Coral fluorescence appearance vs excitation wavelength

Posted On: Tuesday, May 19, 2015

With fluorescence, what you see can depend on how you excite and view the subject. The interaction of the excitation wavelength, the fluorescing substance, and the barrier filter can lead to different appearances in the final image. What is ‘true’ or ‘best’? There is no simple answer to that and it can come down to what your purpose was in taking the image and what looks best to you.

I recently gave a presentation on The Function of Fluorescence on the Reef to the Boston Reefers aquarium club. I brought my stereo microscope, outfitted with the NIGHTSEA Stereo Microscope Fluorescence Adapter, along with me to the meeting to show the attendees first-hand what coral fluorescence looks like at higher magnification. Club Treasurer Tony Diep brought along some small corals that we could look at. One of these, a specimen of Cyphastrea sp., produced an interesting set of images to illustrate the influence of choice in excitation and emission.

In the gallery below are four images, taken under the following conditions:

• White light LED illumination
• Violet (~410 nm) excitation, no barrier filter
• Violet (~410 nm) excitation, with a pale yellow barrier filter that completely blocks the reflected excitation
• Royal Blue (~450 nm) excitation, with a yellow barrier filter that completely blocks the reflected excitation

(Click any image for larger view)

So what’s going on here? The coral seems to contain two main sources of fluorescence, an orange fluorescent protein in the polyp mouths and a cyan fluorescent protein in the surrounding tissue (the coenosarc).

In the first image (white light) you can see the orange tint in the mouths but the cyan fluorescence is overwhelmed by the reflected white light and the coral appears beige/brown. This is the characteristic color of most corals that is due to light absorption by the symbiotic algae (zooxantheallae).

The second image (violet excitation, no barrier filter) is dominated by the reflected excitation light. The combination of that reflected violet and the orange fluorescence in the mouths yields the striking magenta you see there.

In the third image (violet excitation, violet-blocking barrier filter) we can now see the ‘true’ fluorescence of the specimen. The coenosarc fluorescence is what is expected for the well-known cyan fluorescent pigment (CFP) that has a fairly broad emission spectrum with emission peak at about 486 nm.

In the last image (royal blue excitation, blue-blocking barrier filter) the orange fluorescence of the polyp mouths is the same as it was in the third image, but the surrounding tissue fluorescence now appears green rather than cyan. This is not a different protein. It is the same cyan fluorescent protein, but the yellow barrier filter blocks the blue portion of the emission (below about 500 nm), leaving only the portion of the fluorescence that appears green. So from a technical standpoint this is a false-color appearance, not representative of the ‘true’ fluorescence of the subject. But it sure does make a nice image! Note that where the tissue fluorescence meets the mouth fluorescence you see a yellow color. This is an optical effect created by the mixture of green and orange sources, not a true yellow fluorescence.

I also imaged a specimen of Leptoseris sp. that had two fluorescent pigments: a green fluorescent protein at the polyp mouths and a yellow fluorescent protein associated with the tissue at the structural ridges. In this case there was absolutely no difference in the fluorescence when viewed with the Violet or Royal Blue excitation/emission sets. Both were capable of exciting the two sources of fluorescence and of capturing the resulting emission.

Check out our other post from this meeting showing how fluorescence highlights tissue growth extension.

 

4 Responses to “Coral fluorescence appearance vs excitation wavelength”

1. I really appreciate the information you post!

   In the case of the Leptoseris,both excitation wavelengths show the ‘real color of the FPs’ due to the fact that they are not overwhelmed by the excitation light? why is that there is no difference at all if they have different wavelengths?

   Thank you very much

   Reply

2. Hi Erika. In the third and fourth pictures of the Cyphastrea there is no excitation light in either picture because it is blocked by the appropriate barrier filter. In the case of Royal Blue excitation with the Cyphastrea the barrier filter blocks not only the reflected excitation, but also the blue portion of the blue-green (cyan) emission, making it look green.

   In the case of the Leptoseris there are two fluorescent pigments, one that glows green and the other yellow. Both pigments can be excited by Violet and by Royal Blue, but that does not matter – in either case the same light comes out, green and yellow. In this case, though, the fluorescence is at longer wavelengths than either barrier filter. So green fluorescence looks green whether excited by Violet or Royal Blue, and the same for the yellow fluorescence. I hope this makes it clearer.

   Reply

3. What are you using as a violet blocking barrier in pic #3? All yellow filters I’ve seen block both blue and violet wavelengths. You make a point that pic 3 is illuminated with a violet source and pic 4 is with a royal blue source, but the results are really dependent on the barrier filter, not the light source, according to the explanation why one coral appears cyan and other green. Would the results be any different if the same light source was used for both pics, but the barrier filter changed? Thank you.

   Reply

4. Hi Adrian. Good question, and sorry if there is confusion. For the violet source we use a very pale yellow filter that does block some blue (violet is at the end of the blue light spectrum), but not all. That is why you can see the true cyan (blue + green) emission from that specimen.

   Here is what you would expect for different combinations (using VI for Violet, RB for Royal Blue):

   VI light + VI barrier filter – that is what you see in picture #3, lower left

   VI light + RB barrier filter – would look like picture #4. You would be exciting the same emissions, but the deeper yellow barrier filter would cut off the blue portion of the cyan emission. The relative intensities might change a bit, but the colors would be the same.

   RB light + RB barrier filter – that is what you see in picture #4, lower right

   RB light + VI barrier filter – this would look more like picture #2. It would be like using no barrier filter at all since the RB excitation is in the barrier transmission band, not its blocking band. There would be a very strong reflected blue component and a similar color additive effect in the orange polyp mouths, giving them a false color. as in picture #2. The cyan fluorescence would be there, but would be completely overwhelmed by the reflected blue excitation.

   I hope that is clearer!

   Reply