confocal microscope presentation
TRANSCRIPT
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.