Fluorescence Lifetime Imaging of Symbionts and Fluorescent Proteins in Reef Corals

摘要

Reef-building corals are dependent on dinoflagellate algal symbionts (zooxanthellae). Within the range of habitats of any one coral species there can be huge variations in light intensities, so there is a risk of photoinhibition from excess light. In extremes of light and heat, senescent algae are expelled en masse, a phenomenon known as coral bleaching. In freshly isolated tissue the chlorophyll fluorescence has a lifetime of ～1.1ns. 6 hours and 15 hours after isolation the zooxanthellae looked visually healthy, but the lifetimes had increased to 2ns after 6 hours and 2.2ns after 15 hours. Zooxanthellae which were visibly damaged or necrotic had a mean lifetime of 3ns. Lifetime of chlorophyll fluorescence is thus a sensitive indicator, revealing effects in cell metabolism before any structural changes are evident. The occurrence of FRET between fluorescent proteins in corals has already been reported and time-resolved spectra have shown the effect on fluorescent lifetime, but without any spatial resolution. Lifetime confocal microscopy offers lower time resolution but excellent spatial resolution. Lifetimes of the isolated A. millepora pigments amilFP490, amilFP504 and amilFP593 (names indicate emission peaks) were 2.8ns, 2.9ns and 2.9ns respectively. In the coral sample, imaging the entire emission spectrum from 420nm, the mean lifetime was reduced to 1.5ns, implying that FRET was occurring. Looking just at the fluorescence from FRET donors the lifetime was even shorter, at 1.3ns, supporting this interpretation.