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 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 Determine predictive model of cell counting error based on amount of deviance from true endothelial cell shape and size

4. Sample Area: 39981.10μm 2 Cells Counted: 113 Density: 2826 High Resolution Analysis (640x480 pixels)

5. Sample Area: 44843.25μm 2 Cells Counted: 113 Density: 2520 High Resolution Analysis (640x480 pixels)

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

7. Calibration Lens Baseline focal distance = 0.000mm

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

9. Focal Deviance = 0.030mm

10. Focal Deviance = 0.060mm

11. Focal Deviance = 0.100mm

12. Results

13. Focal Deviance (F) Mean Area (A) F=30μmA=9,666μm 2 F=40μmA=9,444μm 2 F=50μmA=9,180μm 2 F=60μmA=9,055μm 2 F=70μmA=8,869μm 2 F=80μmA=8,767μm 2 F=90μmA=8,694μm 2 F=100μmA=8,467μm 2Results

14. Projected cell counting error Conclusion

15. Focal Deviance Cell Density Error 0-29μm Negligible (<100 cells/mm 2 ) 30-59μm Non-negligible (>100 cells/mm 2 ) 60-100μm Problematic (>200 cells/mm 2 ) Conclusion

16. Focal Deviance = 0.100mm

17. Focal Deviance = 0.060mm

18. Focal Deviance = 0.030mm

19. Focal Deviance = 0.000mm

20. Focal Deviance Measured Area Endothelial Cell Density F=0μm42,061μm 2 2377 cells/mm 2 F=30μm39,893μm 2 2507 2507 cells/mm 2 F=60μm37,792μm 2 2646 2646 cells/mm 2 F=100μm36,711μm 2 2724 2724 cells/mm 2 TrueArea Endothelial Cell Density 42,061μm 2 2377 cells/mm 2

21. Discussion Minimizing focal deviance is essential to capturing true area Minimizing focal deviance is essential to capturing true area Higher resolution images allow better judgment of focal deviance Higher resolution images allow better judgment of focal deviance Lower resolution images obscure focal deviance due to compression Lower resolution images obscure focal deviance due to compression

22. Focal Deviance = 0.100mm 640x480160x120

23. Focal Deviance = 0.060mm 640x480160x120

24. Focal Deviance = 0.030mm 640x480160x120

25. Focal Deviance = 0.000mm 640x480160x120

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

27. Focal Deviance = 0.000mm 640x480

28. Focal Deviance = 0.030mm 640x480

29. Focal Deviance = 0.060mm 640x480

30. Focal Deviance = 0.100mm 640x480

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

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