Figure 8
From: Near infra-red labelling and tracking of corneal endothelial cells in-vivo
Labelling of ex vivo human corneas. Human corneas were dual stained with Calcein AM and DIR dyes. (a) After preparation for implantation, the DMEK graft, resting on stroma, is returned to the viewing chamber and imaged using the FFA filter set to detect Calcein AM stained cells. Note the presence of a triangular orientation mark. Uniform green fluorescence is seen as areas of healthy cells and black areas represent dead or absent cells. (b) The same transplant is imaged using the ICG filters to detect DIR staining. There is marked variation in fluorescence across the graft meaning portions become either over or under exposed on global imaging. The yellow arrow points to an area of high DIR fluorescence. Comparison with the Calcein AM staining shows that most of the graft, including this area is cover by viable cells. (c) Global Calcien AM imaging of the graft, in vivo, immediately following transplantation shows a new area of cell death attributable to implantation trauma (blue arrow) (d) Global DIR imaging of the graft, in vivo, immediately following transplantation shows the same variation of fluorescence (e) High magnification imaging acquired by coupling a ×40 air objective microscope lens to the retinal imaging lens of the HFA/Spectralis. Individual Calcein AM negative cells are seen. (d) DIR imaging using the same lens shows DIR fluorescence in the cells that appeared non-fluorescent on the global image, suggesting the level of fluorescence is variable, but present across the entire graft. At this time point, comparison between DIR and Calcein AM imaging shows DIR does not differentiate between living and dead, but attached, cells. (f) At 24 h post transplantation, Calcein AM fluorescence has diminished to the point where now useful images can be obtained. (h) By contrast, at 24 h post-implantation, DIR fluorescence has become more uniform and areas corresponding to pre-identified dead cells can be seen to be devoid of DIR staining, suggesting the cells have been ejected from the monolayer (blue arrow). Scale bar (a–d and g–h): 1 mm, Scale bar (e,f): 250 µm.
