The overall goal of this procedure is to describe how to extract pigments located within the nano-structured granules of freshly dissected squid, Doryteuthis pealeii chromatophores. Cephalopods are complex marine animals that undergo rapid and adaptive changes to their dermal color. and adaptive changes to their dermal color.
However, there is still a lot that is unknown about how these systems work. The main advantage of this technique is that it specifically targets the nano-structured granules in the chromatophore organ. This will enable researchers to elucidate the mechanism for color change originating at the nanoscale.
In addition to insight into cephalopod biology, it can inform design of synthetic systems built to mimic the dynamic range of adaptive color displayed in cephalopods using pre-packaged pigmented nano-particles. Generally, individuals new to this method struggle because collecting pure chromatophore tissue is difficult due to its proximity with the surrounding tissue. Our method is optimized to reduce these contaminants.
To begin dissection, first pour approximately 250 milliliters of filtered sea water into a dissection pan. Place a specimen in the pan and using stainless steel straight fine point scissors, cut from the base of the ventral mantle along the posterior side of the animal, ending at the fin region. Remove all of the internal organs by gently lifting them with dissecting forceps and severing the connective tissue using the scissors.
Discard the organs in a specimen bag. Using dissecting T pins, pin the mantle fin side up into the dissection pad. With forceps, carefully remove the opaque epidermal skin layer, keeping the chromatophore-containing skin layer intact.
Discard the epidermal layer in a specimen bag. Next, remove the chromatophore layer. Cut the chromatophore tissue into small one centimeter by one centimeter sections.
Place the sections into a 1.5 milliliter micro centrifuge tube, filling each tube halfway with dissected tissue. Finally, add 500 microliters of filtered sea water to the tubes. To isolate the pigment granules, first centrifuge the chromatophore tissue sections for five minutes at 14, 000 x g.
Discard the top layer of seawater. Add 500 microliters of papain collagenase solution to the samples and vortex for one to two minutes on the highest setting. Next, sonicate the samples for five minutes at 40 kHz to dissociate cells from the surrounding tissue.
Centrifuge for a further five minutes at 14, 000 x g and then discard the supernatant. Repeat the centrifugation a further two times, discarding the supernatant each time. Then, add the resuspension in collagenase.
Use forceps to manually remove any remaining large tissue sections that did not digest before proceeding. The purity of starting granule solution influences the pigment extraction. So it is important to ensure that the starting chromatophore materials are fully digested.
We find that tissue sections around one centimeter are optimal for collecting the purest population of granules. Prepare the homogenization buffer by taking a 150 milliliter screw cap tube and adding the appropriate amount of HEPES, magnesium chloride, potassium aspartate, and dithiothreitol. Add one commercial mini tablet of protease inhibitor.
Using a graduated cylinder, measure 100ml of deionized water and add it to the tube containing the homogenization salts. Vortex the tube to mix until the solution becomes clear. Centrifuge the tissue samples and then discard the supernatant.
Add 500 microliters of homogenization buffer to each tube and vortex to 2 minutes to thoroughly mix. Sonicate the samples for 30 minutes at 40kHz. And then centrifuge them again.
After discarding the supernatant, repeat the homogenization addition, sonication, and centrifugation two to three times to ensure complete digestion of the extracellular tissue. Finally, the tube should contain a light yellow supernatant and a red colored pellet consisting of the isolated pigment granules. Discard the supernatant.
First, prepare acidic methanol for extraction in a screw cap glass sample vile by adding 5ml of methanol and 25mcL of concentrated hydrochloric acid. Swirl the solution gently to mix. Set aside one sample tube, this will be the end reacted granules treatment.
Remove the homogenization buffer from a second tube containing granules. Add 500mcL of the acidic methanol solution to the remaining tubes and vortex for three to four minutes. Sonicate the samples for 10 minutes at 40kHz and then centrifuge for five minutes at 14000 x g.
Using a micropipette, collect the supernatant in a one-dram screw-top glass vial. This is the pigment, and it should appear dark red in color. Repeat the acidic methanol addition, sonication, and centrifugation steps four to five times collecting the supernatant until no further color is extracted.
The remaining colorless pellet is the pigment extracted granules. To prepare granule samples for imaging, first centrifuge both the unreacted granule aliquot and the pigment extracted granules for five minutes at 14000 x g discard the supernatant, and then resuspend each pellet in 1ml of 95%ethanol. Using a transfer pipette, place 2-3 drops of each granule suspension onto a separate aluminum SEM stub.
Place the samples in desiccator to dry overnight under vacuum. Sputter coat the stubs with gold platinum coating for 3 minutes to achieve a 15 nanometer coating. Image the samples using a scanning electron microscope.
Next, use a spectrophotometer to collect the absorbance profiles for the unreacted granules, extracted granules, and extracted pigment. Take a blank measurement for the granule samples by adding 1ml of deionized water to a clean cuvette. Discard the blank into a waste container.
Next, add the suspension containing the unreacted pigment granules to the cuvette. Collect the UV vis absorption spectra for the granule sample. Take a second blank measurement for the extracted pigment using 1mL of acidic methanol and a clean cuvette.
Finally, take the UV vis absorbent spectrum for the pigment and then determine the maximum wavelength of each sample. Scanning electron microscopy revealed that the pigment extraction process reduces the average diameter of the granules. Prior to extraction, granules have an average diameter of 527.3nm.
After the acidic methanol procedure, this size decreases significantly to around 202.6nm. A reduction thought to be due to the extraction of the pigment. Upon addition of the acidic methanol, the color associated with the unreacted granules also visibly reduces.
Confirmation of this hypothesis was obtained using spectrophotometry. This graph shows a broad absorbance profile for the pre-extracted granules and a similarly broad but lower intensity profile for the post-extracted granules. In contrast, the soluble extracted pigment exhibits a lambda max at around 500nm and a distinct shoulder at around 380nm.
Collectively, these data suggest the successful extraction of visible color from the granules. Once mastered, this technique can be done in approximately four hours from start to finish. While attempting this procedure it's important to remember to fully remove the tissue which contains reflective protein platelets that may interfere with the extraction and analysis.
Epidermal tissue may also interfere with homogenization steps and the pigment extraction. Following this procedure, other methods like thin layer chromatography and mass spectrometry can be used to further purify the pigments and characterize their composition. These steps can help elucidate the molecular contributions to coloration in cephalopods.
This method can help answer key questions for marine biologists, chemists, and engineers including:What is the composition of the pigments used during color change? And how do they contribute to the function and structure of the nano structured granules within the dermal chromatophore organs? After watching this video you should have a good understanding of how to extract pigment from freshly dissected squid, doryteuthis pealeii chromatophores.
