Using Scuba for In Situ Determination of Chlorophyll Distributions in Corals by Underwater Near Infrared Fluorescence Imaging

Studies reporting quantitation and imaging of chlorophyll in corals using visible fluorescent emission in the red near 680 nm can suffer from competing emission from other red-emitting pigments. Here, we report a novel method of selectively imaging chlorophyll distributions in coral in situ using only the near infrared (NIR) fluorescence emission from chlorophyll. Commercially available equipment was assembled that allowed the sequential imaging of visible, visible-fluorescent, and NIR-fluorescent pigments on the same corals. The relative distributions of chlorophyll and fluorescent proteins (GFPs) were examined in numerous corals in the Caribbean Sea, the Egyptian Red Sea, the Indonesian Dampier Strait, and the Florida Keys. Below 2 m depth, solar induced NIR chlorophyll fluorescence can be imaged in daylight without external lighting, thus, it is much easier to do than visible fluorescence imaging done at night. The distributions of chlorophyll and GFPs are unique in every species examined, and while there are some tissues where both fluorophores are co-resident, often tissues are selectively enriched in only one of these fluorescent pigments. Although laboratory studies have clearly shown that GFPs can be photo-protective, their inability to prevent large scale bleaching events in situ may be due to their limited tissue distribution.