CONFOCAL MICROSCOPY OF THE

NORMAL HUMAN CORNEA

MOHD ZHARIF BIN MOHD NORP 82505

Learning Outcomes

Know the optical principle, optical design and construction of confocal microscopes.

Know how to operate confocal microscope and to do patient examination

OPTICAL DESIGN OF THE CONFOCAL MICROSCOPES

resolution, field of view (opposite with slit lamp)

Illuminating a small region of cornea with thousands of tiny spots of light each sec, with each one being synchronously imaged

Single point in the tissue is both optically illuminated from a point light source and simultaneously imaged by a point detector in same focal plane (confocal)

Only one layer of cornea tissue is observed at one time

INSTRUMENT OPERATIONConsist of: Confocal microscope Halogen lamp Camera Light source Objective lens Base unit (x-y-z direction) Adjustable chin and head

rest

Tomey confoScan P4 in-vivo slit-scanning real-time confocal microscope

Halogen lamp:• 12 V/100W => steady, even illumination• Filters => screen out excessive UV and infrared radiation

3 Acroplan immersion objective lens:• 20X/0.4• 40X/0.7 – most suited for general imaging• 63X/0.9

Epiplan 20X/0.5 non-contact dry lens

25 images were captured each second and were viewed in real time computer screen and stored in video tape

PATIENT EXAMINATIONOne drop anaesthetic (benoxinate hydrochloride 0.4%)

Viscotears liquid gel (CIBA vision)

Computer monitor dispayed real time images

Z control-control back and forth slowly and steadily through the entire corneal

thickness

X and Y adjusted-bright, even image

Repeated fellow eye

Gaze straight ahead

RESULTS AND DISCUSSION EPITHELIUM- Superficial, wing and basal ANTERIOR LIMITING LAMINA (Bowman’s

membrane) STROMA POSTERIOR LIMITING LAMINA

(Descemet’s membrane) ENDOTHELIUM

EPITHELIUM (SUPERFICIAL CELL)

40-50 µm in diameter

Large variation in brightness and granularity from cell to cell

Small, bright , rounded nuclei 10 µm in diameter (visible in small number of cell)

EPITHELIUM (WING CELLS) 30-45 µm in diameter Rounded in shape Have 5-10 side Some side are straight

and some are curved Some have light grey

cytoplasm and others dark grey

Small, discrete, bright nuclei (5-8µm)

EPITHELIUM (BASAL CELL) Tightly packed (appear

as a uniform field of bright cell borders and dark cytoplasmic mass

columnar in shape Appear to be smaller in

diameter than superficial and wing cell (approximately 10 µm)

Cell nuclei are not visible

ANTERIOR LIMITTING LAMINA

Featureless Have bright nerve fibres

of subepithelial neural plexus traverse

Odd keratocyte or patch of basal epithelial cell sometimes can be seen

More hazy in older patient

STROMA Collagen fibres and ground substances cannot be

imaged Keratocytes described as discrete bright entities Nerve fibre are slightly thicker and brighter than

in limitting lamina Other cell like monocyte, polymorphonuclear

leukocytes are not observed

ANTERIOR STROMA

The nuclei vary from cigar shape to round

Variation in apparent size of keratocyte nuclei

POSTERIOR STROMA Keratocyte are

less dense packed

Nuclei appear slighty larger and flatter

‘folds’ can be observed in 10, 18,29% of population in 5th, 6th, and 7th decade of life

POSTERIOR LIMITTING LAMINA

Featureless Appear to be more granular in elderly patient

ENDOTHELIUM 5, 6 and 7 sided cell

Lightly reflective cytoplasm

Clearly defined black cell border

Irregularities in endothelium:

- guttae that are observed in 6,12 and 29% of population in 5th, 6th and 7th decade of life

Guttae in the endothelium of a 76 year old female

CONCLUSION

Confocal microscopy can develop a better understanding of normal cornea structure and function and abnormal

cornea condition.