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
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
22. True Endothelial
Area Cell Density
42,061μm2 2377 cells/mm2
Focal Measured Endothelial
Deviance Area Cell Density
F=0μm 42,061μm2 2377 cells/mm2
F=30μm 39,893μm2 2507 cells/mm2
F=60μm 37,792μm2 2646 cells/mm2
F=100μm 36,711μm2 2724 cells/mm2
23. Qualitative Properties
In Focus Out of Focus
Cell borders are bold Cell borders are fuzzy
and clearly visible and poorly visible
High contrast between Low contrast between
cell borders and interiors cell borders and interiors
Cell surfaces are even Cell surfaces are uneven
and consistently lit and/or have hot spots
Cell morphology includes Cell morphology appears
regular polygons flattened or stretched
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
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. 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