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

Ahmad Kheirkhah, Ujwala S. Saboo, Anna Marmalidou, Reza Dana

Research output: Contribution to journalArticlepeer-review

7 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)363-369
Number of pages7
Issue number3
StatePublished - Jan 1 2016
Externally publishedYes


  • Cornea
  • Endothelial cells
  • In vivo confocal microscopy

ASJC Scopus subject areas

  • Ophthalmology


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