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.
Special ThanksSpecial Thanks
for providing research corneafor providing research cornea
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
Sample Area: 39981.10μm2
Cells Counted: 113Density: 2826
High Resolution Analysis(640x480 pixels)
Sample Area: 44843.25μm2
Cells Counted: 113Density: 2520
High Resolution Analysis(640x480 pixels)
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
Calibration LensCalibration Lens
Baseline focal distance = 0.000mmBaseline focal distance = 0.000mm
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
Focal Deviance = 0.030mmFocal Deviance = 0.030mm
Focal Deviance = 0.060mmFocal Deviance = 0.060mm
Focal Deviance = 0.100mmFocal Deviance = 0.100mm
ResultsResults
Focal Deviance (F)Focal Deviance (F) Mean Area (A)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
ResultsResults
Projected cell counting errorProjected cell counting error
ResultsResults
Focal Focal DevianceDeviance
Measured Measured DensityDensity
Difference Difference from Originalfrom Original
F=0μm 2500 +0
F=30μm 2615 +115+115
F=60μm 2750 +250+250
F=100μm 2953 +453+453
Endothelial Cell Endothelial Cell DensityDensity
2500 cells/mm2
Focal Focal DevianceDeviance
Measured Measured DensityDensity
Difference Difference from Originalfrom Original
F=0μm 3000 +0
F=30μm 3137 +137+137
F=60μm 3300 +300+300
F=100μm 3544 +544+544
Endothelial Cell Endothelial Cell DensityDensity
3000 cells/mm2
Focal DevianceFocal Deviance Cell Density ErrorCell Density Error
0-29μmNegligibleNegligible
(<100 cells/mm(<100 cells/mm22))
30-59μm Non-negligibleNon-negligible
(>100 cells/mm(>100 cells/mm22))
60-100μm ProblematicProblematic
(>200 cells/mm(>200 cells/mm22))
ConclusionConclusion
Focal Deviance = 0.100mmFocal Deviance = 0.100mm
Focal Deviance = 0.060mmFocal Deviance = 0.060mm
Focal Deviance = 0.030mmFocal Deviance = 0.030mm
Focal Deviance = 0.000mmFocal Deviance = 0.000mm
Focal Focal DevianceDeviance
Measured Measured AreaArea
Endothelial Endothelial Cell DensityCell Density
F=0μm 42,061μm2 2377 cells/mm2
F=30μm 39,893μm2 2507 2507 cells/mm2
F=60μm 37,792μm2 2646 2646 cells/mm2
F=100μm 36,711μm2 2724 2724 cells/mm2
TrueTrueAreaArea
Endothelial Endothelial Cell DensityCell Density
42,061μm2 2377 cells/mm2
Qualitative PropertiesQualitative PropertiesIn FocusIn Focus Out of FocusOut of Focus
Cell borders are boldand clearly visible
Cell borders are fuzzyCell borders are fuzzy
and poorly visibleand poorly visible
High contrast between cell borders and interiors
Low contrast between Low contrast between cell borders and interiorscell borders and interiors
Cell surfaces are even and consistently lit
Cell surfaces are uneven Cell surfaces are uneven and/or have hot spotsand/or have hot spots
Cell morphology includes regular polygons
Cell morphology appears Cell morphology appears flattened or stretchedflattened or stretched
Focal Deviance = 0.000mmFocal Deviance = 0.000mm
640x480640x480
Focal Deviance = 0.030mmFocal Deviance = 0.030mm
640x480640x480
Focal Deviance = 0.060mmFocal Deviance = 0.060mm
640x480640x480
Focal Deviance = 0.100mmFocal Deviance = 0.100mm
640x480640x480
DiscussionDiscussion
Minimizing focal deviance is Minimizing focal deviance is essential to capturing true area essential to capturing true area and attaining accurate cell densityand attaining accurate cell density
Higher resolution images allow Higher resolution images allow better judgment of focal deviancebetter judgment of focal deviance
Lower resolution images obscure Lower resolution images obscure focal deviance due to compressionfocal deviance due to compression
Focal Deviance = 0.100mmFocal Deviance = 0.100mm
640x480640x480 160x120160x120
Focal Deviance = 0.060mmFocal Deviance = 0.060mm
640x480640x480 160x120160x120
Focal Deviance = 0.030mmFocal Deviance = 0.030mm
640x480640x480 160x120160x120
Focal Deviance = 0.000mmFocal Deviance = 0.000mm
640x480640x480 160x120160x120
RecommendationsRecommendations
Allow donor tissue to warm up to Allow donor tissue to warm up to room temperatureroom temperature
Use both coarse and fine Z-knobs Use both coarse and fine Z-knobs to attain optimum focusto attain optimum focus
If it is difficult to focus, change the If it is difficult to focus, change the angle of the chamber or vialangle of the chamber or vial
RecommendationsRecommendations
Capture specular images at the Capture specular images at the highest possible resolution (e.g. highest possible resolution (e.g. 640x480 pixels)640x480 pixels)
Select flat areas of endothelial Select flat areas of endothelial cells to perform density analysiscells to perform density analysis
http://www.hailabs.com/specular-microscopyhttp://www.hailabs.com/specular-microscopy