effect of specular focal distance on endothelial cell density accuracy
TRANSCRIPT
Effect of Specular Focal DistanceEffect of Specular Focal Distanceon Endothelial Cell Counting Accuracyon Endothelial Cell Counting Accuracy
Jackie Hai and Vivian XueJackie Hai and Vivian XueHAI Laboratories, Inc.HAI Laboratories, Inc.
PurposePurpose
Quantify the relationship between Quantify the relationship between focal distance and image distortion focal distance and image distortion in specular microscopyin specular microscopy
Determine predictive model of cell Determine predictive model of cell counting error based on amount of counting error based on amount of deviance from true endothelial cell deviance from true endothelial cell shape and sizeshape and size
MethodMethod
High resolution specular image of High resolution specular image of standard calibration lensstandard calibration lens
Baseline pachymetry of 0.000mm Baseline pachymetry of 0.000mm established for focused imageestablished for focused image
Images captured 30Images captured 30μm, 40, 40μm, 50, 50μm, , 6060μm, 70, 70μm, 80, 80μm, 90, 90μm and 100 and 100μm from baseline focal distancefrom baseline focal distance
10 sample area measurements per 10 sample area measurements per image (single blind trial)image (single blind trial)
Determine mean area of each imageDetermine mean area of each image
Simple linear regression modelSimple linear regression model
MethodMethod
A=8,869μm2 F=70μmA=8,767μm2 F=80μmA=8,694μm2 F=90μm
A=9,666μm2 F=30μmA=9,444μm2 F=40μm
A=8,467μm2 F=100μm
A=9,055μm2 F=60μmA=9,180μm2 F=50μm
Mean Area (A)Focal Deviance (F)
ResultsResults
2953
2750
2615
2500
Measured Density
+0 F=0μm
+115 F=30μm
+453 F=100μm
+250 F=60μm
Difference from Original
Focal Deviance
2500 cells/mm2
Endothelial Cell Density
3544
3300
3137
3000
Measured Density
+0 F=0μm
+137 F=30μm
+544 F=100μm
+300 F=60μm
Difference from Original
Focal Deviance
3000 cells/mm2
Endothelial Cell Density
Problematic(>200 cells/mm2)
60-100μm
Non-negligible(>100 cells/mm2)
30-59μm
Negligible(<100 cells/mm2)
0-29μm
Cell Density ErrorFocal Deviance
Conclusion
36,711μm2
37,792μm2
39,893μm2
42,061μm2
Measured Area
2377 cells/mm2 F=0μm
2507 cells/mm2 F=30μm
2724 cells/mm2 F=100μm
2646 cells/mm2 F=60μm
Endothelial Cell Density
Focal Deviance
2377 cells/mm242,061μm2
Endothelial Cell Density
TrueArea
Qualitative Properties
Cell surfaces are uneven and/or have hot spots
Cell surfaces are even and consistently lit
Cell morphology appears flattened or stretched
Cell morphology includes regular polygons
Low contrast between cell borders and interiors
High contrast between cell borders and interiors
Cell borders are fuzzyand poorly visible
Cell borders are boldand clearly visible
Out of FocusIn Focus
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
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