Overestimation of corneal endothelial cell density in smaller frame sizes in In Vivo confocal microscopy

Purpose: To evaluate the effect of frame size on the calculated corneal endothelial cell density (CECD) in images of laser scanning in vivo confocal microscopy (IVCM). Methods: Forty-nine corneal endothelial images acquired by laser scanning 1VCM (Heidelberg Retina Tomograph 3 with Rostock Corneal Module) with different endothelial cell densities were analyzed. In each image (160,000 μm²), die CECD was calculated using the fixed-frame method by counting cells in the following frame sizes: 80,000 μm², 40,000 μm², 20,000 μm², 10,000 μm², 5000 μm², and 2500 μm². The calculated CECD was then compared with that of the variable-frame method as the reference value. Results: There was no significant difference in die calculated CECD between the variable-frame method (2004 ± 832 cells/mm²), and the fixed-frame method using a 40,000-μm² frame (2023 ±810 cells/mm²). On the other hand, the calculated CECD showed significant overestimations in frame sizes of 20,000 jinr (2066 ± 820 cells/mm²), 10,000 μm² (2156 ± 785 cells/mm²), 5000 \μm² (2352 ± 783 cells/mm²), and 2500 \μm² (2715 ± 754 cells/mm²), with P < 0.001 in all. This resulted in overestimations of 4.8 ± 9.8%, 11.9 ± 16.2%, 24.9 ± 23.1%, and 49.1 ± 38.8% for these frame sizes, respectively. Images with lower CECD demonstrated higher overestimations of cell density in smaller frame sizes. Conclusions: In laser scanning IVC'M images, there is significant overestimation of CECD if the celLs are counted in frames smaller than 25% of the image. Similar frame sizes should be used when monitoring CECD over time.