A brief explanation about confocal microscope concept and findings

1. CONFOCAL MICROSCOPY
OF THE
NORMAL HUMAN CORNEA
MOHD ZHARIF BIN MOHD NOR
P 82505

2. Learning Outcomes
 Know the optical principle, optical design
and construction of confocal
microscopes.
 Know how to operate confocal
microscope and to do patient examination

3. 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

4. INSTRUMENT OPERATION
Consist 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

5.  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

6. PATIENT EXAMINATION
One 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

7. RESULTS AND DISCUSSION
 EPITHELIUM
- Superficial, wing and basal
 ANTERIOR LIMITING LAMINA (Bowman’s
membrane)
 STROMA
 POSTERIOR LIMITING LAMINA (Descemet’s
membrane)
 ENDOTHELIUM

8. 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)

9. 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)

10. 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

11. 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

12. 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

13. ANTERIOR STROMA
 The nuclei
vary from
cigar shape
to round
 Variation in
apparent size
of keratocyte
nuclei

14. 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

15. POSTERIOR LIMITTING
LAMINA
 Featureless
 Appear to be more granular in elderly patient

16. ENDOTHELIUM
 5, 6 and 7 sided cell
 Lightly reflective
cytoplasm
 Clearly defined black
cell border

17.  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

18. CONCLUSION
Confocal microscopy can develop a better
understanding of normal cornea structure and
function and abnormal cornea condition.