Effect of Specular Focal Distance on Endothelial Cell Density Accuracy

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Effect of Specular Focal Distance on Endothelial Cell Density Accuracy

  1. 1. Effect of Specular Focal Distanceon Endothelial Cell Counting Accuracy Jackie Hai and Vivian Xue HAI Laboratories, Inc.
  2. 2. Special Thanksfor providing research cornea
  3. 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. 4. High Resolution Analysis Sample Area: 39981.10μm2(640x480 pixels) Cells Counted: 113 Density: 2826
  5. 5. High Resolution Analysis Sample Area: 44843.25μm2(640x480 pixels) Cells Counted: 113 Density: 2520
  6. 6. Method High resolution specular image of standard calibration lens Baseline pachymetry of 0.000mm established for focused image
  7. 7. Calibration LensBaseline focal distance = 0.000mm
  8. 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. 9. Focal Deviance = 0.030mm
  10. 10. Focal Deviance = 0.060mm
  11. 11. Focal Deviance = 0.100mm
  12. 12. Results
  13. 13. ResultsFocal 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. 14. ResultsProjected cell counting error
  15. 15. Endothelial Cell Density 2500 cells/mm2 Focal Measured DifferenceDeviance Density from Original F=0μm 2500 +0F=30μm 2615 +115F=60μm 2750 +250F=100μm 2953 +453
  16. 16. Endothelial Cell Density 3000 cells/mm2 Focal Measured DifferenceDeviance Density from Original F=0μm 3000 +0F=30μm 3137 +137F=60μm 3300 +300F=100μm 3544 +544
  17. 17. ConclusionFocal Deviance Cell Density Error Negligible 0-29μm (<100 cells/mm2) Non-negligible 30-59μm (>100 cells/mm2) Problematic 60-100μm (>200 cells/mm2)
  18. 18. Focal Deviance = 0.100mm
  19. 19. Focal Deviance = 0.060mm
  20. 20. Focal Deviance = 0.030mm
  21. 21. Focal Deviance = 0.000mm
  22. 22. True Endothelial Area Cell Density 42,061μm2 2377 cells/mm2 Focal Measured EndothelialDeviance Area Cell Density F=0μm 42,061μm2 2377 cells/mm2F=30μm 39,893μm2 2507 cells/mm2F=60μm 37,792μm2 2646 cells/mm2F=100μm 36,711μm2 2724 cells/mm2
  23. 23. Qualitative Properties In Focus Out of FocusCell borders are bold Cell borders are fuzzyand clearly visible and poorly visibleHigh contrast between Low contrast betweencell borders and interiors cell borders and interiorsCell surfaces are even Cell surfaces are unevenand consistently lit and/or have hot spotsCell morphology includes Cell morphology appearsregular polygons flattened or stretched
  24. 24. Focal Deviance = 0.000mm 640x480
  25. 25. Focal Deviance = 0.030mm 640x480
  26. 26. Focal Deviance = 0.060mm 640x480
  27. 27. Focal Deviance = 0.100mm 640x480
  28. 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. 29. Focal Deviance = 0.100mm 640x480 160x120
  30. 30. Focal Deviance = 0.060mm 640x480 160x120
  31. 31. Focal Deviance = 0.030mm 640x480 160x120
  32. 32. Focal Deviance = 0.000mm 640x480 160x120
  33. 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. 34. Recommendations  Capture specular images at the highest possible resolution (e.g. 640x480 pixels)  Select flat areas of endothelial cells to perform density analysishttp://www.hailabs.com/specular-microscopy

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