Journal: The British Journal of Ophthalmology
Article Title: Global cell-by-cell evaluation of endothelial viability after two methods of graft preparation in Descemet membrane endothelial keratoplasty
doi: 10.1136/bjophthalmol-2015-307534
Figure Lengend Snippet: (A) TRITC/ethidium channel image showing manual demarcation of graft edge using the polygon selection toll in Image J (yellow outline). (B) Yellow line defined in the TRITC/ethidium image is copied and applied to the FITC/calcein channel image. The edge of calcein-positive area is defined in the same way as the graft edge (blue outline). The difference in the areas of the yellow and blue selections was defined as the trephination damage area. (C) The FITC/calcein channel image is thresholded and segmented. The area of living cells (red area) is used to create a selection mask (green lines). (D) Ultraviolet/Hoechst channel images were colour inverted and the viable graft area selection applied (green outline). Each nucleus (black dot) within the selection mask is counted. Zoomed in area (top right) showing all nuclei within the mask have been counted, as shown by the presence of a red dot in the centre of nucleus. Nuclei outside the mask are not counted (no red dot in centre of nucleus). (E) TRITC/ethidium channel image is inverted, and the living area selection is applied (green outline). Each nucleus (black dot) within the selection mask is counted. Zoomed in area (top right) showing ethidium-staining nuclei within the mask have been counted, as shown by the presence of a red dot in the centre of nucleus.
Article Snippet: Each sample was covered with 250 μL of BSS containing Hoechst 33342 (10 μM), ethidium homodimer II(4 μM) and calcein-AM (2 μM) (Invitrogen, Carlsbad, California, USA) and incubated at 37°C for 30 min. Hoechst 33342 and ethidium homodimer II are nuclear binding dyes that do not fluoresce until they are bound to nuclear DNA.
Techniques: Selection, Staining
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