Automated assessment of keratocyte density in stromal images from the ConfoScan 4 confocal microscope

Purpose. To develop a program to determine cell densities in images from the ConfoScan 4 (Nidek, Inc., Freemont, CA) confocal microscope and compare the densities with those determined in images obtained by the Tandem Scanning confocal microscope (Tandem Scanning Corp., Reston, VA). Methods. A program was developed that used image-processing routines to identify stromal cell nuclei in images from the ConfoScan 4 confocal microscope. Cell selection parameters were set to match cell densities from the program with those determined manually in 15 normal corneas of 15 volunteers. The program was tested on scans from 16 other normal volunteers and 17 volunteers 3 years after LASIK. Cell densities were compared to densities determined by manual assessment and to those in scans by the Tandem Scanning confocal microscope in the same corneas. Results. The difference in cell density between the automatic and manual assessment was -539 +/- 3005 cells/mm(3) (mean +/- SD, P = 0.11) in the 16 test corneas. Densities estimated from the ConfoScan 4 agreed with those from the Tandem Scanning confocal microscope in all regions of the stroma except in the anterior 10%, where the ConfoScan 4 indicated a 30% lower density. Conclusions. Differences in anterior stromal cell density between the ConfoScan 4 and the Tandem Scanning confocal microscope can be explained by the different optical designs. The lower spatial resolution of the ConfoScan 4 limits its ability to resolve thin layers. The adaptation of our earlier cell-counting program to the ConfoScan 4 provides a timesaving, objective, and reproducible means of determining stromal cell densities in images from the ConfoScan 4.