With a blocking filter the Olympus BH2/BHS can be modified to view a limited range of light frequencies. In this instance, the microscope can be set to illuminate the subject with royal blue light (460nm) from above and below the stage. If the subject of interest is bioluminescent, it will glow green. If the blue light illuminates the subject, the predominant color will be blue. With a yellow blocking filter in the light path, the blue light will be completely blocked. The image will appear completely yellow except for the green fluorescent protein (GFP). Hence the term “blocking filter.”
This is part of a series of articles related to the Olympus HBH2/BHS Microscope. Please reference the previous articles LED Illumination and Fiberoptic Illumination.
Fig 1. Side view of the microscope turret area showing polarized tube with the slider in position.
Fig 2. Slider removed from its carrier in polarized tube in the microscope. Note that the aperture on the left side is vacant but has a diameter of 22mm with an end stop on its inside surface.
Fig 3. Slider inverted with vacant aperture on left end. This will accommodate the filter with a 21.9mm diameter. It is prevented from falling out by the end stop.
Fig 4. 21.9mm diameter filter custom made to fit into the slider. The 515nm Long-Pass (LP) filter has an arrow on the side of the metal ring of the filter indicating the direction of light passage. The arrow points away from the eye.
Fig 5. Simply place the filter into the empty hole space. Gravity is sufficient to retain it in place. Note that the slider must be used in the inverted position while this filter is in place.
Fig 6. Filter in place. There is no need to use the polarizing filter at this time. When the blocking filter is not needed, remove it and place into its original package, then return the slider to its normal side up and reinsert the polarizing filter.
Fig 7. 460nm blue illumination of the Micro-Aquarium Closed System Flow Cell on stage. The blue will be blocked in the images.
Fig 8 Non fluorescing subject with white illumination. No blocking filter
Fig 9. Non fluorescing subject with blue illumination. No blocking filter.
Referencing Fig 8,9,10,11
Fig 9 is totally blue because the blue light has no blocking filter. Fig 10 is totally yellow because the white light passes through the yellow blocking filter. Fig 11 is illuminated by blue light but passes through the yellow blocking filter. On very close examination Fig 11 does have a very slight green cast when compared with Fig 10 confirming that the blocking filter is 95% effective.
Fig 10. Non fluorescing subject with white illumination with yellow blocking filter.
Fig 11. Non fluorescing subject with blue illumination with yellow blocking filter.
Assembly and use of a blocking filter with 460nm blue illumination:
The light path in operation for biofluoresence:
The Light Path: The 460nm blue light sits at the base of the microscope. It passes through the condenser, excites the GFP in the specimen, and travels up into the objective lens. [1, 2]. Direct supra-stage illumination does not pass through the condenser but excites the GFP and then is reflected up from the surface of the subject traveling into the objective lens and or sensor. The GPS is excited by the 460nm blue and will show as a bright green in a background of yellow, black or red.
The Filter Placement: The yellow barrier filter is placed in the polarizer tube (the intermediate tube located between the nosepiece and the viewing head, normally used for the Olympus BH2/BHS-KP analyzer) to put it in the observation path. The filter is acting on emitted light as it blocks the blue light after it leaves the specimen but before it reaches the eyes. [1, 2, 3, 4, 5]
Specimen Thickness: Transmitted fluorescence works best for highly transparent, intensely fluorescing specimens (like thin tissue slices, bacteria, or clear larvae). Thicker tissues will scatter the blue light and produce a high-background green haze. [1] See the above description regarding direct lighting.
Blocking Filter Purpose:
For microscopy applications the best blocking filter choices depend on the specific application. In this application preparations are in process for in-vivo coral polyps photo-microscopy. The polyps contain green fluorescing protein (GFP). The polyps host zooanthlea (dinoflagellates) in a symbiotic relationship. It is the physiologic relationship that will be observed. White light will be used for anatomic exploration. Blue light will excite and highlight the GFP, an important contributor to the photosynthetic production of metabolic products that sustain the lives of the community.
Choice of a Standard Long-Pass Filter vs a Band Pass Filter: The filter of choice is the 515nm Long-Pass (LP) filter (such as a 515IF or DM505/LP515 combination) with an optical density of 4.0 (OD4) or higher at 460nm. It completely blocks the reflected 460nm blue excitation light and allows green, yellow, and red fluorescent signals to pass to the optical sensor of the camera. [1, 2]. As a alternative, a Band-Pass Filter may be used if needed to isolate a specific emission color (like green or yellow/green GFP) while blocking longer red wavelengths, an emission band-pass filter centered around 510nm to 535nm (e.g., EM510 LP or EM535 BP) may be used. [1, 2, 3, 4]
The slider for the intermediate polarizer/analyzer tube on a BH2/BHS typically requires a 22mm circular disc filter. To source the yellow barrier filter for the Olympus BH2/BHS microscope intermediate tube, a custom, optical-grade component sized to 21.9mm diameter was custom fabricated to passively fit in the slider housing. Here are additional dimensions of the blocking filter lens: For the standard intermediate slider on the Olympus BH2/BHS series used in this study, the standard diameter for a mounted glass custom lens a 1.5mm to 2.0mm thick, cut on, 21.9mm diameter, 500nm to 515nm filter is placed flat side toward the subject. The filter is placed into the empty hole space in the microscope slider with the arrow pointed away from the viewer. It is retained in place by inverting the slider and placing the filter into the unused aperture of the slider.
Meetoptics supplies this filter with a metal ring ($190.00).
Effect:
This arrangement is 95% effective. Post processing of images using PhotoShop to remove scatter may be needed to filter out any stray blue. Although this is in development to study marine biology it may be used on a number of non marine subjects that include insects and mushrooms. It would be wonderful, if you could post some of your findings on the discussion board.
The next posting on this subject will be a description of the Micro Aquarium Closed System Flow Cell which was developed by Knapp for this marine biology research.
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This section presents a structured account of observations made in the reef system surrounding Roatan, Honduras. This is not unique to this location but the conditions are consistent with other findings in the reef systems of the Gulf. These findings and identifications are based on personal experience and the efforts of amateur naturalists. These observations extend a continuing investigation into coral reef health, previously initiated in the post titled “Death in Paradise.” See past posts. This is presented is made to support my application to the Water School at Florida Gulf Coast University to study for a Master’s in Environmental Sciences. The goal is to learn more about coral reefs, coal polyps, and polyp disease.
These observations are findings revealed by white light flash photography during the day and ultra blue illumination lighting with amber filtration to show fluorescence at night. It was not possible to superimpose photos of identical structures due to instability caused by rough sea conditions.
IMAGES WITH FLUORESCENT LIGHT
Figure 1: Fluorescent Green Goniopora Coral
Photographed using an ultra-blue light source and an amber filter, this image depicts a fluorescent green Goniopora coral, also known as the flowerpot coral. Under white light, these corals appear dull brown, but under blue or UV light, their fluorescent pigments—produced by dinoflagellate algae within the polyp—create a vivid green glow. This species is a Large Polyp Stony (LPS) coral, notable for long, fleshy polyps resembling a bouquet of flowers. The coral may belong to either the Goniopora or Alveopora genus, which can be differentiated by tentacle count—Goniopora polyps have 24 tentacles, while Alveopora have 12.
Figure 1. Photo with ultra blue light source and amber filter on camera
Figure 2: Tubastraea faulkneri (Orange Cup Coral)
This image, also captured with ultra blue lighting and an amber filter, shows a coral likely to be Tubastraea faulkneri, the orange cup coral. Typically orange, this species may display green or yellow variations under specific lighting. Tubastraea faulkneri is a non-reef building LPS coral, often inhabiting cryptic environments such as caves, overhangs, and shipwrecks. Notably, it is a zooxanthellate carnivore, lacking symbiotic algae and feeding by extending its polyps at night to capture plankton. Its distribution spans the Atlantic Ocean and Caribbean Sea with origins in the Indo-Pacific.
Figure 2. Photo with ultra blue light source and amber filter on camera
Figure 3. Montastraea cavernosa (Great Star Coral)
This photograph features a coral with a bright green base and purple-rimmed polyps or tips, possibly a Neon Green Cyphastrea, Montastraea cavernosa (Great Star Coral), Goniopora, or Fimbriaphyllia (Frogspawn/Hammer coral).
Figure 3: Neon Green Cyphastrea or Similar Species
Figure 4.
An image taken with blue light and an amber filter shows a central region of dead coral polyps highlighting loss within the colony.
Figure 4: Central Area of Dead Coral Polyps
Figure 5: Montipora Coral (“Shamrock Monti” or “Rainbow Montipora”)
This photo reveals a Montipora coral, recognized for vibrant green polyps on a purple or blue base under actinic lighting. As an SPS coral, it displays a bumpy, encrusting growth form and is valued by reef keepers for its resilience and fast growth. The coloration varies with lighting conditions, often intensifying under different spectrums.
Figure 5. Photo with ultra blue light source and amber filter on camera
Figures 6–8: Disease Progression in Montipora Coral
Figure 6 shows a Montipora coral under blue light. Figure 7, an enlargement, illustrates zones of normal tissue, dysfunction, and tissue loss, suggesting the possible progression of Stony Coral Tissue Loss Disease (SCTLD). Figure 8 highlights an encrusting Montipora structure glowing green under blue/UV light, with blue/purple areas indicating disease and tissue death.
Figure 6. Photo with ultra blue light source and amber filter on cameraFigure 7. This enlargement of the Figure 6 image shows the possible progress of SCTLD on the surface of the coral reef. Indicated are the various zones of normal, dysfunctional and tissue loss zone.Figure 8. Photo with ultra blue light source and amber filter on camera
Figure 9: Surviving Fluorescent Polyp Coral
This image depicts a fluorescent polyp coral barely surviving amidst lifeless surfaces devoid of reflective polyps.
Figure 9. Photo with ultra blue light source and amber filter on camera
Figure 10: WWC Electric Daisy (Stylocoeniella armata)
This specimen is a small polyp stony (SPS) coral, forming a dense, textured mat and known to compete with other corals upon contact. (often called a “thorn coral”).
Figure 10. Photo with ultra blue light source and amber filter on camera
Figure 11: Maze Brain or Worm Brain Coral
Likely a Platygyra or Favia species, these coral display neon green and purple fluorescence due to specialized proteins. It forms dome-shaped colonies with maze-like channels and can be semi-aggressive, extending sweeper tentacles at night. Nutrition is primarily derived from symbiotic algae.
11. Photo with ultra blue light source and amber filter on camera Figure
Figure 12: Encrusting Montipora or Cyphastrea
This image shows an encrusting coral, possibly Montipora or Cyphastrea, with green and purple fluorescence. The coral spreads as a thick mat, but species identification requires microscopic skeleton examination.
Figure 12. Photo with ultra blue light source and amber filter on camera
imagesWITHWhite light with photoFlash
Figure 13: Mustard Hill Coral or Related Species
Using white light flash, this photo captures a yellow, bumpy stony coral, likely a mustard hill coral (Porites astreoides), star coral (Orbicella spp.), or brain coral. The coral features a massive, uneven surface covered in small polyps. An encrusting goup of organism is growing in the cavity of the stony skeleton devoid of polyps. These corals are native to shallow, warm Caribbean reefs and play a critical role in reef-building.
Figure 13. Photo illuminated by white light flash.
Figure 14: Diseased Brain Coral
This image shows a brain coral suffering from disease with evident demineralization and erosion.
Figure 14. Photo illuminated by white light flash.
This photograph shows Colpophyllia natans, a common and large brain coral in the Caribbean. The coral displays wide, meandering ridges and valleys with contrasting colors. Fine, narrow lines (septa) running from ridges to valleys help distinguish this species. Boulder brain coral forms massive colonies and is a dominant reef-builder in shallow environments. It is currently considered vulnerable due to climate change, acidification, and disease. The central area in the image shows disease-related polyp loss.
This image likely features a Symmetrical Brain Coral, forming a dome-shaped colony with convoluted valleys and rounded ridges. The coral is yellowish-tan with darker areas, lacking the groove found in some related species. Pseudodiploria strigosa is widespread in Caribbean shallow waters, slow-growing, and foundational to reef habitats, supporting symbiotic algae (zooxanthellae) for photosynthesis.
Note the irregular loss of polyp growth on the surface of central and right side of the specimen. This is consistent with the result of spreading SCTL disease. Additionally, the adjacent coral is completely overrun by the opportunistic algae.
Figure 16. Photo illuminated by white light flash
Figure 17: Lettuce Coral (Agaricia spp.) and Codium Macroalgae
The image features a ruffled lettuce coral (Agaricia spp.) (Agaricia agaricites or Agaricia tenuifolia} and green branching macroalgae (Codium). The coral forms undulating plates or branches, often yellow-green or brown.
Substantial polyp loss and algal overgrowth are evident. The accompanying Chlorophyta Codium macroalgae, known as green sea fingers, have a distinctive branching structure absorb nutrients like nitrates and phosphates from the water.
Figure 17. Photo illuminated by white light flash
Figure 18: Field of Broken Coral
White light photography reveals an expanse of broken, lifeless coral, a few healthy finger corals, and one small fish.
Figure 18. Photo illuminated by white light flash.
Figure 19: Pillar Coral Disease Progression
This image of one of the pillar coral species shows a pattern of disease spread from the bottom to the top where the top appears relatively healthy in this daylight exposed photograph. The lower two thirds are bare of polyps. This area has been overrun by algae.
Figure 19. Photo illuminated by white light flash
Figure 20:
This image of a collective variety of stony corals in various states of disease progress. A patch of polyp covered coral is on the lower left. Exposed stone is seen in the central area. The remainder is covered by several opportunistic algae species. The images in the distance are overrun by SCTLD and covered with algae.
The organism shown in Figure 21 is likely a Snakelocks anemone. This finding is new to me. It is an invasive species out of its eastern Atlantic / Mediterranean range. Its tentacles are long and flowing, colored green, grey, or light brown, and tipped with violet or pink. Symbiotic algae embedded in its tissues supply nutrients through photosynthesis. Predatory by nature, it uses stinging cells to immobilize prey, although its sting is mild for humans. There were no observable mutualistic anemone fish. This supports the suggestion that these are indeed invasive.
Figure 21. Photo illuminated by white light flash
Figure 22: Chocolate Brittle Star (Ophiuroidea cinereum)
The organism in this image is likely a brittle star, endemic to the Gulf of Mexico and known as the Chocolate Brittle Star.
Figure 22. Photo illuminated by white light flash.
Figure 23 – 24: Finger Coral (Porites spp.)
This image displays a branching finger coral, common in the Caribbean, with short, blunt, finger-like lobes. Porites typically have thicker branches, while Porites furcata features thinner, rounded tips. Both can appear green, yellow, or greyish, with color and growth form influenced by habitat conditions.
Figure 23. Photo illuminated by white light flash
This enlarged view of Figure 23 depicts relatively healthy coral with no signs of Stony Coral Tissue Loss Disease (SCTLD).
Figure 24 This enlargement image of Figure 23 shows relatively healthy coral without signs of SCTLD
Conclusion:
All stony coral varietals seem to be subject to the same disease. The progress of the disease is unchecked and is advancing. The algae may be flourishing because of increased nutrients in the areas resulting from decomposing polyps and because they are optimizing an echo-niche left vacant by the polyps. The algae are not rebuilding the reefs. The fish population is also in great decline probably secondary to the degradation of the reefs.
Personal Commentary and Reef Health Summary
After thirteen dives across six reef sites in Roatan, Honduras, the overall impression was a declining reef system. Notably absent were turtles, large fish, sharks, lobsters, tunicates, and nudibranchs. The reef, once vibrant, now stretches for miles as broken, gray-brown expanses covered by algae. Invasive species observed included lionfish, a single anemone, Tubastraea faulkneri coral, and two spiny urchins. Only small fragments of some coral species persist, with no Elkhorn, Pillar, or Staghorn forms present and much of the previously widespread fluorescence now largely gone. While some sponges and fans remain, the images presented here document the few surviving patches of live coral polyps amid non-fluorescent, algae-covered stone skeletons.
Plan for remediation:
Problem Statement
The Stony Coral Tissue Loss Disease is relentlessly advancing and destroying the vital surface of the stony reefs. The cause of this disease is not yet discovered. This is a continuing catastrophic event. Work is in progress and includes maping, determining the progress rate and direction of spread. The causality and cure are not yet determined.
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4 responses to “Fluorescent Photography Highlights Coral Health Issues”
Cindy
You examined so many varities of coral. It is very disheartening to learn how badly the disease is treating the reef. If you can determine the primary cause that will be wonderful, and a first step to treatment.
Cindy
There is so much unknown about diseases in the ocean. This one is devistating and the cause is unknown. Hopefully the project that I planned will make some difference.
It is devastating seeing the reefs dying and nearly everything else with them. I stopped scuba diving some years ago and I am happy to have seen beautiful reefs in many parts of the world.
This is a follow-up publication on Stony Coral Tissue Loss Disease. It describes the findings in the coral reefs in the Cayman Islands during a week in mid April, 2024. The travel to the island was reasonably easy and the accommodations onboard the live-aboard cruise were good. The conditions of the reef life and tropical fish however was disheartening. We have a big problem.
The featured image was made from a view of the sunset from the port on Grand Cayman island. The sunset may be symbolic of the future diving in the Caribbean. I hope that there is a recovery in the future.
As reported in four previous posts (Death in Paradise), there is a continuance of even greater proportions seen during this trip. I reported on the findings on the reefs around Roatan Island off the coast of Honduras two years ago. The condition of the reefs showed considerable loss of the polyp life because of Stony Coral Tissue Loss Disease (SCTLD). It was quite extensive and showed nearly 70% loss of life. At night the coral showed a significant loss of fluorescence because of the large dead areas of coral polyps. During the day one could see the areas of exposed stony skeleton of the reef.
The reef at multiple sites of the Cayman Islands including Grand, Little and Brack, has been devastated by the disease. The population of the reef fish is also significantly diminished. I did not make a count of the fish or of the areas of dead coral. Because of the extent of the dead coral there was no reasonable way for me to make an accurate comparison. It was very distressing. With the option to make 20 dives I made only 12. There was nothing worthwhile to see. I would estimate more than 80% to 90% loss of polyp life.
In conversation with other experienced divers on board the ship they said that they saw similar conditions across many of the Caribbean islands from Bonaire to the Mayan coast. Generally, they were unaware of the cause of the polyp depletion but said that it looked the same as in the Caymans. The less experienced divers were unaware of the problem and were happy to be in the warm water (85.5 degrees F). There was still some interesting wildlife but is was very reduced when compared to the concentration I had seen as little as 5 years ago.
50% of central coral head is dead. All of the surrounding coral is dead.
Coral completely over run by SCTLD.
70% of brain coral is dead. Surrounding coral is lifeless.
nderwaterOther areas typical of the reefs showing the discruction of the polyps.
Lion fish (Invasive)
Caribbean Spiny Lobster (Panulirus argus) It was good to see a lobster but formerly there were dozens.
Rough file clam (Ctenoides scaber). Similar to the disco-clam seen in the Palau reefs.
There was still life in the Caribbean but it may be below the 100-foot depth accessible for divers and also for much of the live that requires light. The life forms that were readily apparent were mostly bottom feeding animals. Sponges and soft fans were still alive and did not seem to be affected by the SCTLD.
The effects upon the economy that are a result of reef polyp loss are not readily available, however, many of the recreational hospitality workers are questioning their future employment opportunities. I suspect that there may be some resistance in these businesses to make these issues more public.
There has been some publication of the research into the cause of the disease and it seems that researchers have identified an infection. “Results suggest that the bacterial community may be involved in SCTLD, but other members of the holobiont (i.e., viruses and Symbiodiniaceae) may contribute to lesion progression”.1,2
Vectors and process of viral action is not yet publicized 3. One should not expect a recommended solution for a cure until much further research will be done. I anticipate that it may take decades and that the reef corals will not survive at the current rate of disease progress. This has a direct impact on the southwest coast of Florida.
If you have observed the effects of SCTLD in other areas of the Caribbean or other places in the world please engage in the SCTLD dialogue in the discussion board.
References: Where 2. and 3. are specifically related to work done in Florida reefs.
4 responses to “Fluorescent Photography Highlights Coral Health Issues”
You examined so many varities of coral. It is very disheartening to learn how badly the disease is treating the reef. If you can determine the primary cause that will be wonderful, and a first step to treatment.
Cindy
There is so much unknown about diseases in the ocean. This one is devistating and the cause is unknown. Hopefully the project that I planned will make some difference.
It is devastating seeing the reefs dying and nearly everything else with them. I stopped scuba diving some years ago and I am happy to have seen beautiful reefs in many parts of the world.
Yes. Caribbean diving was my first salt water experience now I am reluctant to return. I am so anxious to see some reversal of marine life there.