Confocal microscopy

CONFOCAL MICROSCOPY

1. CONFOCAL MICROSCOPY PREPARED BY: NURUL AKIMI BINTI ABDULLAH P82506

2. INTRODUCTION Confocal  a single point of tissue can be illuminated by a point light source and simultaneously imaged by a camera in the same plane able to serially produce images of thin layers from the cornea A confocal microscope creates sharp images of a specimen Image had better contrast & less hazy

3. Principal of CM

4. It was pioneered by marvin Minsky in 1955 Illuminating a single point at a time – avoid most of the unwanted scatter light that obscure image a non invasive technique for in vivo imaging of the living cornea to investigate numerous corneal diseases

5. rapidly produce images of cell morphology without the need to process the tissue Very high mag & resolution with small field of view All corneal layers can be seen in a single cross-section images comparable to in-vitro histochemical techniques delineating all 5 corneal layers

6. Corneal epithelium consists of three layers – the superficial cells, wing cells and basal cells  Confocal microscopic images of epithelial layer of cornea: (a) the superficial epithelial cells, (b) wing cells, (c) basal cells.

7. Corneal epithelium Superficial cells polygonal, 40–50 μm in diameter and approximately 5 μm thick  exhibit small bright rounded nuclei, surrounded by a darker cytoplasm with a perinuclear hypo reflective dark ring and a well defined cell border darker cells being those about to desquamate

8. Corneal epithelium Wing cells the intermediate level of epithelial cells variable in size and shape ( 30–45 μm ) bright cell borders and nucleus Basal cells 10–15 μm in diameter nuclei not visible uniformly bright cell border with a dark cytoplasmic mass

9. Bowman’s membrane (anterior limiting lamina ) posterior basal epithelium approximately 10 μm thick made of collagen fibers and contains unmyelinated c-nerve fibers  featureless and grey discrete beaded nerve bundles of the sub-basal nerve plexus traversing the field of view increasing age results in a decrease in nerve fiber density

10. Confocal microscopic images of Bowman’s membrane with nerve fibres

11. Corneal stroma 90% of corneal thickness composed of collagen fibers, interstitial substance and keratocytes  collagen fibers and interstitial substance form grey background   Keratocyte nuclei varied shape bean-like in anterior stroma oval shaped in posterior stroma discrete bright entities against a grey background

12. Confocal microscopic images of the stroma

13. Descemet’s membrane ( posterior limiting lamina ) basement membrane of corneal endothelium generalized hazy appearance no identified cellular structures more visible with increasing age

14. Corneal endothelium single layer of endothelial cells 4–6 μm thick and 20 μm in diameter hexagonal or polygonal shape bright cell bodies with dark cell borders Increasing age causes a reduction in endothelial cell density by approximately 0.6% a year and an increase in polymegathism

15. Confocal microscopic image of Endothelium.

16. THANK YOU