Figure 4

Ambient light subtraction.

When imaging fluorescence during daytime, subtraction of the pixel values of an image that contains only ambient light from an image taken with the blue strobes on produces a derived fluorescence image, similar to a nighttime image taken directly with no ambient light. As the strobe duration is fixed (on the order of a few ms), increasing the exposure time increases the intensity of the ambient illumination in the image, while keeping the same fluorescence intensity. Thus, imaging the same scene with different shutter speeds allowed us to test how varying contamination levels from ambient light affected the resulting fluorescence image. In the daytime images shown, the shutter speed was 1/80s, three times the minimum possible shutter speed (1/250s), to allow high levels of ambient light in the image. For both these exposure times (1/80s and 1/250s) the accuracy of the ambient light subtraction method was 85% compared to nighttime GFP/CHL ratios for the coral on the left and 90% for the coral on the right. However, when the exposure time was increased to 1/50s the accuracies dropped significantly, suggesting that there was a point where the fluorescence values relative to ambient light dropped below noise levels. Images were taken in Bocas Del Toro, Panama at a depth of 5 m.