Effect of Specular Focal Distance on Endothelial Cell Density Accuracy

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

ResultsResults

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

Projected cell counting errorProjected cell counting error

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


Problematic(>200 cells/mm2)

60-100μm

Non-negligible(>100 cells/mm2)

30-59μm

Negligible(<100 cells/mm2)

0-29μm

Cell Density ErrorFocal Deviance

Conclusion

Focal Deviance = 0.100mm

36,711μm2

37,792μm2

39,893μm2

42,061μm2

Measured Area

2377 cells/mm2 F=0μm





Focal Deviance

2377 cells/mm242,061μm2


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


640x480

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


640x480 160x120

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

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

Effect of Specular Focal Distance on Endothelial Cell Density Accuracy

Health & Medicine

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