Classification results for empirically-measured, fluorescence spectra.

<p>Water-column attenuation effects were applied for distances of 1 m and 3 m. Different numbers of example field spectra were used for each functional group. Example spectra from multiple genera were used for each group when possible. Erroneous classifications are indicated by the corresponding functional-group number. Asterisks denote groups for which classification success changed with increasing attenuation effects.</p

Water-column attenuation of fluorescence.

<p>The wavelength dependence of the diffuse attenuation coefficient (black) differentially attenuates the emission spectra of the pigments designated 486, 515, 575, and 685 (gray).</p

Classification results for <i>MAHAL</i>.

<p>Highlighted values indicate the minimum score for each band configuration.</p

Fluorescence functional groups.

<p>These 15 spectra denote the most common fluorescent emissions we have measured on Caribbean reefs. Note: All of the stony coral functional groups (1–7) also include a chlorophyll component. Fluorescence intensities are plotted in arbitrary units, and the spectra indicate typical peak-to-peak ratios. The spectra also illustrate actual inter-functional-group differences. For example, corals with pigments 486 and 515 (group 4) tend to fluorescence ‘brighter’ than the other groups, and chlorophyll fluorescence in soft corals (group 8) is approximately 4 times more intense than that in green or brown algae (group 9). The spectra used in the classification comparisons included the effects of water-column attenuation and observed peak shifts, as described in the text.</p

Classification results for <i>SAM</i>.

<p>Highlighted values indicate the minimum score for each band configuration.</p

Functional groups and the pigment-mixing formulations used in the fluorescence model.

<p>Numbers indicate relative fluorescence-peak intensities. A value within the specified range was randomly chosen for each of the 10,000 synthesized spectra for each functional group.</p

Fluorescence endmember library.

<p>Based on our measurements, most fluorescent signals observed on Caribbean reefs are attributable to one or more of these spectra.</p

Different spectral band sets used in the classification tests.

<p>Each band is designated by a center wavelength and half bandwidth, both in nm.</p

Photoswitching of the chromophore in cerFP505.

<p>A) Absorption spectra after irradiation with 400 nm (on-state) or with 450 nm light (off-state). Deactivation gives rise to a second peak around 390 nm. B) Ground state equilibrium. Irradiation at 400 nm drives the protein to the on-state, 450 nm light turns off the “activatable” fraction of the protein. Bars above the x-axis specify the light treatment, 400 nm light (white bar), 450 nm light (grey bar) or no light (black bar). C) Fluorescence emission of the protein in the on- or off-state was recorded for ≥30 min in the dark after 5 min irradiation with 400 or 450 nm light. Bars above the x-axis specify the light treatment as described above. D) The reversibility of the reaction is shown for eight cycles of activation/deactivation. Bars above the x-axis specify the light treatment as described in (B).</p

Expression of cerFP505 in mammalian cells.

<p>A) cerFP505, cmFP512 and EGFP were expressed in HEK293 cells. Photographs were taken 12 h after transfection. B) The upper panels show the expression and maturation of cerFP505 <i>in vivo</i> in HEK293 cells. Images were excised from a time-lapse movie. The time-points of image acquisition are indicated in the panels as minutes after transfection. The increase in fluorescence intensity of cells in the green channel was analyzed for each image of the time-lapse movie. The graph displays the mean of the individual measurement normalized to 1.0, error bars denote standard deviations. C) Photobleaching of cerFP505, cmFP512 and EGFP in HEK293 cells under continuous irradiation with blue light under the fluorescence microscope. The upper panels are images from a time-lapse movie showing the photobleaching of cells expressing cerFP505. Imaging time-points are given and correspond to minutes of irradiation. The graph displays the mean values of the green fluorescence emission of blue-light irradiated cells expressing cerFP505, cmFP512 and EGFP. Error bars denote standard deviations. <i>Inset</i>: Photobleaching measured <i>in vitro</i> using purified recombinant protein samples.</p