The health and long-term dynamics of coastal ecosystems such as kelp forests, mangroves, sea grass beds, and especially coral reefs, are significantly driven by processes and organisms that occur on scales of a millimeter or less. These organisms indeed are primary producers, allowing transformation of sunlight energy into molecular energy, which is what flows through food chains and drives ecosystems.  Despite the importance of small-scale events, the availability of tools to address a variety of problems on the appropriate scales has been severely lacking.  In this research we accomplished the fabrication and successful deployment of a high-resolution underwater microscope that possesses the spatial and temporal requirements to fill this gap in measurement technology.  Accordingly, in the case of corals that nurture a symbiotic photosynthetic symbiont, a ~10 mmdinoflagellate, in situ images of the individual organisms and their photosynthetic transfer efficiency were obtained.  

            Figure 1 illustrates both the inside of the instrument as well as the underwater setup for deployment.  As illustrated, panel A and B show the locations of the illumination ring that has two colors (white and blue); a linear actuator that allows the system to scan focused images as a variety of ranges; a wi-fi antenna that permits communication; the camera; objective lens; and a main computer board.  In operation, the system uses the linear actuator to obtain focused images inside of a volume at successive ranges.  The images are then stacked to obtain a 3D volume.    Figure 2 illustrates a "compressed" focal scan.  We refer to it as compressed because the set of range focused images has been integrated in the vertical using open ware software, that preserves the in-focus components. The image portrays a single coral polyp that is 1 mm across.  We note the small fluorescent red spheres inside the finger-like tentacles.  These are the symbiodinium, the photosynthetic autotrophic dinoflagellates that contain chlorophyll that fluoresces red under blue illumination.  These symbionts play an important role in providing the coral with the food stuffs that it needs to stay healthy when there is enough light to power photosynthesis.

            An interesting question about coral-dinoflagellate morphology can be addressed by determining the 3D locations of the dinoflagellates. Using images from a sequential scan by our microscope, we can estimate the three-dimensional positions of the dinoflagellates, both inside the coral and in the coenosarc, the living tissue that overlays the stony skeleton.  Our method consists of first carefully calibrating both the lateral and range resolution of the system using standard targets in a focal scan mode.  Subsequent processing of the set of focal scanned images then yields an estimate of 3D location.  Figure 3 contains a projected image of a 3D point cloud of a coral polyp and the underlying coenosarc.  Volumetric density estimation can then follow.

A special feature of the microscope is that it can deliver a set of precisely determined amplitude timed pulses of blue light to implement a technology known as "Pulse Amplitude Modulation (PAM)".  This permits an estimate of the photosynthetic efficiency of the symbionts, an important quantity that measures the fraction of light that is converted into chemical energy, hence providing some estimate of overall health.  Current underwater PAM instruments integrate their measurements over volumes and surface areas that are large compared to the size of the dinoflagellates.  This provides useful information, however nothing about heterogeneity of the organisms that can be important for health assessment.  Figure 4 contains an image of a coral polyp where we used the PAM methodology to estimate photosynthetic efficiency over a sequence of images that were recorded with the special modulation that PAM uses.  The blue areas such as the coral tips are low efficiency as they have low concentrations of the symbiodinium.  Other areas have higher efficiency, due to higher concentrations.

This unique instrument will, thus, provide a beacon for further development while, at the same time, allowing us to make this important measurement at a resolution that provides information on a scale that relates to the physiology of the important organisms that mediate coral health.

Last Modified: 01/02/2024
Modified by: Jules S Jaffe