ul><li>Before implants were universally considered, he was possibly the </li></ul>first to fit soft lenses immediately after cataract surgery<br /><ul><li>Kersley was President of the British Contact Lens Association 1978-9</li></ul>'Modern Anterior Segment Surgery and advancing contact lens technology'<br />
  7. 7. Historical prospective<br /><ul><li> 1957 Minsky describes confocal microscope principle
  8. 8. 1980In vitro confocal microscopy well established
  9. 9. 1985 Lemp et al apply in vivo technique to rabbit cornea
  10. 10. 1989 Cavanaugh et al publish first human data
  11. 11. 1995 Slit-scanning in vivo confocal microscopy
  12. 12. 2003 Laser-scanning in vivo confocal microscopy</li></ul>Jonathan Kersley Lecture 2011<br />
  13. 13. Tandem-scanning in vivo confocal microscopy<br />White light<br />Scanning spot<br />or scanning slit<br />Incident light is<br />focussed in same <br />plane as objective <br />lens i.e. confocal<br />Objective<br />Nipkow <br /> lens<br />Beam<br /> Disk<br />Light<br />splitter<br />source<br />Video<br />camera<br />CORNEA<br />Dual light path technique (Tandem scanning)<br />Single light path technique<br />Jonathan Kersley Lecture 2011<br />
  14. 14. Slit-scanning in vivo confocal microscopy<br />Light <br />source<br />Condenser <br />lens<br />Front<br />lens<br />Scanning<br />slits<br />Condenser <br />lens<br />Video <br />camera<br />Cornea<br />Jonathan Kersley Lecture 2011<br />
  15. 15. Confoscan 2 (model 2000)<br />improved NAVIS software<br />Confoscan 2 (model 1999)<br />In vivo confocal microscopy<br />Jonathan Kersley Lecture 2011<br />
  16. 16. Tandem scanning confocal microscopy<br /> Precise movement of the disk<br /> Applanation technology ?<br />Small round area<br />Low illumination<br />One scan per examination<br />Time consuming<br />Slit- scanning confocal <br />microscopy<br />Greater illumination<br />Adjustable parameters<br />Relatively quick<br />Non-applanation technology<br /> Small oblong area<br /> Moving objective lens<br />In vivo confocal microscopy<br />Jonathan Kersley Lecture 2011<br />
  17. 17. Laser-scanning in vivo confocal microscopy<br />Jonathan Kersley Lecture 2011<br />
  18. 18. In vivo confocal microscopy<br />Light-scanning confocal microscopy<br />Non applanation<br />Clearer images of the endothelium<br />Depends on transparency<br />Low resolution<br />Artefacts<br />Time consuming<br />Laser- scanning confocal <br />microscopy<br />Greater resolution<br />Greater depth of focus<br />More compact, stabile objective<br />Precise z location ?<br />Small area<br />Low reproducibility<br />Jonathan Kersley Lecture 2011<br />
  19. 19. Research & clinical applications<br />Clinical:<br /> Description<br /> Differentiation<br /> Diagnosis<br /> Prognosis<br /> Dynamic follow up<br />Research:<br /> Descriptor<br /> Discriminator<br /> Dissector<br /> 3D reconstructor<br /> 4D reconstructor<br />
  20. 20. 2<br />3<br />1<br />1<br />2<br />3<br />4<br />5<br />4<br />5<br />6<br />6<br />7<br />8<br />7<br />8<br />Research applications: <br />Descriptor & Discriminator<br />Epithelium<br />Anterior stroma<br />Posterior stroma<br />Endothelium<br />
  21. 21. Research applications: <br />Descriptor & Discriminator<br />2<br />3<br />1<br />1<br />Epithelium<br />2<br />3<br />Anterior stroma<br />4<br />5<br />4<br />5<br />6<br />6<br />Posterior stroma<br />7<br />8<br />7<br />Endothelium<br />8<br />
  22. 22. Research applications: <br />Descriptor & Discriminator<br />Epithelium<br />Anterior stroma<br />Posterior stroma<br />Endothelium<br />
  23. 23. Quantitative <br />analysis<br />Research applications: <br />Descriptor & Discriminator<br />Basal epithelium<br />Qualitative analysis:<br />Honey-comb appearance<br />Bright borders<br />Dark homogenous bodies<br />Jonathan Kersley Lecture 2011<br />
  24. 24. Quantitative <br />analysis<br />Research applications: <br />Descriptor & Discriminator<br />Wing epithelial cells<br />Qualitative analysis:<br />50% presence <br />resembling “fried eggs”<br />bright borders<br />homogenous cytoplasm<br />bright nuclei<br />Occasionally:<br />very bright bodies<br />grouped in duplets,<br />bigger groups<br />up to 10 cells <br />Jonathan Kersley Lecture 2011<br />
  25. 25. Research applications: <br />Descriptor & Discriminator<br />Sub-basal nerve plexus<br />Qualitative analysis:<br />Bright, linear<br />Branching<br />Beadings<br />Jonathan Kersley Lecture 2011<br />
  26. 26. ...thickness of the sample...<br />5 mm<br />3 mm<br />… however, confocal microscopy is not perfect... <br />...optical shadowing...<br /> … ghost images...<br />...artefacts…...<br />?<br />
  27. 27. Research applications: <br />Descriptor & Discriminator<br />AnalySIS<br />Nerve density:<br />- calibration<br />- outlining <br />- reading<br />Jonathan Kersley Lecture 2011<br />
  28. 28. Research applications: <br />Descriptor & Discriminator<br />AnalySIS<br />Nerve diameter:<br />10 randomly<br />distributed <br />readings/slide<br />Jonathan Kersley Lecture 2011<br />
  29. 29. Research applications: <br />Descriptor & Discriminator<br />AnalySIS<br />Nerve beadings:<br />N of beadings<br />per 100 mm<br />Jonathan Kersley Lecture 2011<br />
  30. 30. Research applications: <br />Descriptor & Discriminator<br />Group 1 Group 2 Statistical <br /> significance <br />Nerve 632.35287.57 582.39327.13 P<0.005 <br />density m/mm2 m/mm2<br />Nerve <br /> 0.520.23m 0.560.27m P=0.133<br />fiber <br />diameter<br />Beadings 213123/mm 201192/mm P=0.078 <br />
  31. 31. degeneration (13.5 h PM)*<br />artefacts<br />static evaluation<br />availability of tissue <br />*Muller LJ, et al Invest Ophthamol Vis Sci 1997;38:985-94<br />Research applications: <br />Descriptor & Discriminator<br />Any considerations ?<br />Why in vivo analysis?<br />movement<br />corneal transparency<br />optical phenomena <br />Jonathan Kersley Lecture 2011<br />
  32. 32. Research applications: <br />Descriptor & Discriminator<br />
  33. 33. 3 D reconstructor<br />Allows:<br />precise sectioning<br /> no “movement”artefacts<br /> labelling of the structures<br /> different lasers<br />Ex vivo confocal microscopy<br />...but:<br /> static examination<br /> tissue processing artefacts<br /> preparation difficulties<br /> expensive<br />Jonathan Kersley Lecture 2011<br />
  34. 34. 3 D reconstructor<br />Epithelium <br />(DAPI)<br />Keratocytes<br /> (cell tracker)<br />X=500mm<br />Y=500mm<br />Z= 10mm<br />Red-green 3D <br />Jonathan Kersley Lecture 2011<br />
  35. 35. Z<br />Optical dissector - a probe which samples <br />isolated particles with uniform probability,<br />in three dimensional space irrespective <br />of their size and shape*<br />Z<br />*Gundersen, H.J., et al., Apmis, 1988. 96(10): p. 857-81.<br />3 D reconstructor<br /> X<br />Y<br />
  36. 36. 4mm<br />5mm<br />X-Y measurement<br />3 D reconstructor<br />Accuracy of the slit-scanning in vivo confocal microscope….<br />Artificial cornea:<br />Agarose (refractive index 1.3345)<br />Latex calibration spheres <br />Size: 7.5, 10, 15.5 and 20.1m <br />Jonathan Kersley Lecture 2011<br />
  37. 37. 3 D reconstructor<br />Jonathan Kersley Lecture 2011<br />
  38. 38. *<br />n<br />*<br />N/vol=<br />*<br />*<br />*<br />*<br />x.y.z<br />*<br />*<br />*<br />*<br />*<br />*<br />*<br />*<br />*<br />*<br />3 D reconstructor<br />How to select the cells?<br />Z distance?<br />z=16 mm<br />x/y=0.03 mm2<br />n=15<br />Jonathan Kersley Lecture 2011<br />
  39. 39. 4 D reconstructor<br />Time<br />Structure of a rat cornea<br />Female Wistar rats (d33, n=4)<br />Experimental model of<br />post-traumatic corneal oedema<br />Dynamics were followed by <br />in vivo confocal microscopy<br />Ex vivo confocal microscopy <br />performed as a control<br />
  40. 40. Rat cornea - 48 hours after trauma:<br /> “epithelialisation of the endothelium” <br />rounded, non-hexagonal, cells <br />with prominent nuclei <br />Fully restored normal endothelial <br />anatomy 72 hours after trauma<br />4 D reconstructor<br />A human cornea with clinical <br />and in vivo microscopical <br />diagnosis of ICE <br />
  41. 41. 4 D reconstructor<br />H&E staining of the rat cornea (right), in comparison to the ex vivo in situ <br />confocal microscopical observations of the cornea labeled with cytokeratin <br />AE1/AE3 (left). Note the intense labeling of the epithelium and absence of <br />staining at the level of the endothelium (arrows).<br />
  42. 42. Clinical applications<br />
  43. 43. *Gahl WA, et a.l Corneal crystals <br />in nephropathic cystinosis: natural <br />history and treatment with cysteamine <br />eyedrops. Mol Genet Metab 2000;71:100-20.<br />Clinical applications: <br />Description & Discrimination<br />Case of nephropathic cystinosis<br />Photophobia<br />Discomfort<br />VA 6/9 OU<br />IOP 12&14 mm/Hg<br />Slit-lamp biomicroscopy<br />Gahl grade 2-3<br />Normal fundus<br />C/D=0.2 OU<br />
  44. 44. b, c, d) <br />Middle stroma - <br />fewer larger & <br />smaller needle <br />shaped crystals<br />e, f)<br />Anterior stroma - <br />criss-crossing crystals <br />(5741m to 2117m)<br />In vivo confocal microscopy<br />a) <br />Pre-Descemet’s region<br />crystals measured <br />8537m to 4329m <br />
  45. 45. Clinical applications: <br />Description & Discrimination<br />UBM of advanced cystinosis<br />In vivo confocal microscopy <br />of advanced cystinosis<br /><ul><li>Same age groups
  46. 46. Transverse vs. a/p sections
  47. 47. Resolution: UBM<IVCF
  48. 48. Measurement options: UBM<IVCF
  49. 49. Area covered: UBM>IVCF</li></li></ul><li>Clinical applications: <br />Description & Discrimination<br />Case of Scheie’s syndrome<br />Photophobia<br />Discomfort<br />VA 6/12 OU (HM glasses)<br />Slit-lamp biomicroscopy<br />Altered transparency<br />Obscuration of the iris details<br />Granular haze (high mag.)<br />Normal fundus<br />C/D=0.1 OU<br />Jonathan Kersley Lecture 2011<br />
  50. 50. Pre-Descemet’s stroma:<br />“cellular conglomerations” (a), stromal matrix<br />brighter than normal cornea (b) <br />Normal endothelium <br />In vivo confocal microscopy<br />Posterior corneal stroma:<br />round or elliptical in shape <br />keratocytes with <br />prominent dark centers (a, c)<br />normal stroma, at corresponding <br />levels (b, d)<br />
  51. 51. Clinical applications: <br />Description & Discrimination<br />Clinico-pathological correlation<br /> round regular shape<br /> fibrillogranular material*<br /> increased reflectivity<br /> mucopolysaccharide deposits**<br /> prominent nuclei<br /> collagen fibers hypertrophy **<br /> increased background reflectivity<br /> electron clear/dense inclusions**<br />* Zabel RW et al. Ophthalmology 1989, 96:1631-8<br />** Tabone E et al. Virchows Archiv. 1978, 27:63-7<br />
  52. 52. Clinical applications: <br />Description & Discrimination<br />Differential diagnosis:<br />...linear structures at endothelial level...<br />Case 1<br />Haab’sstriae<br />PPD<br />Birth trauma<br />Unusual lattice<br />Keratoconus<br />Traumatic rupture<br />CHED<br />Jonathan Kersley Lecture 2011<br />
  53. 53. Clinical applications: <br />Description & Discrimination<br />Differential diagnosis:<br />…guttata - like changes...<br />Case 2<br />Fuchs’ endothelial dystrophy<br />Irido-corneal endothelial s-m<br />Posterior polymorphous dystrophy<br />Jonathan Kersley Lecture 2011<br />
  54. 54. Case 1<br />Case 2<br />endothelial vesicular <br />lesions, composed of <br />optically dense material<br />Elevated elliptical areas,<br />small ( 6-16 m) cells <br /> 5238155 cells/ mm2<br />well-demarcated <br />curvilinear bands <br />“optical shadowing” with<br /> no detectable details <br />vesicles protruding into AC<br />posterior concavity <br />small, dark,<br /> vesicle-like structures <br />In vivo confocal microscopy<br />
  55. 55. Clinical applications: <br />Description & Discrimination<br />In vivo confocal microscopy:<br />PosteriorPolymorphousDystrophy (PPD)<br />
  56. 56. “beaten metal appearance”<br />(retro-illumination) <br />Clinical applications: <br />Description & Discrimination<br />20 corneae<br />Fuchs’ ED<br />clinically<br />in vivo confocal microscopy<br />Slit-lamp photograph <br />of<br />Fuchs’ endothelial<br />dystrophy<br />Jonathan Kersley Lecture 2011<br />
  57. 57. In vivo confocal classification<br />A Grade 1 Classic guttata with <br />endothelial mosaic (more than 50 %)<br />A<br />B<br />B Grade 2 Guttata predominance <br />(more than 50 %)<br />C Grade 3 Corrugated endothelium <br />&some areas with detectable <br />endothelial pattern <br />C<br />D<br />DGrade 4 “Strawberry-like” <br />endothelial structure is replaced <br />by acellular areas<br />Clinical applications: <br />Description & Discrimination<br />Jonathan Kersley Lecture 2011<br />
  58. 58. Clinical applications: <br />Description & Discrimination<br />A Fine dark lines at the level of <br />Descemet’s membrane presumed <br />to be folds characteristic of <br />early an stage<br />B Wider and deeper bands <br />believed to be sign of more<br />advanced stages<br />C Fibrosis at the level of Bowman’s <br />layer <br />D Cystic spaces thought to <br />correspond with epithelial oedema<br />Jonathan Kersley Lecture 2011<br />
  59. 59. 25000<br />20000<br />15000<br />10000<br />5000<br />0<br />1<br />2<br />3<br />4<br />Group<br />Clinical applications: <br />Description & Discrimination<br />Three-dimensional analysis of the Fuchs’ endothelial dystrophy<br />Apoptosis<br />Initial stress<br />
  60. 60. Clinical applications: Prognosis<br />Prognostic markers of Fuchs’ endothelial dystrophy<br />Endothelial appearance (Grade 1-4)<br />Keratocyte density (?apoptosis)<br />Bownan’s layer fibrosis<br />Cystic basal epithelial oedema<br />Corneal tickness<br />
  61. 61. Clinical applications: Follow up<br />Subject 1 - coincidental <br />observation<br />Two children of subject 1<br />
  62. 62. Clinical applications: Follow up<br />Subject 4<br />Subject 5<br />
  63. 63. Subject 4<br />Subject 5<br />In vivo confocal microscopy <br />Subject 1<br />GRUPCHEVA CN, MALIK TY1, CRAIG JP, SHERWIN T, MCGHEE CNJ. <br />Microstructural assessment of rare corneal dystrophies using real-time in vivo confocal microscopy'.<br /> Clinical and Experimental Ophthalmology 2001;29:281-5.<br />
  64. 64. 28/02/00<br />20/08/00<br />over time<br />In vivo confocal microscopy <br />Subject 1<br />
  65. 65. Clinical applications: <br />Description & Discrimination<br />MDF<br />Jonathan Kersley Lecture 2011<br />
  66. 66. Clinical applications: <br />Description & Discrimination<br />Jonathan Kersley Lecture 2011<br />
  67. 67. 'Modern Anterior Segment Surgery and advancing contact lens technology'<br />Jonathan Kersley<br />(1939-2000)<br />
  68. 68. Vogt’s striae <br />Fleisher ring <br />Acute hydrops <br />Munson’s sign <br />Scarring <br />Keratoconus<br />Clinical signs<br />
  69. 69. 12.5%<br />75%<br />12.5%<br />Orbscan II topography <br />Keratometric map classification according to Rabinowitz: <br />class 3<br />class 1<br />class 2<br />increased area of <br />corneal power<br />inferior/superior <br />asymmetry<br />skewing or <br />lazy-eight configuration<br />
  70. 70. RR-50%<br />RS-37.5%<br />R-12.5%<br />R-100 %<br />Orbscan II topography <br />Elevation maps<br />
  71. 71. In vivo confocal microscopy<br />
  72. 72. Ex vivo confocal microscopy<br />
  73. 73. Ex vivo confocal microscopy<br />
  74. 74. Provides aids to the diagnosis, prognosis and <br />follow up <br />Must be interpreted in the clinical context<br />Conclusions<br />In vivo confocal microscopy is а readily <br />available, reliable non-invasive clinical tool <br />
  75. 75. In vivo confocal microscopy is ideal for optical<br />dissection, however, some considerations for<br />living subjects apply<br />In vivo confocal microscopy can be used for<br />4D analyses and dynamic follow up<br />Conclusions<br />In vivo confocal microscopy is an useful tool<br />for instantaneous observation and <br />measurement of corneal micro-structural <br />elements<br />
  76. 76. The future…...<br />Improve hardware<br />Clinico-pathological <br />correlations<br />Topographic repeatability<br />Decreased examination time<br />Optimise 4D analysis<br />Other AS pathology<br />Combined with aberrometry<br />Improved software<br />
  77. 77. Jonathan Kersley<br />(1939-2000)<br />“…spending most time developing and nurturing a network of friendships and personal relationships that would lead naturally to Scientific co-operation between the various National Societies. Co-operation and communication can only flourish in such an atmosphere . <br />If I have achieved this to any degree, <br />I am happy"<br />
  78. 78. Big thank you !!!<br />ECLSO friends<br />European friends and collaborators<br />All international friends and collaborators<br />My Bulgarian friends and employee <br />…all of you who keeping me alive and creative<br />
  79. 79. ECLSO London 2005<br />

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