Micro Aquarium Closed Cell System for In-Vivo Coral Research

Introduction:

This presentation details the materials and method for observation of coral reef polyps at a microscopic level in a controlled laboratory setting. It was designed and fabricated to significantly reduce operational complexity, maintenance, and overall cost. Research on coral reefs is traditionally conducted in large, multi-gallon, open-tank aquariums housed in designated isolation rooms to strictly control atmospheric conditions. Those systems are supplied with hundreds of gallons of carefully formulated and monitored artificial seawater, allowing for macro- and microscopic in-vivo and in-vitro observations.

For researchers interested in in-vivo marine life microscopy, this document describes a highly integrated component and support system. The microscope lighting and viewing modifications can be found on these previous posts. Substage lighting, fiberoptic direct lighting, blocking filter.

Distributed Support Network:

This is part of research of reef coral polyps. It utilizes a distributed support model that relies heavily on a collaborative partnership with a well-established local commercial aquarium facility¹. Aquariums Inc is located within five miles of the research laboratory. Mr. Ryan Bollinger with 20 years of experience and his cooperative staff are highly capable. The facility acted as the specimen procurement agent and managed the long-term housing and husbandry of the primary brain coral sample within their controlled systems.(Indo-Pacific “Maze Brain” Coral (Platygyra species) or “Favites” Brain Coral)

Additionally, the partner provided essential supplies, including specialized artificial seawater and reagents for experimental materials, alongside consultation on coral physiology and behavior. The facility continuously monitors and logs environmental data for their large-scale systems. Notably, while housed at this facility, the experimental subject coral was separated from, but cohabitated with, a diverse variety of other coral species.

System Architecture and Spatial Zoning:

An upright Olympus BH2/BHS microscope was utilized to study coral polyp physiology. The standard orientation for the specimen within this system is negative geotropic (upright). This also facilitates natural water flow consistent with open-ocean environments.

To ensure the system fits comfortably on the Olympus BH2/BHS stage and clears the 20x objective lens, the apparatus was split into two functional zones:

1. The Micro-Chamber: Located directly on the microscope stage.
2. The Life Support Package: Positioned adjacent to, but separate from, the microscope workspace.

1. The Micro-Chamber (Stage Component):

• Base Material: A Nunc 27 mm glass-bottom petri dish. Approximate volume ~ 5cc.
• Viewing Window: The chamber floor consists of #1.5 cover glass (0.17 mm thickness). Because the Olympus BH2/BHS optics are precisely corrected for this specific thickness, using standard thick plastic petri dishes would introduce severe spherical aberration and degrade image sharpness.
• Chamber Height: 2.0 mm to 3.5 mm. This dimension provides sufficient vertical clearance for the polyps to extend their tentacles without contacting the upper ceiling, while remaining thin enough to minimize total fluid volume and stay within the optical working distance.
• Fluid Ports: Barbed miniature Luer fittings (1/16″ ID) are pressed and sealed directly into opposite sides of the chamber walls to serve as inflow and outflow ports.
• Port Modification Process: Two or more 3/32-inch holes were drilled into the side of the glass side of the petri dish. Before insertion, a 1.5-mm length of 1/16-inch silicone tubing was fitted over the barbed end of a male Luer lock fitting. This assembly was then pressed through the 3/32-inch hole. Once seated inside the chamber wall, a second 1.5- mm length of silicone tubing was secured onto the interior section of the barbed fitting. This sandwiched configuration creates a completely watertight seal while allowing a slight range of motion. This will be described in detail later*

2. The Life Support Package (Off-Stage Component):

The life support package* consists of the artificial seawater reservoir, a peristaltic pump and connecting silicone tubing. To eliminate mechanical vibration, the pumping apparatus was isolated on a surface completely decoupled from the microscope workbench. Light and ambient temperature are centrally controlled and not further discussed in detail here.

The laboratory environment was temperature-controlled to 74°F ± 1°F. Indirect lighting was strictly regulated for both duration and intensity to preserve natural behaviors during protracted time-lapse photography sessions.

3. Flow Cell Stage Support Assembly}

To ease manipulation of the Micro-Chamber on the Olympus microscope stage, a custom support assembly was designed and fabricated. The design criteria required a low profile to fit the narrow clearance between the lens and the stage, a watertight seal while inverted, and straightforward fabrication. This assembly allows the specimen to be precisely positioned using the microscope’s standard slide holder.

• Base Plate: Fabricated from flat PVC sheet stock cut to fit the microscope stage, featuring a central aperture slightly smaller than the diameter of the Nunc petri dish. Fig 1
• Stabilizing Ring: A short cylinder of 2 inch PVC pipe, with a 39mm internal diameter matching the outer diameter of the dish, was bonded concentrically over the base aperture to align and predictably stabilize the chamber. Fig 3,4,5.
• Compression Mechanism: Orthodontic brackets were bonded to the surrounding PVC base using cyanoacrylate adhesive. Fig 2, 5. A silicone gasket was placed on the resin floor of the dish. Fig 8. A custom metal split-ring was fitted against the outer cover glass surface of the dish Fig 9. Elastic bands were hooked onto the metal ring and stretched to engage the orthodontic brackets. Fig 10 Spacing six elastic bands equally distributed the downward pressure evenly, resulting in a completely leak-free chamber. Fig 11

Optics, Imaging, and Post-Processing:

When using a standard 20x lens on the BH2/BHS microscope, fracturing the flow cell’s cover slip would compromise the living coral, disrupt time-lapse imaging, spill corrosive saltwater onto the mechanical stage, and risk immersing the objective lens. To mitigate these risks, an Olympus LWD CD Plan 20X objective was selected. This flat-field, long-working-distance achromat lens features a correction collar (optimized for viewing upward through culture vessels) and is ideal for navigating the flow cell height and protective cover slip.

To maximize data extraction, crisp, low-noise RAW images are captured at 20x magnification using a Canon EOS R5 camera. During post-processing in Adobe Photoshop, these images are tightly cropped. This method allows for the inspection of micro-anatomical structures at a digital 40x-level detail while preserving the superior depth of field and framing advantages inherent to the 20x optical path. Furthermore, multi-layer image stacking via Photoshop or Helicon Focus software is utilized to dramatically improve the final depth of field.

Significance:

The miniaturization of the research down to the microscopic level vastly reduces the scale of the project while preserving the in-vivo observation of the subject in a very controlled environment. Further reports can detail the use of effluent filtration to collect zooxanthella and other constituents of the  metabolic processes. Additional opportunities may include micro dosing of the polyps to precipitate specific behaviors such as bleaching, antibiotic treatment for disease control and behavioral change in response to micro environmental alterations.

If you are interested in collaboration on marine biology research at a microscopic level, please feel free to email me. john@evergladesark.com

*In an upcoming post the circulating water supply will be presented in detail.

Don’t forget to like us!

Please use the comment box to submit your remarks.

Reference:

1. Aquariums Inc., 1585 Pine Ridge Rd, Unit 4, Naples, FL 34109, United States

If you wish to receive notification of new posts please enter your email address and click on subscribe.

#coral #polyps #microscopy #aquarium #micro #closed cell #Olympus #