1. Effect of Specular Focal Distance
on Endothelial Cell Counting Accuracy
Jackie Hai and Vivian Xue
HAI Laboratories, Inc.

2. Special Thanks
for providing research cornea

3. Purpose
 Quantify the relationship between
focal distance and image distortion
in specular microscopy
 Determine predictive model of cell
counting error based on amount of
deviance from true endothelial cell
shape and size

4. High Resolution Analysis Sample Area: 39981.10μm2
(640x480 pixels) Cells Counted: 113
Density: 2826

5. High Resolution Analysis Sample Area: 44843.25μm2
(640x480 pixels) Cells Counted: 113
Density: 2520

6. Method
 High resolution specular image of
standard calibration lens
 Baseline pachymetry of 0.000mm
established for focused image

7. Calibration Lens
Baseline focal distance = 0.000mm

8. Method
 Images captured 30μm, 40μm, 50μm,
30 40 50
60μm, 70μm, 80μm, 90μm and 100μm
60 70 80 90 100
from baseline focal distance
 10 sample area measurements per
image (single blind trial)
 Determine mean area of each image
 Simple linear regression model

9. Focal Deviance = 0.030mm

10. Focal Deviance = 0.060mm

11. Focal Deviance = 0.100mm

12. Results

13. Results
Focal Deviance (F) Mean Area (A)
F=30μm A=9,666μm2
F=40μm A=9,444μm2
F=50μm A=9,180μm2
F=60μm A=9,055μm2
F=70μm A=8,869μm2
F=80μm A=8,767μm2
F=90μm A=8,694μm2
F=100μm A=8,467μm2

14. Results
Projected cell counting error

15. Endothelial Cell Density
2500 cells/mm2
Focal Measured Difference
Deviance Density from Original
F=0μm 2500 +0
F=30μm 2615 +115
F=60μm 2750 +250
F=100μm 2953 +453

16. Endothelial Cell Density
3000 cells/mm2
Focal Measured Difference
Deviance Density from Original
F=0μm 3000 +0
F=30μm 3137 +137
F=60μm 3300 +300
F=100μm 3544 +544

17. Conclusion
Focal Deviance Cell Density Error
Negligible
0-29μm
(<100 cells/mm2)
Non-negligible
30-59μm
(>100 cells/mm2)
Problematic
60-100μm
(>200 cells/mm2)

18. Focal Deviance = 0.100mm

19. Focal Deviance = 0.060mm

20. Focal Deviance = 0.030mm

21. Focal Deviance = 0.000mm

22. True Endothelial
Area Cell Density
42,061μm2 2377 cells/mm2
Focal Measured Endothelial
Deviance Area Cell Density
F=0μm 42,061μm2 2377 cells/mm2
F=30μm 39,893μm2 2507 cells/mm2
F=60μm 37,792μm2 2646 cells/mm2
F=100μm 36,711μm2 2724 cells/mm2

23. Qualitative Properties
In Focus Out of Focus
Cell borders are bold Cell borders are fuzzy
and clearly visible and poorly visible
High contrast between Low contrast between
cell borders and interiors cell borders and interiors
Cell surfaces are even Cell surfaces are uneven
and consistently lit and/or have hot spots
Cell morphology includes Cell morphology appears
regular polygons flattened or stretched

24. Focal Deviance = 0.000mm
640x480

25. Focal Deviance = 0.030mm
640x480

26. Focal Deviance = 0.060mm
640x480

27. Focal Deviance = 0.100mm
640x480

28. Discussion
 Minimizing focal deviance is
essential to capturing true area
and attaining accurate cell density
 Higher resolution images allow
better judgment of focal deviance
 Lower resolution images obscure
focal deviance due to compression

29. Focal Deviance = 0.100mm
640x480 160x120

30. Focal Deviance = 0.060mm
640x480 160x120

31. Focal Deviance = 0.030mm
640x480 160x120

32. Focal Deviance = 0.000mm
640x480 160x120

33. Recommendations
 Allow donor tissue to warm up to
room temperature
 Use both coarse and fine Z-knobs
to attain optimum focus
 If it is difficult to focus, change
the angle of the chamber or vial

34. Recommendations
 Capture specular images at the
highest possible resolution (e.g.
640x480 pixels)
 Select flat areas of endothelial
cells to perform density analysis
http://www.hailabs.com/specular-microscopy