it is the ppt on confocal mictoscopy of eye

1. IN VIVO CONFOCAL
MICROSCOPY
Presented by
Dr Rohit rao

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.  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. Con – focal

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. The confocal microscope
Detector
Scan excitation spot pointby-point to build up image
Pinhole
Tube lens
Emission light
Objective lens
Sample
Excitation light

7. Types of confocal microscopy
 Tandem scanning-based confocal microscopy
 Scanning slit confocal microscopy
 Laser scanning confocal microscopy

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. 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. 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.  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).

14.  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.

15. Confocal images of corneal layers
 Tear Film

16. Epithelium

18.  Corneal erosion.
 Confocal images of corneal
 Note the visible anterior stroma

19.  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

20.  Filamentous keratitis in a case
of Sjögren’s syndrome.

21.  Vernal keratoconjunctivitis.
 Trantas dots.
 Confocal in vivo microscopy of Trantas
dots: microcysts and inflammatory
 cells (hyperreflective cells).

22.  Calcific band keratopathy. Hyperreflective
 areas in the corneal epithelium

23. 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.

24. 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.

25.  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

26. 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

27. 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.

30.  Pigment dispersion: pigment deposits on the endothelium

31.  Trabeculum.
 Confocal in vivo microscopy image of Schwalbe’s ring and
trabecular meshwork.
 Ex vivo histologic image of the same area

32. 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).

34. 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.

35.  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.

36. 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.

38. 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.

39.  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.

41. 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

42. Epithelial basement membrane dystrophy
 Epithelial basement membrane dystrophy (map-dot-fingerprint
dystrophy) with fingerprint like corneal lesions.
 Linear hyperreflective

43. 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.

44. 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

45. 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

46. Fuch’s Corneal Dystrophy
 Corneal guttata with stromal edema•
 Central beaten metal-like endothelial changes with or without
pigment dusting

47. 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 .

48.  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

50. Lens

51. Iris

52. PEX

53. Limbus

54. Conjunctiva

56.  Pterygium.
 Superficial confocal
in vivo microscopy
image of pterygium:
microcysts between
conjunctival epithelial
cells.
 Reflective stroma of
pterygium

57. Nonfunctioning blebs.
Functioning blebs

58. Thank You