Fig 1.
A: The imaging set-up includes the digital holographic microscope (D3HM) and its computer interface, with a flow chamber placed on the microscope stage. Arrows indicate the direction of the water flow. B: Example of a coral fragment in a flow chamber positioned on the microscope stage ready for imaging. The red light dot is generated by the incident LED beam of the D3HM. C: Sketch of one of the corals used in this study, exemplifying the observation paths that were followed before and after exposure to different stimuli.
Fig 2.
Example of a coral polyp not exposed to stimuli.
A merged hologram image is shown for one polyp along the observation path. Dark areas = polyp (opaque to the DHM LED beam), light area = surrounding seawater medium (transparent to the DHM LED beam). No mucus layer or strings were detected along any part of the observation path.
Fig 3.
Mucus release after addition of charcoal particles.
String-like (A-B) and sheath-like (C) formation of mucus occurs along theca of the polyp. Entrapped activated charcoal particles render the mucus strings visible to the naked eye (D-F). Black arrows in E-F highlight the change in position of particles along the theca. Light intensity image (G), phase image (H) and zoomed in false-colour phase image (I) of the selected area in H of particle-laden strings placed on a glass slide. The position of two particle-laden strings running from the top to the bottom of the image are indicated with red arrows. Grey arrows in G-I point at some of the activated charcoal particles of the left mucus string as examples, whilst the white arrows indicate where mucus is likely to be present connecting particles, although the OPD is not different enough from the surrounding seawater to visualize the mucus with the D3HM (I) (i.e. OPD remains at the background level). Blue bars (numbered 1 and 2) depict where profiles of optical path length differences (OPD) were measured. Blue bar ‘1’ crosses a particle of the string, whilst blue bar ‘2’ crosses the mucus string at a position where we would expect the presence of mucus. The scale bar shown in (I) depicts the range in OPD applicable to the image, from its lowest values in the blues to its highest values in the red in μm.
Fig 4.
Mucus release after addition of food particles.
(A) Artemia salina individuals entrapped in mucus strings. The arrows indicate strings extending from the polyp calice towards the surface of the water in the flow chamber. (B) The alignment of trapped A. salina and small particles clearly hint at the presence of a mucus string (white arrow) in the light intensity image (B-I), yet the optical path length differences (OPD) of these strings were not sufficiently different from the surrounding seawater to visualize them using DHM as shown in the phase image (B-II) and (zoomed in) false-coloured phase (B-III) image, respectively. The polyp and shrimp are outlined in B-II for clarity. The scale bar shown in (B-III) depicts the range in OPD applicable to the image, from its lowest values in the blues to its highest values in the red in μm.
Fig 5.
Detailed observation of mucus strings connecting entrapped brine shrimp.
(A) Two Artemia salina nauplii (As) were connected by mucus strings that contain mucus similar in refractive index to the surrounding seawater, but also particles (black arrows) and mucoid substances having a higher refractive index compared to the surrounding seawater (indicated by white arrows) in the light intensity image. The A. salina on the left is also attached to a polyp of Lophelia pertusa (Lp). (B) A close-up section of the light intensity image (B-I, white and black arrows are in the same position as in A) is already able to discern the presence of some mucoid substance (white arrows), yet it is only in the phase (B-II) and false-coloured phase image (B-III) that we see the full extent of mucus present (see grey arrows). Blue bars (1 and 2) depict where profiles of optical path length differences (OPD) were measured. The scale bar shown in (B-III) depicts the range in OPD applicable to the image, from its lowest values in the blues to its highest values in the red in μm.
Fig 6.
Mucus release associated with digestive processes.
(A) An Artemia salina nauplius (As) that had been ingested was pushed out of the mouth opening of a Lophelia polyp and evidently had mucus attached to it (arrows). (B) Mucus (arrow) associated with the entrapment and potential digestion of the same A. salina individual now found more directly in the mouth area at a later time point. Subfigures I, II and III are the light intensity, phase and false-coloured phase image, respectively. The polyp and nauplius’ outlines are presented in A-II and B-II for clarity. Note that the phase images here show an uneven background in the seawater medium due to artefacts on the flow chamber base found at a different focus plane. The scale bars shown in (A-III) and (B-III) depict the range in optical path length differences applicable to the images, from its lowest values in the blues to its highest values in the greens in μm.
Fig 7.
Examples of mucus ‘string balls’.
(A) A string ball seen floating among A. salina individuals. (B) A string ball (white arrow) next to a particle-laden mucus sheath (red arrow). Subfigures I, II and III are the light intensity, phase and zoomed in false-coloured phase image, respectively. The polyp and nauplius outlines are presented in A-II and B-II for clarity. The scale bars shown in (A-III) and (B-III) depict the range in optical path length differences applicable to the images, from its lowest values in the blues to its highest values in the reds (for A-III) and in the greens (for B-III) in μm.
Fig 8.
Mucus release after exposure of Lophelia pertusa to air and re-introduction into the seawater medium.
(A) Hologram image of mucus observed close to the mouth of a polyp. Mucoid material is also shown along a thecal part of a polyp in image series (B) where (B-I) is the initial hologram, (B-II) the phase image, and (B-III) the zoomed-in false-coloured phase image. The white arrow highlights the mucus, which stands out more clearly in the phase information image and false-coloured phase image compared to the hologram. Blue bars (1, 2) depict where profiles of optical path length differences (OPD) were measured. The scale bar shown in (B-III) depicts the range in OPD applicable to the image, from its lowest values in the blues to its highest values in the red in μm.