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Effect of Specular Focal Distance on Endothelial Cell Counting Accuracy

This study by Jackie Hai and Vivian Xue explores the relationship between focal distance and image distortion in specular microscopy to create a predictive model for cell counting errors. Results indicate the importance of minimizing focal deviance for accurate cell counting. Recommendations include capturing images at high resolution and adjusting focus techniques. For more information, visit http://www.hailabs.com/specular-microscopy.

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Effect of Specular Focal Distance on Endothelial Cell Counting Accuracy

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  1. Effect of Specular Focal Distanceon 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 (640x480 pixels) Sample Area: 39981.10μm2 Cells Counted: 113 Density: 2826

  5. High Resolution Analysis (640x480 pixels) Sample Area: 44843.25μm2 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, 60μm, 70μm, 80μm, 90μm and 100μm 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

  14. Conclusion Projected cell counting error

  15. Conclusion

  16. Focal Deviance = 0.100mm

  17. Focal Deviance = 0.060mm

  18. Focal Deviance = 0.030mm

  19. Focal Deviance = 0.000mm

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

  21. Focal Deviance = 0.100mm 640x480 160x120

  22. Focal Deviance = 0.060mm 640x480 160x120

  23. Focal Deviance = 0.030mm 640x480 160x120

  24. Focal Deviance = 0.000mm 640x480 160x120

  25. Qualitative Properties

  26. Focal Deviance = 0.000mm 640x480

  27. Focal Deviance = 0.030mm 640x480

  28. Focal Deviance = 0.060mm 640x480

  29. Focal Deviance = 0.100mm 640x480

  30. 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

  31. 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

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