To demonstrate the universality of this methodology and to examine the relative distribution of GFPs and Chl simultaneously, collections of visible, visible fluorescent and NIR fluorescent images were collected from corals in four worldwide locations over a 9-month period at various depths, temperatures, and times of day. Hundreds of corals were imaged, and thousands of images collected. A select few are shown in
Figure 6 from three locations: (1) Carriacou, Grenada, (2) Marsa Alam in Egyptian Red Sea, and (3) Key Largo, FL. All of these images were collected at night so that a comparison of GFP and Chl distributions could be made.
Figure 6a–c are images of
Diploria strigosa, Figure 6d–f are of
Acropora sp., and
Figure 6g–i are of
Montastraea cavernosa. Consistent with laboratory observations, most corals responded to blue light much more rapidly than they did to white or red LED illumination. Some species began rapidly retracting tentacles within 30 s when continuously illuminated with the blue LED video lights. Since blue LEDs were used to select corals expressing predominantly GFPs, as soon as corals were selected, the blue LEDs were shut off and one red LED source was used to illuminate the area during set up. In
Figure 6a, extension of the tentacles is clearly seen in this coral, the tips of the tentacles are opaque relative to the majority of the tentacle shaft. The center portion of the ridges in this brain coral have very little visible pigmentation. The oral disks of a few polyps can be seen between the ridges. In
Figure 6b, the vast majority of the GFP is located in regions where red fluorescence from Chl is not located, that is, on the central ridge and in the coenosarc between ridges. While some red fluorescence from Chl is seen in the tentacle tips corresponding to the opaque regions in
Figure 6a, by far the vast majority of red Chl fluorescence is seen in the mesentery tissues on the sides of the ridges co-localizing with the yellow pigmentation seen in the visual images, and it is virtually absent from the coenosarc. This drastically different, almost mutually exclusive distribution of GFP and Chl is confirmed in
Figure 6c with NIR fluorescence. The center of the ridge has virtually no Chl, and most is in the tips of the tentacles or in the mesentery on the sides of the ridges. Co-localization of the visual yellow pigmentation with Chl is seen in the next coral as well. It is also noteworthy that the red fluorescence in the small fish on the surface of the coral in
Figure 6b is not displayed in
Figure 6c. In
Figure 6d, the tentacles from many of the polyps of this
Acropora sp. can be seen, but most remain retracted. The coenosarc on the theca shows the familiar striped yellow pattern characteristic of many
Acropora sp. corals. In contrast the tops of the corallites near the polyp are relatively lacking in yellow pigmentation, especially near the actively growing tip of the coral. In
Figure 6b, the GFP is distributed mostly on the theca and tops of the polyp, and enriched in the actively growing ends of the coral. Although there is more overlap in this species than the last, it appears to be most intense in regions where Chl and the yellow pigmentation are not present. In contrast, most the red fluorescence from Chl appears to be co-distributed within the yellow stripes seen in the visible images, as noted for the brain coral. This is confirmed in the NIR fluorescent images as seen in
Figure 6e. Unlike the previous species, very little NIR fluorescence is seen within the tentacles. Additionally, in the actively growing tips where GFP expression is highest in the fluorescent images, Chl is all but absent. In
Figure 6g, the tentacles of this coral are just beginning to emerge from some polyps. It is worth noting the sponge in front of the coral (which casts shadows from illuminating light sources), as well as the
Dictyota sp. algae at the bottom of the image. If
Figure 6h, GFP is much more broadly distributed in this coral, and is much brighter relative to the red Chl fluorescence seen in the other two samples. GFP is also seen enriched in the newly emerging tentacles, as is some red Chl fluorescence. The bright red fluorescence from the
Dictyota sp. also acts as a positive control since these algae are rich in Chl. The sponges are generally not fluorescent, and in this case again acts as a negative control; the few red spots that can be seen are most likely from an adhering algae, and there appears to be a fine web of algal material over the end of the sponge in the visible image.
Figure 6i shows distribution of photosynthetic zooxanthellae by the NIR fluorescence from the Chl. There is some punctate emission in the emerging tentacles, but it is much brighter in the mesentery tissue. Comparing
Figure 6b,h, it is relatively difficult to see the distribution of zooxanthellae from the visual red fluorescence when GFP expression is higher, again providing value for the NIR fluorescence imaging methodology. Two difficulties in this imaging are noteworthy in
Figure 6. First, the shallow depth of focus with wide apertures is apparent, and second, a shadow from the left light source that is not readily apparent in the visible fluorescence image, is clearly seen in
Figure 6i.
A semi-quantitative analysis of the relative distributions of chlorophyll and GFP was undertaken from each pair of fluorescent images in
Figure 6 (
Figure 6b,c,e,f,h,i). That analysis is shown in
Figure 7. First, the pixel intensities from GFP green emission, red emission (presumably mostly from Chl), and from Chl NIR emission were analyzed on a single line across each of the images (lines in
Figure 6a,d,g). In
Figure 7a–c, the relative intensities of GFP, red emission and Chl NIR emission are seen along a single line across the image. Although pixel intensities are not linearly correlated with pigment fluorescence (
Supplementary Figure S2), an increase in pixel intensity is indicative of increased concentration. Within any given coral sample there is a 3–4-fold difference in pixel intensity versus position on a polyp. Likewise, there is a 4–10-fold difference in pixel intensity for Chl NIR fluorescence. As noted above by visual inspection, it is clear from
Figure 7a–c, that there are many tissues in every coral examined that indicate green GFPs and Chl diverge in nearly opposite directions (maxima for GFPs and minima for Chl, and vice versa). That there is little correlation between GFP and Chl distribution is verified in
Figure 7d–f, where mean GFP and mean Chl pixel intensities within the same 60 × 60 square pixel ROI (roughly 2 × 2 mm) are compared. The extremely small values of the correlation coefficients indicate no relationship between GFP and Chl distributions. While the first two corals in
Figure 6 have overall negative slopes in this analysis suggesting the possibility of reduced Chl fluorescence as GFP pigmentation increases, the opposite is seen for the last coral. Given the insignificant correlation coefficients, the large data scatter, and the lack of linearity in fluorescence imaging signals, this data should not be over interpreted but does provide an objective view of data in the
Figure 6 images. Of note, the red channel fluorescence, while much more closely aligned with Chl NIR fluorescence in all three cases, does have some slight variation suggesting that part of that signal could come from red GFPs or other red-emitting pigments.