This study was supported by the State Key Development Programs for Basic Research of China (2021YFC3100502).

The corals used for the present study were Seriatopora caliendrum, which are cultured in our lab. All the corals were kindly provided by the South China Sea Institute of Oceanology, University of Chinese Academy of Sciences.
1. Inspection and startup
NOTE: Each module must be tested for tightness and function individually before assembling the system. Deionized water should be used to test the module&#39;s tightness. Commercial details of all the module components are provided in the Table of Materials.
<ol>
	<li>Tightness test of the inter-module connection
	<ol>
		<li>Connect all the modules and the pumps (Figure 1), and ensure the water flows circularly over the system for at least 30 min.</li>
		<li>Check all the seams for possible leakage issues. If any leaking occurs in any of the bonding seams, apply bonding glue from the outside. If any leaking occurs in any of the connection seams, tighten the connection again, and check whether the sealing gasket needs to be changed.</li>
	</ol>
	</li>
	<li>Loading
	<ol>
		<li>After the tightness test, evacuate and dry the water inside.</li>
		<li>Load the appropriate contents. 
		NOTE: For example, the bacterial culture substrate is loaded into the microbial purification module, and the Chaetomorpha Algae is loaded into the algae purification module.</li>
	</ol>
	</li>
	<li>Assembling and testing the whole system
	<ol>
		<li>After loading, fix the modules onto the baseboard using the screws.</li>
		<li>Connect the inner circulation modules with the outer circulation ones (without the coral culture module).</li>
		<li>For the seawater perfusion, inject the seawater through the water inlet in the seawater store module. When the water level is 3 cm higher than the water inlets of the pumps, switch on the pumps, and continue the injection of the seawater until the inner circulation modules are full of the seawater, with room for air (3 cm high) in the seawater store module. 
		NOTE: The seawater is prepared using pure water and sea salt (see the Table of Materials).</li>
	</ol>
	</li>
	<li>System test
	<ol>
		<li>Turn on all the switches and set both the seawater pump voltages to 9 V. Set the water temperature to 25 &#176;C.</li>
		<li>Set the cycle timer to &#34;1 minute on and 1 minute off&#34;. Set all three channels of the three-channel timer to &#34;9:00 am on&#34; and &#34;5:00 pm off&#34;.</li>
		<li>Monitor the system for at least 24 h for any malfunction. If no problem is found, then the system is ready for the next step of the operation. 
		&#8203;NOTE: It is important to clear all the bubbles in all the modules except for the seawater store module. The whole system can be slightly lifted and shaken to move the bubbles from the module inlet to the outlet.</li>
	</ol>
	</li>
</ol>
2. Establishment of the microbial environment
NOTE: Establishing a coral-friendly microbial environment is necessary before coral transplantation. In order to culture microorganisms in the system, especially in the microbial purification module, the diluted probiotic solution should be added as the microbial source for the nitrification system.
<ol>
	<li>Adding the microbiome source
	<ol>
		<li>Add 1 mL of the commercial microbiome source solution (see the Table of Materials) into 500 mL of seawater with stirring.</li>
		<li>Inject 50 mL of the above diluted solution and 10 &#181;L of the commercial coral nutrition solution (see the Table of Materials) into the circulation system.</li>
	</ol>
	</li>
	<li>Microbiome culture
	<ol>
		<li>Switch on the inner circulation pump and an air pump to culture the microbiome for 21 days. The microbiome oxygen content requirements determine the on-time and off-time proportions of the air pump. 
		&#8203;NOTE: This step aims to culture the seawater purification microbiome and promote coral-beneficial microbiome growth in the system. In this process, the seawater starts to become muddy from the second day to the fourth day after the microbiome injection. After this microbiome culture process, the nutrient degradation capacity in the system should be established. It should be noted that to meet different experimental requirements, different microbiome sources can be used to establish the microbiome environment.</li>
	</ol>
	</li>
</ol>
3. Coral transplantation and growth
<ol>
	<li>Coral transplantation
	<ol>
		<li>Cut the raw coral branches with length scales of 3-5 cm, and then adhere these coral branches onto 3D-printed coral support bases (Supplementary Coding File 1).</li>
		<li>Put these coral branch samples back into the original seawater tank for at least 7 days for recovery.</li>
		<li>Fix the coral support base onto the rotation unit with glue. Assemble the coral culture module, and connect it to the outer circulation loop.</li>
	</ol>
	</li>
	<li>Imaging the coral growth 
	NOTE: The coral images over time need to be acquired to evaluate the coral growth. Using a demountable connection makes it convenient to remove the coral culture module from the whole system for imaging. To this end, a mini-photo studio with appropriate illuminating conditions is built. A camera with a macro lens (see the Table of Materials) is used to capture the coral surface morphologies in different periods. The coral rotation unit in the culture module can be operated outside the module using non-contact mode. By rotating the magnetic handle adjacent to the module, full-angle coral images can be captured.
	<ol>
		<li>Place the camera on the top of the studio, and capture the images from a vertical view.</li>
		<li>Place the coral culture module in the mini-photo studio with the coral positioned in the center and at the bottom.</li>
		<li>Capture the coral images by rotating the outside handle. 
		&#8203;NOTE: For coral survival reasons, the time period of imaging should be limited to 15 min.</li>
	</ol>
	</li>
</ol>
4. System routine maintenance
NOTE: The routine maintenance includes leakage inspection, malfunction inspection, additive addition, and seawater exchange.
<ol>
	<li>Leakage inspection
	<ol>
		<li>Inspect the baseboard for water stains or droplets. As the system&#39;s cover shell is transparent, visually inspecting the water leakage is easy and convenient. This inspection must be performed every day.</li>
	</ol>
	</li>
	<li>Malfunction inspection
	<ol>
		<li>Ensure that this step includes the inspection of the water temperature, pumps, light voltages, air pump status, and timer status, including visually checking and recording the set water temperature, real-time temperature, transformer output voltages, UV lamp settings, and timer working status. This inspection should be performed every day. 
		NOTE: Certain system malfunctions can be diagnosed based on abnormal sounds or unusual temperatures.</li>
	</ol>
	</li>
	<li>Additive addition 
	NOTE: Additive addition is the process of adding nutrients and other reagents into the system.
	<ol>
		<li>For example, extract 10 mL of the seawater using a syringe from the three-way valve between the activated charcoal module and seawater module.</li>
		<li>Dissolve the additives in the extracted seawater.</li>
		<li>Inject the solution back into the system through the three-way valve. In real cases, the types, amounts, and addition frequencies of the additives are decided by the system seawater quality, considering the experimental requirements.</li>
	</ol>
	</li>
	<li>Water exchange 
	NOTE: Routine water exchange can reduce toxic concentration and eutrophication in the culture system. If the experimental conditions allow, exchanging the seawater can be a routine operation.
	<ol>
		<li>Switch off the power for the whole system, and unplug the power cable for safety reasons.</li>
		<li>Remove the coral culture module.</li>
		<li>Connect the outside wastewater pipeline to the outlet in the seawater store module.</li>
		<li>Rotate the system, and place the system front-side down.</li>
		<li>Switch on the outlet. Let the inside seawater flow out of the system. 
		NOTE: Do not use any pump to draw the water out since the inside negative pressure may damage the system.</li>
		<li>Discharge an appropriate amount of seawater, and switch off the outlet. The amount of discharged seawater is decided by the physiological state of the corals.</li>
		<li>Reset the system, and inject the newly prepared seawater into the system through the water inlet.</li>
		<li>Install the coral culture module back into the system.</li>
		<li>Switch on the system power, and wait until the whole system goes back to normal.</li>
	</ol>
	</li>
</ol>
5. Module replacement
NOTE: If any module needs to be replaced due to malfunction or according to the experimental arrangement, it is important to change the module without suspending or negatively affecting the culture experiment.
<ol>
	<li>For the seawater store module, algae purification module, microbial purification module, or activated charcoal purification module, switch off the inner circulation pump, and loosen the fixing screws for the module.</li>
	<li>Disconnect the pipelines between the two joined modules, and disassemble the module to be replaced from the system. The final step is to assemble the new module into the system by connecting the pipelines and retightening the fixing screws. 
	NOTE: The replacement of the temperature control module is somehow different. First, all the wires need to be disconnected from the module. The fixing bolts are then unscrewed, and the pipelines are disconnected. Afterward, the heating panel is dismounted, and the module is disassembled from the system. The installation process for the temperature control module is the reverse process.</li>
</ol>
6. Shutting down the system and restoring the system to its initial state
NOTE: The system will eventually be shut down after the necessary coral culture experiment. The system needs to be restored to its original state.
<ol>
	<li>Shutting down the system
	<ol>
		<li>Switch off the system power, and unplug the power cable.</li>
		<li>Evacuate the seawater inside the system.</li>
		<li>Disassemble the modules in the following order: the coral culture module, the activated charcoal purification module, the seawater store module, the algae purification module, the microbial purification module, the UV lamp, the two circulation pumps, and the temperature control module.</li>
	</ol>
	</li>
	<li>System restoration
	<ol>
		<li>Clean all the modules with pure water and surface active agents (see the Table of Materials).</li>
		<li>Sterilize the modules with 3% hydrogen peroxide solution. 
		&#8203;NOTE: Do not use any organic solvent to wash the modules.</li>
		<li>Dry the modules at 40 &#176;C for 12 h. Ensure that all the water inside the system is evaporated.</li>
		<li>Clean all the pipelines and valves using the same surface active agents.</li>
	</ol>
	</li>
</ol>
7. Modification for the controlled microorganism environment
NOTE: Aside from the coral culture experiment, for certain special experiments, such as acquiring a controlled microorganism environment in the system, the microbiome species and abundance must be strictly controlled. The most innovative feature of our coral culture system is that the coral&#39;s physiological activity can be explored in a specific microbial environment in a relatively closed micro-ecosystem. Performing this function requires a different operating procedure.
<ol>
	<li>Pre-sterilization
	<ol>
		<li>Sterilize all the modules, pipelines, and valves using 3% hydrogen peroxide solution before assembling the system.</li>
		<li>Sterilize the bacterial culture substrate via autoclaving.</li>
		<li>Sterilize Chaetomorpha algae with 75% ethanol solution, and dry it using sterile paper.</li>
	</ol>
	</li>
	<li>System modification and sterilization
	<ol>
		<li>In assembling the system, add an air sterilizing filter (see the Table of Materials) between the air pump and the seawater store module.</li>
		<li>Add a water sterilizing filter between the inlet and the three-way valve. This step ensures the air and water injected into the system are sterilized.</li>
		<li>Introduce ozone into the system to eliminate the remaining microbiome.</li>
		<li>Wash off the remaining disinfectant agents with sterile seawater three times, and inject the sterile seawater into the system.</li>
		<li>For only the establishment of the microbial environment, inject the microbiome source solution through the water outlet. 
		NOTE: Do not use the water inlet to inject the microbiome source. Other reagents and seawater are still injected through the water inlet.</li>
	</ol>
	</li>
</ol>

A Combined Micro-Device System for Promoting and Monitoring Coral Growth

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The authors declare no competing financial interest.

This coral culture system is designed to simulate and provide a relatively natural or customized microenvironment for corals to be transplanted into and survive. Meanwhile, as a self-developed equipment, this system needs to be reliable, user-friendly, and safe. For example, in terms of temperature control, the seawater temperature should be controlled appropriately based on the daily environmental circumstances. The system was tested by culturing the coral for 1 month, confirming the system's reliability.
<p class="...

The deterioration of coral reef ecosystems has been occurring worldwide over the past 70 years. Considering all the major coral areas across Central America1, Southeast Asia2,3,4,5,6, Australia7,8, and East Africa9, the global coverage of coral reefs has halved since the 1950s10. This mass loss of coral reefs has resulted in ecological and economic problems. For example, by tracing the presence/absence and abundance of all kinds of coral-dependent fishes for 8 years, researchers concluded that the coral decline has directly caused a substantial decrease in fish biodiversity and abundance in Papua New Guinea11. This result proved that the coral decline can not only undermine coral reef-based biological systems but also reduce fishery incomes.
Over decades of field surveys, including direct monitoring, remote sensing, and data comparison, the scientific community has identified several factors causing the mass coral decline. One major reason for the mass coral decline is coral bleaching caused by high seawater temperatures12,13. By combining bleaching and meteorological records, scientists have concluded that coral bleaching is happening more frequently in El Ni&#241;o-Southern Oscillation phases14. Another reason for the coral decline is ocean acidification. Owing to the increased CO2 concentration in both the atmosphere and seawater, calcium carbonate dissolves faster than before, causing downscale net coral reef calcification15. Indeed, it has been concluded that when the CO2 concentration in the atmosphere reaches above 500 ppm, tens of millions of people will suffer, and the coral reefs will be at risk of significant deterioration and symbiodinium detachment16,17. There are other factors that can also affect coral survival, such as inshore pollutants causing or accelerating coral decline. Researchers in Hawaii measured the carbon, oxygen, and nitrogen isotopes in corals, along with the dissolved inorganic carbonate and the related nutrients (NH4+, PO43-, NO2&#8722;, and NO3&#8722;), and concluded that pollution from the land magnified the coastal acidification and bioerosion of corals18. Further to pollution, urbanization also endangers coral survival and causes relatively low architectural complexity in corals, as revealed by a study on the coral survival status in Singapore, Jakarta, Hong Kong, and Okinawa. Thus, the impact of anthropogenic stressors and the superimposed effects of climate change are leading to widespread reduced biodiversity on coral reefs and an associated decline in coral ecological function and resilience19.
It should also be noted that a large number of microorganisms participate in the physiological functions of corals, including nitrogen fixation, chitin decomposition, the synthesis of organic compounds, and immunity20, and these microorganisms should, thus, be included when considering coral reef deterioration. In natural environments, such as coral reefs, many factors cause hypoxic or anoxic conditions, including insufficient water circulation, algal exudate, and algal overgrowth. This phenomenon negatively affects the population distributions of coral and coral-related microorganisms. For example, Vietnamese scientists found that in Nha Trang, Phu Quoc, and Ujung Gelam, bacterial composition in the coral Acropora Formosa could be affected by dissolved oxygen at different locations21. Researchers in the United States explored hypoxic or anoxia conditions in corals and found that algal exudates can mediate microbial activity, leading to localized hypoxic conditions, which may cause coral mortality in the direct vicinity. They also found that corals could tolerate reduced oxygen concentrations but only above a given threshold determined by a combination of the exposure time and oxygen concentration22. Researchers in India found that when Noctiluca scintillans algae bloomed, the dissolved oxygen decreased to 2 mg/L. Below this concentration, about 70% of Acropora montiporacan died because of hypoxic conditions23.
All the abovementioned facts and factors suggest that environmental change leads to the deterioration of coral reefs. To culture and study reef corals under certain conditions, it is important to accurately and comprehensively build up a controllable microscopic environment for reef corals to inhabit. Normally, scientists focus on temperature, light, water flow, and nutrients. However, other features, such as the dissolved oxygen concentration, microorganism abundance, and microorganism diversity in the seawater, are commonly ignored. To this end, our group has explored the possibility of applying small equipment to culture coral polyps in a relatively controlled environment24,25. In this work, we designed and built up a modular micro-device system for coral culture. This modular micro-device system can provide a controllable micro-environment in terms of the temperature, light spectrum, dissolved oxygen concentration, nutrients, and microorganisms, etc., and has the capacity for expansion and upgrade.
Modules and functions of the device 
The micro-device system was inspired by the Berlin system26, but no live rocks are used in the current system. As shown in Figure 1, the current system comprises six main modules, two brushless motor pumps, one gas pump, one flow-through UV lamp, one power supply, certain electronic control components, and the related wires and screws. The six main modules include a seawater store module (with an air pump and temperature sensor), a temperature control module, an algae purification module, a microbial purification module, an activated charcoal purification module, and a coral culture module.
Device architecture 
As shown in Figure 2 and Figure 3, the overall micro-device system can horizontally be divided into two compartments with a temperature control module in between. For safety reasons, all the seawater-containing modules and parts are placed in the left compartment, named the culture compartment. The other electronic parts are placed in the right compartment, named the electronic compartment. Both compartments are sealed or packaged within shells. The temperature control module is fixed in a divider plate in between. The shell of the culture compartment includes a baseboard and three screw-fixing plates. This design ensures compartment tightness and facilitates the operation of the system. Additionally, the tightness favors accurate temperature control. The shell of the electronic compartment includes a baseboard, two screw-fixing plates, and one front control panel.
Water circulation 
An inner and outer seawater circulation loop connected to the seawater store module was pre-designed. The inner circulation loop successfully connects the seawater store module, temperature control module, flow-through UV lamp, algae purification module, and microbial purification module. This circulation loop aims to provide suitable physiochemical and physiological seawater conditions for the corals, and no frequent maintenance is needed. The algae purification module contains Chaetomorpha algae, which absorbs the extra nutrients (nitrate and phosphate) in the water. The microbial purification module contains the bacterial culture substrate, which cultivates the microbiome to transfer nitrite and ammonium into nitrate for water purification. All these modules need to be replaced only under critical circumstances.
The outer circulation loop successively connects the seawater store module, coral culture module, and activated charcoal module. This circulation loop aims to provide light, tightness, water current, and high seawater quality to the corals. The seawater can be refreshed through a water inlet and a water outlet. Additives are added through a three-way valve, and the seawater sample can also be extracted from this valve for inspection. Air can be pumped in through an air inlet and discharged from an air outlet.
Electronic design 
A 220 V AC power supply with a switch and a fuse is used for the whole system. The input power is divided into four branches. The first branch goes to a 12 V DC power supply, which directly powers the heating panel, cooling panel, and cooling fan. This branch also indirectly powers two pumps and two lighting panels through a four-channel DC transformer. The second branch goes to a PID temperature controller. The third branch goes to an air pump power supply. The last branch connects to a UV lamp power supply. A solid-state relay connects the PID temperature controller and the cooling panel in the temperature control module. A regular relay is used to connect the PID temperature controller and the heating panel. The four-channel DC transformer converts the voltage to that required.
There are two control panels on the right part of the system. There are four switches and one controller for the UV lamp on the top panel, including a main power switch, a UV lamp power switch, an air pump switch, and a temperature control switch. The main power switch controls the 12 V power supply of the system.
A PID temperature controller, a cycle timer, a four-channel DC transformer, and a three-channel timer are on the front panel. The PID temperature controller adjusts the water temperature by controlling the heating and cooling panels in the temperature control module. The temperature control module only works when the inner circulation pump is working and the water is flowing past the temperature control module. The cycle timer is connected to the air pump power line. Its purpose is to assign the working time period to the air pump. There is a three-channel timer deployed in the electronic compartment too. This timer controls the work time period for the air pump, coral light, and algae light.

<table><tbody><tr><td>12V DC power supply</td><td>Delixi Electric Co., Ltd.</td><td>CDKU-S150W</td><td>12V12.5A</td></tr><tr><td>3% hydrogen peroxide solution</td><td>Shandong ANNJET High tech Disinfection Technology Co., Ltd</td><td>NULL</td><td>NULL</td></tr><tr><td>75% ethanol solution</td><td>Shandong ANNJET High tech Disinfection Technology Co., Ltd</td><td>NULL</td><td>NULL</td></tr><tr><td>Air pump</td><td>Chongyoujia Supply Chain Management Co., Ltd.</td><td>NHY-001</td><td>NULL</td></tr><tr><td>Air sterilizing filter</td><td>Beijing Capsid Filter Equipment Co., Ltd</td><td>S593CSFTR-0.2H83SH83SN8-A</td><td>NULL</td></tr><tr><td>Camera</td><td>SONY</td><td>&amp;Alpha;7r4-ILCE-76M4A</td><td>NULL</td></tr><tr><td>Coral nutrition solution</td><td>Red Sea Aquatics Co., Ltd.</td><td>22101</td><td>Coral nutrition</td></tr><tr><td>Coral pro salt (sea salt)</td><td>Red Sea Aquatics Co., Ltd.</td><td>R11231</td><td>NULL</td></tr><tr><td>Cycle timer</td><td>Leqing Shangjin Instrument Equipment Co., Ltd.</td><td>CN102A</td><td>220V version</td></tr><tr><td>Double closed quick connector</td><td>JOSOT Co., Ltd</td><td>NL4-2103T</td><td>NULL</td></tr><tr><td>Flow-through UV lamp</td><td>Zhongshan Xinsheng Electronic technology Co., Ltd.</td><td>211</td><td>NULL</td></tr><tr><td>Four-channel transformer</td><td>Dongguan Shanggushidai Electronic Technology Co., Ltd</td><td>LM2596</td><td>NULL</td></tr><tr><td>Macro lens</td><td>SONY</td><td>FE 90mm F2.8 Macro G OSS</td><td>NULL</td></tr><tr><td>Microbiome source solution</td><td>Guangzhou BIOZYM Microbial Technology Co., Ltd.</td><td>303</td><td>NULL</td></tr><tr><td>Mini-photo studio</td><td>Shaoxing Shangyu Photography Equipment Factory</td><td>CM-45</td><td>NULL</td></tr><tr><td>PID temperature controller</td><td>Guangdong Dongqi&amp;nbsp; Electric Co., Ltd.</td><td>TE9-SC18W</td><td>SSR version</td></tr><tr><td>Pump (for water)</td><td>Zhongxiang Pump Co., Ltd.</td><td>ZX43D</td><td>Seaswater version</td></tr><tr><td>Pure water machine</td><td>Kemflo&amp;nbsp;(Nanjing) environmental technology Co, ltd</td><td>kemflo A600</td><td>NULL</td></tr><tr><td>Solid-state relay</td><td>Delixi Electric Co., Ltd.</td><td>DD25A</td><td>NULL</td></tr><tr><td>Surface active agents</td><td>Guangzhou Liby Group Co., Ltd.</td><td>Libai detergent</td><td>NULL</td></tr><tr><td>Three-channel timer</td><td>Leqing Changhong Intelligent Technology Co., Ltd.</td><td>CHE325-3</td><td>220V version</td></tr><tr><td>Water sterilizing filter</td><td>Beijing Capsid Filter Equipment Co., Ltd</td><td>S593CSFTR-0.2H83SH83SN8-L</td><td>NULL</td></tr></tbody></table>

an integrated micro-device system for coral growth and monitoring

Corals are fundamental organisms in marine and coastal ecosystems. With the advancement of coral protection research in recent years, precise control of the coral culture environment is highly in demand for coral conservation and study. Here, we developed a semi-closed coral culture micro-device system as a multi-functional platform, which can provide accurate and programmable temperature control, a sterile initial environment, long-term stable water quality, an adjustable dissolved oxygen concentration, and a customized light spectrum for corals. Owing to the modular design, the coral culture system can be upgraded or modified by installing desirable new modules or removing existing ones. Currently, under appropriate conditions and with proper system maintenance, the sample corals can survive for at least 30 days in a healthy state. Furthermore, due to the controllable and sterile initial environment, this coral culture system can support research into the symbiotic relationship between corals and associated microorganisms. Therefore, this micro-device system can be applied to monitor and investigate sea corals in a relatively quantitative manner.

Temperature control accuracy 
The system temperature is normally set to 23-28 &#176;C depending on the coral species. However, as one of the most important factors, temperature fluctuation can strongly affect coral survival. Hence, temperature control accuracy is a decisive factor for the coral culture system. A temperature sensor and an independent data collector with a temperature range from 9 &#176;C to 32 &#176;C can be used to test the temperature control accuracy in the coral culture module. W...

Watch this Scientific Journal Video about Advancing Coral Culture — Creating a Semi-Quantitatively Controlled Microenvironment System to Counter Current Limitations at JoVE.com

Author Spotlight: Advancing Coral Culture - Creating a Semi-Quantitatively Controlled Microenvironment System to Counter Current Limitations 

This protocol describes the development of a modular controllable micro-device system that can be applied for the long-term culturing and monitoring of sea corals.

An Integrated Micro-Device System for Coral Growth and Monitoring

Corals are fundamental organisms in marine and coastal ecosystems. With the advancement of coral protection research in recent years, precise control of ...

an-integrated-micro-device-system-for-coral-growth-and-monitoring

Research

JoVE Journal

Bioengineering

1.7K Views.  Southeast University. Het doel van ons onderzoek is het ontwikkelen van een kunstmatig micro-omgevingssysteem voor koraalkweek en -studie. De micro-omgeving in het micro-apparaatsysteem kan op zijn minst semi-kwantitatief worden gecontroleerd. Op dit moment ontbreekt het de bestaande apparatuur en het systeem dat wordt gebruikt voor koraalkweek aan nauwkeurige controle over temperatuur en andere omgevingsfactoren.Bovendien zijn deze systemen open, waardoor ze vatbaar zijn voor besmetting door een stof in de...

Video: Auteur in de schijnwerpers: Bevordering van de koraalcultuur - Het creëren van een semi-kwantitatief gecontroleerd micro-omgevingssysteem om de huidige beperkingen tegen te gaan 

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps&#160;and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter&#160;scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation,&#160;(2) superior optical sectioning ability,&#160;and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.

An integrated suite of imaging techniques has been applied to determine polyp morphology and tissue structure in the Caribbean corals Montastraeaannularis and M. faveolata. Fluorescence, serial block face,&#160;and two-photon confocal laser scanning microscopy have identified lobate structure, polyp walls, and estimated chromatophore and zooxanthellae densities and distributions.

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues ...

Environment

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

Developmental Biology

Coral Reef Arks: An In Situ Mesocosm and Toolkit for Assembling Reef Communities

Coral reefs thrive and provide maximal ecosystem services when they support a multi-level trophic structure and grow in favorable water quality conditions that include high light levels, rapid water flow, and low nutrient levels. Poor water quality and other anthropogenic stressors have caused coral mortality in recent decades, leading to trophic downgrading and the loss of biological complexity on many reefs. Solutions to reverse the causes of trophic downgrading remain elusive, in part because efforts to restore reefs are often attempted in the same diminished conditions that caused coral mortality in the first place. 
Coral Arks, positively buoyant, midwater structures, are designed to provide improved water quality conditions and supportive cryptic biodiversity for translocated and naturally recruited corals to assemble healthy reef mesocosms for use as long-term research platforms. Autonomous Reef Monitoring Structures (ARMS), passive settlement devices, are used to translocate the cryptic reef biodiversity to the Coral Arks, thereby providing a "boost" to natural recruitment and contributing ecological support to the coral health. We modeled and experimentally tested two designs of Arks to evaluate the drag characteristics of the structures and assess their long-term stability in the midwater based on their response to hydrodynamic forces. 
We then installed two designs of Arks structures at two Caribbean reef sites and measured several water quality metrics associated with the Arks environment over time. At deployment and 6 months after, the Coral Arks displayed enhanced metrics of reef function, including higher flow, light, and dissolved oxygen, higher survival of translocated corals, and reduced sedimentation and microbialization relative to nearby seafloor sites at the same depth. This method provides researchers with an adaptable, long-term platform for building reef communities where local water quality conditions can be adjusted by altering deployment parameters such as the depth and site.

Coral Reef Arks: An In Situ Mesocosm and Toolkit for Assembling Reef Communities

Moored midwater geodesic structures called Coral Arks provide a modular, scalable, and vertically adjustable research platform that can be used to build, monitor, and perturb coral reef communities in previously inoperative areas, including offshore.

Coral reefs thrive and provide maximal ecosystem services when they support a multi-level trophic structure and grow in favorable water quality conditions ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such ...

Inducing Polyp Bail-out in Coral Colonies to Obtain Individualized Micropropagates for Laboratory Experimental Use

Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to the rest of the colony and ultimately detach from the skeleton to continue living as separate individuals. Coral biologists have acknowledged the process of polyp bail-out for years, but only recently the micropropagates generated by this process have been recognized as a model system for coral biology studies. The use of polyp bail-out can create a high number of clonal units from a single coral fragment. Another benefit is that single polyps or patches of polyps can be easily visualized under a microscope and maintained in highly standardized low-cost environments such as Petri dishes, flasks, and microfluidic chips. The present protocol demonstrates reproducible methods capable of inducing coral micropropagation and different approaches for maintaining the single polyps alive in the long term. This methodology was capable of successfully cultivating polyps of the coral species Pocillopora verrucosa for up to 8 weeks after bail-out, exhibiting the practicality of using individual coral polyps for coral research.

Polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to their colony and detach from it to live as individuals. The present protocol describes how to induce coral micropropagation by bail-out using hypersaline or calcium-free seawater treatments.

Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of ...

Biology

Physiological Characterization of the Coral Holobiont Using a New Micro-Respirometry Tool

Metabolic activity, defined as the sum of organismal processes that involve energy, is of critical importance in understanding the function and evolution of life on earth. Measuring organismal metabolic rates is, therefore, at the center of explaining the physiological states of organisms, their ecological roles, and the impact of environmental change on species within terrestrial and aquatic ecosystems. On coral reefs, measures of metabolism have been used to quantify symbiosis functioning between corals and their obligate algal symbionts (Symbiodiniaceae), as well as assess how environmental stressors, including climate change, will impact coral health. Despite this significance, there is a lack of methods, and therefore data, relating to metabolic rate measurements in coral offspring, likely due to their small size. To address this gap, this study aimed to develop a custom setup for measuring the respiration of small (millimeter size range) marine animal ecologies. This low cost and easy setup should allow for the improved measurement of metabolic rate. This will be essential for applied ecological research utilizing the sexual production of corals for reef restoration.

This protocol describes the setup and running of a micro-respirometry system that can be employed to investigate the physiological features of the coral holobiont.

Metabolic activity, defined as the sum of organismal processes that involve energy, is of critical importance in understanding the function and evolution ...

An Integrated Micro-Device System for Coral Growth and Monitoring

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