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