11. 1995 Slit-scanning in vivo confocal microscopy
12. 2003 Laser-scanning in vivo confocal microscopyJonathan Kersley Lecture 2011
13. Tandem-scanning in vivo confocal microscopy White light Scanning spot or scanning slit Incident light is focussed in same plane as objective lens i.e. confocal Objective Nipkow lens Beam Disk Light splitter source Video camera CORNEA Dual light path technique (Tandem scanning) Single light path technique Jonathan Kersley Lecture 2011
14. Slit-scanning in vivo confocal microscopy Light source Condenser lens Front lens Scanning slits Condenser lens Video camera Cornea Jonathan Kersley Lecture 2011
15. Confoscan 2 (model 2000) improved NAVIS software Confoscan 2 (model 1999) In vivo confocal microscopy Jonathan Kersley Lecture 2011
16. Tandem scanning confocal microscopy Precise movement of the disk Applanation technology ? Small round area Low illumination One scan per examination Time consuming Slit- scanning confocal microscopy Greater illumination Adjustable parameters Relatively quick Non-applanation technology Small oblong area Moving objective lens In vivo confocal microscopy Jonathan Kersley Lecture 2011
23. Quantitative analysis Research applications: Descriptor & Discriminator Basal epithelium Qualitative analysis: Honey-comb appearance Bright borders Dark homogenous bodies Jonathan Kersley Lecture 2011
24. Quantitative analysis Research applications: Descriptor & Discriminator Wing epithelial cells Qualitative analysis: 50% presence resembling “fried eggs” bright borders homogenous cytoplasm bright nuclei Occasionally: very bright bodies grouped in duplets, bigger groups up to 10 cells Jonathan Kersley Lecture 2011
25. Research applications: Descriptor & Discriminator Sub-basal nerve plexus Qualitative analysis: Bright, linear Branching Beadings Jonathan Kersley Lecture 2011
26. ...thickness of the sample... 5 mm 3 mm … however, confocal microscopy is not perfect... ...optical shadowing... … ghost images... ...artefacts…... ?
27. Research applications: Descriptor & Discriminator AnalySIS Nerve density: - calibration - outlining - reading Jonathan Kersley Lecture 2011
28. Research applications: Descriptor & Discriminator AnalySIS Nerve diameter: 10 randomly distributed readings/slide Jonathan Kersley Lecture 2011
29. Research applications: Descriptor & Discriminator AnalySIS Nerve beadings: N of beadings per 100 mm Jonathan Kersley Lecture 2011
30. Research applications: Descriptor & Discriminator Group 1 Group 2 Statistical significance Nerve 632.35287.57 582.39327.13 P<0.005 density m/mm2 m/mm2 Nerve 0.520.23m 0.560.27m P=0.133 fiber diameter Beadings 213123/mm 201192/mm P=0.078
31. degeneration (13.5 h PM)* artefacts static evaluation availability of tissue *Muller LJ, et al Invest Ophthamol Vis Sci 1997;38:985-94 Research applications: Descriptor & Discriminator Any considerations ? Why in vivo analysis? movement corneal transparency optical phenomena Jonathan Kersley Lecture 2011
33. 3 D reconstructor Allows: precise sectioning no “movement”artefacts labelling of the structures different lasers Ex vivo confocal microscopy ...but: static examination tissue processing artefacts preparation difficulties expensive Jonathan Kersley Lecture 2011
34. 3 D reconstructor Epithelium (DAPI) Keratocytes (cell tracker) X=500mm Y=500mm Z= 10mm Red-green 3D Jonathan Kersley Lecture 2011
35. Z Optical dissector - a probe which samples isolated particles with uniform probability, in three dimensional space irrespective of their size and shape* Z *Gundersen, H.J., et al., Apmis, 1988. 96(10): p. 857-81. 3 D reconstructor X Y
36. 4mm 5mm X-Y measurement 3 D reconstructor Accuracy of the slit-scanning in vivo confocal microscope…. Artificial cornea: Agarose (refractive index 1.3345) Latex calibration spheres Size: 7.5, 10, 15.5 and 20.1m Jonathan Kersley Lecture 2011
38. * n * N/vol= * * * * x.y.z * * * * * * * * * * 3 D reconstructor How to select the cells? Z distance? z=16 mm x/y=0.03 mm2 n=15 Jonathan Kersley Lecture 2011
39. 4 D reconstructor Time Structure of a rat cornea Female Wistar rats (d33, n=4) Experimental model of post-traumatic corneal oedema Dynamics were followed by in vivo confocal microscopy Ex vivo confocal microscopy performed as a control
40. Rat cornea - 48 hours after trauma: “epithelialisation of the endothelium” rounded, non-hexagonal, cells with prominent nuclei Fully restored normal endothelial anatomy 72 hours after trauma 4 D reconstructor A human cornea with clinical and in vivo microscopical diagnosis of ICE
41. 4 D reconstructor H&E staining of the rat cornea (right), in comparison to the ex vivo in situ confocal microscopical observations of the cornea labeled with cytokeratin AE1/AE3 (left). Note the intense labeling of the epithelium and absence of staining at the level of the endothelium (arrows).
43. *Gahl WA, et a.l Corneal crystals in nephropathic cystinosis: natural history and treatment with cysteamine eyedrops. Mol Genet Metab 2000;71:100-20. Clinical applications: Description & Discrimination Case of nephropathic cystinosis Photophobia Discomfort VA 6/9 OU IOP 12&14 mm/Hg Slit-lamp biomicroscopy Gahl grade 2-3 Normal fundus C/D=0.2 OU
44. b, c, d) Middle stroma - fewer larger & smaller needle shaped crystals e, f) Anterior stroma - criss-crossing crystals (5741m to 2117m) In vivo confocal microscopy a) Pre-Descemet’s region crystals measured 8537m to 4329m
50. Pre-Descemet’s stroma: “cellular conglomerations” (a), stromal matrix brighter than normal cornea (b) Normal endothelium In vivo confocal microscopy Posterior corneal stroma: round or elliptical in shape keratocytes with prominent dark centers (a, c) normal stroma, at corresponding levels (b, d)
52. Clinical applications: Description & Discrimination Differential diagnosis: ...linear structures at endothelial level... Case 1 Haab’sstriae PPD Birth trauma Unusual lattice Keratoconus Traumatic rupture CHED Jonathan Kersley Lecture 2011
53. Clinical applications: Description & Discrimination Differential diagnosis: …guttata - like changes... Case 2 Fuchs’ endothelial dystrophy Irido-corneal endothelial s-m Posterior polymorphous dystrophy Jonathan Kersley Lecture 2011
54. Case 1 Case 2 endothelial vesicular lesions, composed of optically dense material Elevated elliptical areas, small ( 6-16 m) cells 5238155 cells/ mm2 well-demarcated curvilinear bands “optical shadowing” with no detectable details vesicles protruding into AC posterior concavity small, dark, vesicle-like structures In vivo confocal microscopy
55. Clinical applications: Description & Discrimination In vivo confocal microscopy: PosteriorPolymorphousDystrophy (PPD)
56. “beaten metal appearance” (retro-illumination) Clinical applications: Description & Discrimination 20 corneae Fuchs’ ED clinically in vivo confocal microscopy Slit-lamp photograph of Fuchs’ endothelial dystrophy Jonathan Kersley Lecture 2011
57. In vivo confocal classification A Grade 1 Classic guttata with endothelial mosaic (more than 50 %) A B B Grade 2 Guttata predominance (more than 50 %) C Grade 3 Corrugated endothelium &some areas with detectable endothelial pattern C D DGrade 4 “Strawberry-like” endothelial structure is replaced by acellular areas Clinical applications: Description & Discrimination Jonathan Kersley Lecture 2011
58. Clinical applications: Description & Discrimination A Fine dark lines at the level of Descemet’s membrane presumed to be folds characteristic of early an stage B Wider and deeper bands believed to be sign of more advanced stages C Fibrosis at the level of Bowman’s layer D Cystic spaces thought to correspond with epithelial oedema Jonathan Kersley Lecture 2011
59. 25000 20000 15000 10000 5000 0 1 2 3 4 Group Clinical applications: Description & Discrimination Three-dimensional analysis of the Fuchs’ endothelial dystrophy Apoptosis Initial stress
63. Subject 4 Subject 5 In vivo confocal microscopy Subject 1 GRUPCHEVA CN, MALIK TY1, CRAIG JP, SHERWIN T, MCGHEE CNJ. Microstructural assessment of rare corneal dystrophies using real-time in vivo confocal microscopy'. Clinical and Experimental Ophthalmology 2001;29:281-5.
69. 12.5% 75% 12.5% Orbscan II topography Keratometric map classification according to Rabinowitz: class 3 class 1 class 2 increased area of corneal power inferior/superior asymmetry skewing or lazy-eight configuration
74. Provides aids to the diagnosis, prognosis and follow up Must be interpreted in the clinical context Conclusions In vivo confocal microscopy is а readily available, reliable non-invasive clinical tool
75. In vivo confocal microscopy is ideal for optical dissection, however, some considerations for living subjects apply In vivo confocal microscopy can be used for 4D analyses and dynamic follow up Conclusions In vivo confocal microscopy is an useful tool for instantaneous observation and measurement of corneal micro-structural elements
76. The future…... Improve hardware Clinico-pathological correlations Topographic repeatability Decreased examination time Optimise 4D analysis Other AS pathology Combined with aberrometry Improved software
77. Jonathan Kersley (1939-2000) “…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 . If I have achieved this to any degree, I am happy"
78. Big thank you !!! ECLSO friends European friends and collaborators All international friends and collaborators My Bulgarian friends and employee …all of you who keeping me alive and creative