Confocal microscopy - a new method for evaluation of cornea

1. CONFOCAL MICROSCOPY

2. Introduction
• 1st developed by Marvin& Minsky
• Advantages over conventional light microscope
• Provides high resolution images with better
rejection of out of focus images
• Optical sectioning ability allows image to be
obtained from different depths within a thick
tissue specimen
• Eliminates need for processing & sectioning
specimens

3. Advantages over specular microscpe
• It is useful even in presence of corneal edema
& scarring
• It permits a 3D optical sectioning of the
cornea where as specular is restricted to a
single cell plane

4. Why it is called confocal
• Minskys microscope had a condensor that
focussed light source within a small area of
the tissue with concomitant focussing of
microscope objective lens on same area
• Since both condensor&objective had same
focal point it was called confocal

5. Types of confocal systems in clinical
use
• 1.Tandem scanning confocal microscope/TSCM
• PRINCIPLE
• Uses a modified Nipkow disc containing
thousands of optically conjugate pinholes
arranged in Archemedian spirals
• Light from a broad light source passes through
source pinholes on one side of the disc& is
focussed on to the specimen

6. • Detector pinholes on opposite side of the disc
prevent light from outside the optical volume
determined by objective lens & pinhole
diameter from reaching the eye piece
• Rotation of disc allows even scanning of tissue
in real time
• Because illumination& detection of light
through conjugate pinholes occurs in tandom
this microscope is called as TSCM
• ADV-it uses a broad light source which causes
less tissue damage compared to laser
• It provides a shallower depth of field

7. • 2.Scanning slit confocal microscope
• More user friendly& commonly used
• 3.Laser scanning confocal microscope
• 670 nm laser beam
• Highest resolution of 1-2

8. CORNEAL STRUCTURES SEEN
• TSCM-Epithelial cells&subepithelial fine nerve
plexus
• SSCM-Epithelium,subepithelial nerve plexus&
endothelium
• LSCM-wing cells,langherhan cells,stromal
cells&endothelium

10. Clinical application
• 1.infectious keratitis
• Localisation of acanthamoeba cysts &
trophozoites in living eye
• Detection of fungal keratitis
• Filaments of fusarium,aspergillus
• Bacterial contact lens induced keratitis
• Microsporidial keratitis
• Borrelia keratitis
• To asess corneal response after refractive
surgeries like PRK& LASIK