IN VIVO CONFOCAL
MICROSCOPY
Presented by
Dr Rohit rao
References
1. Clinical applications of corneal confocal microscopy by Tavakoli M, Hossain P,
and Malik R A , Clin Ophthalm...
 In 1968, the same year that Maurice described the first highpowered specular microscope, the first scanning confocal
mic...
Con – focal
 While using light biomicroscopy, the resolution is decreased by
interference of light reflected from structures above an...
The confocal microscope
Detector

Scan excitation spot pointby-point to build up image

Pinhole

Tube lens

Emission light...
Types of confocal microscopy

 Tandem scanning-based confocal microscopy
 Scanning slit confocal microscopy
 Laser scan...
Tandem scanning-based confocal microscopy

The tandem scanning confocal microscope (TSCM). (A) An illustration of the opti...
Scanning slit confocal microscopy
 Techniques based on the
principles of the rotating Nipkow
disk or tandem slit-scanning...
Laser scanning confocal microscopy

 As an alternative to confocal slit-scanning microscopes,a confocal
laser scanning mi...
 The HRT II has been modified with a lens system attachment known
as the Rostock Cornea Module
 The distance from the co...
 The primary advantage of laser scanning confocal microscopy is the
ability to serially produce images of thin layers fro...
Confocal images of corneal layers
 Tear Film
Epithelium
 Corneal erosion.
 Confocal images of corneal
 Note the visible anterior stroma
 Keratoconjunctivitis sicca.
 Severe punctate keratitis on fluorescein staining
 Confocal in vivo microscopy images sho...
 Filamentous keratitis in a case
of Sjögren’s syndrome.
 Vernal keratoconjunctivitis.
 Trantas dots.
 Confocal in vivo microscopy of Trantas
dots: microcysts and inflammatory
...
 Calcific band keratopathy. Hyperreflective
 areas in the corneal epithelium
Bowman’s layer
 Amorphous membrane located immediately posterior to the basal
epithelium
 10 μm thick and is made of col...
Corneal stroma
 90% of the thickness of the cornea and is composed of collagen
fibers, interstitial substance and keratoc...
 Keratocyte nuclei are 5–30 μm in diameter.
 Discrete bright entities against a grey background.
 Cytoplasm, cell walls...
Descemet’s membrane
 Basement membrane of the corneal
endothelium
 Images of Descemet’s membrane
have a generalized hazy...
Corneal endothelium
 Single layer of endothelial cells which are 4–6 μm thick and 20 μm
in diameter with a hexagonal or p...
 Pigment dispersion: pigment deposits on the endothelium
 Trabeculum.
 Confocal in vivo microscopy image of Schwalbe’s ring and
trabecular meshwork.
 Ex vivo histologic image o...
Measuring corneal thickness with corneal
confocal microscopy
 One of the most important advances in confocal imaging has ...
Infectious keratitis

 In microbial keratitis, early diagnosis is of major importance as delay
in appropriate treatment c...
 PCR tests take only one day before results become available.
 IVCM, on the other hand, has the potential to identify
Ac...
Acanthamoeba keratitis
 Acanthamoeba keratitis is suspected when a ring infiltrate and radial
perineuritis have developed...
Fungal keratitis

 Gold standard for diagnosis is corneal smear or culture.
 Because both have a varying sensitivity and...
 At IVCM, hyphae of filamentous fungi appear as hyperreflective,
interlocking white lines of 5-10 μm in diameter and 200-...
Corneal dystrophies
 Meesmann’s corneal dystrophy.

 Multiple epithelial cystic lesions.
 Confocal in vivo microscopy
i...
Epithelial basement membrane dystrophy
 Epithelial basement membrane dystrophy (map-dot-fingerprint
dystrophy) with finge...
Thiel-Behnke dystrophy
 Thiel-Behnke corneal dystrophy. Honeycomb-shaped gray opacities
were observed at the level of Bow...
Reis-Bücklers
 Slit-lamp biomicroscopic photograph of Reis-Bücklers corneal
dystrophy. Bilateral gray-white, amorphous op...
Lattice dystrophy
 Slit-lamp biomicroscopic photograph of lattice corneal dystrophy
showed radially oriented thick lattic...
Fuch’s Corneal Dystrophy
 Corneal guttata with stromal edema•
 Central beaten metal-like endothelial changes with or wit...
Contact lens-induced corneal changes

 Confocal microscopy has been used to study contact lens-induced
corneal changes
 ...
 Contact lens induces the release of inflammatory mediators that
may cause keratocyte dysgenesis or apoptosis.
 A reduct...
Lens
Iris
PEX
Limbus
Conjunctiva
 Pterygium.
 Superficial confocal
in vivo microscopy
image of pterygium:
microcysts between
conjunctival epithelial
cell...
Nonfunctioning blebs.

Functioning blebs
Thank You
Confocal microscopy of the eye
Confocal microscopy of the eye
Confocal microscopy of the eye
Confocal microscopy of the eye
Confocal microscopy of the eye
Confocal microscopy of the eye
Confocal microscopy of the eye
Confocal microscopy of the eye
Confocal microscopy of the eye
Confocal microscopy of the eye
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Confocal microscopy of the eye

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Confocal microscopy of the eye

  1. 1. IN VIVO CONFOCAL MICROSCOPY Presented by Dr Rohit rao
  2. 2. References 1. Clinical applications of corneal confocal microscopy by Tavakoli M, Hossain P, and Malik R A , Clin Ophthalmol. 2008 June; 2(2): 435–445. 2. In vivo confocal microscopy, an inner vision of the cornea – a major review by Guthoff R F & et al, Clinical & Experimental Ophthalmology Volume 37, Issue 1,pages 100–117, January/February 2009 3. Atlas of Confocal Laser Scanning In-vivo 4. Microscopy in Opthalmology – Principles and Applications in Diagnostic and Therapeutic Ophtalmology by R.F.Guthoff 5. Confocal Microscopy: When Is it Helpful to Diagnose Corneal and Conjunctival Disease? By Elisabeth M. Messmer, Medscape 6. In vivo confocal microscopy of the human cornea by I Jalbert & et al, Br J Ophthalmol 2003;87:225–236 7. Cornea, 3rd Edition By Jay H. Krachmer 8. In vivo confocal microscopy Expanding horizons in corneal imaging by Toine Hillenaar 9. In Vivo Biopsy of the Human Cornea By Akira Kobayashi, Hideaki Yokogawa and Kazuhisa Sugiyama
  3. 3.  In 1968, the same year that Maurice described the first highpowered specular microscope, the first scanning confocal microscope was proposed  Minsky (1988) developed the original confocal microscope in 1955 to image brain cells and study neural networks in the living brain.
  4. 4. Con – focal
  5. 5.  While using light biomicroscopy, the resolution is decreased by interference of light reflected from structures above and below the plane of examination.  Principle  Single point of tissue can be illuminated by a point light source and simultaneously imaged by a camera in the same plane, ie, it is “confocal”. This produces an image with a very high resolution but it has virtually no field of view due to a single point of illumination and detection.  To solve this problem, the instrument instantaneously illuminates and synchronously images, ie, scans, a small region of tissue with thousands of tiny spots of light which are reconstructed to create a usable field of view with high resolution and magnification.
  6. 6. The confocal microscope Detector Scan excitation spot pointby-point to build up image Pinhole Tube lens Emission light Objective lens Sample Excitation light
  7. 7. Types of confocal microscopy  Tandem scanning-based confocal microscopy  Scanning slit confocal microscopy  Laser scanning confocal microscopy
  8. 8. Tandem scanning-based confocal microscopy The tandem scanning confocal microscope (TSCM). (A) An illustration of the optical pathway used in TSCM. Light from a broadband source (1) passes through the pinholes on one side of a Nipkow disk (2) and a beam splitter (3), and is focused by an objective lens (4) into the specimen (5). The reflected or emitted signal is then reflected by the beam splitter (3) and front surface mirror (6) to the conjugate pinholes on the opposite side of the disk, which prevent light from outside the optical volume from reaching a camera or eyepiece. Rotation of the disk results in even scanning of the tissue in real time.
  9. 9. Scanning slit confocal microscopy  Techniques based on the principles of the rotating Nipkow disk or tandem slit-scanning  Uses of a halogen lamp.  The slit height can be adjusted, which allows the user to vary the thickness of the optical section, and the slit width adjustment allows control of the amount of light that reaches the cornea.
  10. 10. Laser scanning confocal microscopy  As an alternative to confocal slit-scanning microscopes,a confocal laser scanning microscope for the anterior segment of the eye was developed at the Rostock Eye Clinic (Germany)  The original functions of the basic HRT II device for evaluating the optic nerve head in glaucoma are fully retained when the system is modified into the confocal laser microscope.
  11. 11.  The HRT II has been modified with a lens system attachment known as the Rostock Cornea Module  The distance from the cornea to the microscope is kept stable by use of a single-use contact element in sterile packaging (TomoCap®).  Polymethyl methacrylate (PMMA).
  12. 12.  The primary advantage of laser scanning confocal microscopy is the ability to serially produce images of thin layers from the cornea.  According to this the depth of focus for the TSCM (Tandem scanning confocal microscope) is 7–9 μm, and in slit scanning systems it is 26 μm whilst it is 5–7 μm using the laser confocal microscope.
  13. 13. Confocal images of corneal layers  Tear Film
  14. 14. Epithelium
  15. 15.  Corneal erosion.  Confocal images of corneal  Note the visible anterior stroma
  16. 16.  Keratoconjunctivitis sicca.  Severe punctate keratitis on fluorescein staining  Confocal in vivo microscopy images showing corneal epithelial metaplasia with enlarged cells, activated nuclei, and decreased nucleus/cytoplasm ratio
  17. 17.  Filamentous keratitis in a case of Sjögren’s syndrome.
  18. 18.  Vernal keratoconjunctivitis.  Trantas dots.  Confocal in vivo microscopy of Trantas dots: microcysts and inflammatory  cells (hyperreflective cells).
  19. 19.  Calcific band keratopathy. Hyperreflective  areas in the corneal epithelium
  20. 20. Bowman’s layer  Amorphous membrane located immediately posterior to the basal epithelium  10 μm thick and is made of collagen fibers and contains unmyelinated c-nerve fibers.  Confocal microscopic images are featureless and grey, with discrete beaded nerve bundles of the sub-basal nerve plexus traversing the field of view.
  21. 21. Corneal stroma  90% of the thickness of the cornea and is composed of collagen fibers, interstitial substance and keratocytes.  Collagen fibers and interstitial substance are transparent and form the grey amorphous background.
  22. 22.  Keratocyte nuclei are 5–30 μm in diameter.  Discrete bright entities against a grey background.  Cytoplasm, cell walls and processes cannot be visualized.  Myelinated nerve fibers can also be seen in the anterior stroma
  23. 23. Descemet’s membrane  Basement membrane of the corneal endothelium  Images of Descemet’s membrane have a generalized hazy appearance and no cellular structures can be identified.  Normal Descemet’s membrane is not visible in young subjects but becomes more visible with increasing age
  24. 24. Corneal endothelium  Single layer of endothelial cells which are 4–6 μm thick and 20 μm in diameter with a hexagonal or polygonal shape  Bright cell bodies with dark cell borders.  Cell nuclei are rarely recognizable, and the cellular body is homogeneously bright with clearly defined borders.  Increasing age causes a reduction in endothelial cell density and increase in polymegathism.
  25. 25.  Pigment dispersion: pigment deposits on the endothelium
  26. 26.  Trabeculum.  Confocal in vivo microscopy image of Schwalbe’s ring and trabecular meshwork.  Ex vivo histologic image of the same area
  27. 27. Measuring corneal thickness with corneal confocal microscopy  One of the most important advances in confocal imaging has been the development of confocal microscopy ‘through focusing’ (CMTF) (also known as Z-Scan mode) which enables the measurement of corneal thickness.  As all points of the CTMF curve correlate directly with high resolution images,i.e epithelial surface, the sub epithelial nerve plexus, and the endothelium  These are used to precisely calculate the distance between the different corneal layers.  In a Z-Scan profile curve the percentage reflected light intensity (yaxis) is plotted against the distance in the cornea in μm (x-axis).
  28. 28. Infectious keratitis  In microbial keratitis, early diagnosis is of major importance as delay in appropriate treatment can lead to bad outcome.  To ascertain the causative agent, culture of corneal scrape specimens remains the gold standard.  Culture however,often takes three or more days before definite results become available.
  29. 29.  PCR tests take only one day before results become available.  IVCM, on the other hand, has the potential to identify Acanthamoeba and fungal keratitis, immediately  Also, differentiation between bacterial and viral keratitis has been suggested, based on a pathogen specific immune response.
  30. 30. Acanthamoeba keratitis  Acanthamoeba keratitis is suspected when a ring infiltrate and radial perineuritis have developed.  Acanthamoeba cysts and, to a lesser extent, trophozoites can be distinguished from the corneal cellular structures using IVCM.  Double-walled Acanthamoeba cysts appear as coffee bean-shaped hyperreflective structures 15-28 μm in diameter, whereas the trophozoites are larger measuring 25-40 μm.
  31. 31. Fungal keratitis  Gold standard for diagnosis is corneal smear or culture.  Because both have a varying sensitivity and fungal cultures take 2 or more weeks to become positive  IVCM has an important role in early detection of fungal keratitis.
  32. 32.  At IVCM, hyphae of filamentous fungi appear as hyperreflective, interlocking white lines of 5-10 μm in diameter and 200-400 μm in length, which branch dichotomously at a 45 degree angle (Aspergillus) or at a 90 degree angle (Fusarium).  To monitor and guide antifungal therapy.  Only method to determine the depth of invasion, prognostic factor in fungal keratitis.
  33. 33. Corneal dystrophies  Meesmann’s corneal dystrophy.  Multiple epithelial cystic lesions.  Confocal in vivo microscopy images. Microcysts are seen as hyporeflective areas in the basal epithelial layer.  Hyperreflective dots are observed inside most of the microcysts
  34. 34. Epithelial basement membrane dystrophy  Epithelial basement membrane dystrophy (map-dot-fingerprint dystrophy) with fingerprint like corneal lesions.  Linear hyperreflective
  35. 35. Thiel-Behnke dystrophy  Thiel-Behnke corneal dystrophy. Honeycomb-shaped gray opacities were observed at the level of Bowman’s layer.  In vivo laser confocal microscopy showed focal deposition of homogeneous reflective materials with round-shaped edges in the basal epithelial layer. All deposits accompanied dark shadows.
  36. 36. Reis-Bücklers  Slit-lamp biomicroscopic photograph of Reis-Bücklers corneal dystrophy. Bilateral gray-white, amorphous opacities of various sizes at the level of Bowman’s layer were observed.  In vivo laser confocal microscopy showed focal deposition of highly reflective irregular and granular materials in the basal epithelial layer. No deposits accompanied dark shadows
  37. 37. Lattice dystrophy  Slit-lamp biomicroscopic photograph of lattice corneal dystrophy showed radially oriented thick lattice lines in the stroma.  In vivo laser confocal microscopy showed highly reflective latticeshaped materials in the stromal layer
  38. 38. Fuch’s Corneal Dystrophy  Corneal guttata with stromal edema•  Central beaten metal-like endothelial changes with or without pigment dusting
  39. 39. Contact lens-induced corneal changes  Confocal microscopy has been used to study contact lens-induced corneal changes  Confocal microscopy has led to the identification of a new type of chronic stromal change in patients who wear contact lenses  Extended contact lens wear causes a loss of keratocytes .
  40. 40.  Contact lens induces the release of inflammatory mediators that may cause keratocyte dysgenesis or apoptosis.  A reduction in corneal sensitivity occurs in patients with long term contact lens wear  But neither short-term (overnight wear) nor long-term (12 months extended wear) soft contact lens wear appears to affect the morphology and/or distribution of corneal nerves viewed using confocal microscopy
  41. 41. Lens
  42. 42. Iris
  43. 43. PEX
  44. 44. Limbus
  45. 45. Conjunctiva
  46. 46.  Pterygium.  Superficial confocal in vivo microscopy image of pterygium: microcysts between conjunctival epithelial cells.  Reflective stroma of pterygium
  47. 47. Nonfunctioning blebs. Functioning blebs
  48. 48. Thank You
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