ICP Analysis Update

We have results from the Inductively Coupled Plasma Mass Spectronomy (ICP-MS), but have not yet processed the data. (Click here for a short explanation of what the ICP-MS is). Two things that seem promising, though, are the facts that the Calcium Carbonate was so saturated that the machine couldn't obtain an accurate measurement and aluminum was present in the ossicles. The first thing is unusual due to the fact that the sample size consisted of milligrams (not even grams) of ossicles which, like all bones, contain lots of calcium carbonate, but usually not in such high saturation. Also, the fact that aluminum is embedded in the tiny bones is strange in light of recent findings that aluminum is a neurotoxin that, though abundant in nature, is not an essential element and poses a threat to organisms. However, in an analysis of iridescent fish scales it was found that there were high concentrations of aluminum that might play a part in the scales' light refraction. Since we already found out that the ossicles are specialized for fluorescent light transmission by transmitting green visible light wavelengths, it is possible that aluminum also enhances the ossicles' light enhancing properties.

Since we were unable to get a calcium reading with our previous samples, I'll be sorting and prepping new samples (about 1/10 of the amount we tested last time) for another ICP analysis. Hopefully there won't be a saturation of calcium in these readings since the proportion of Calcium Carbonate is what we compare all the other measurements to in order to create ratios of metals to calcium. (I.e. there are 10 parts Calcium and 1 part Magnesium.)  Once that's done we'll have to make sense of all the measurements and numbers and proportions.

Prepping for the Plasma Machine (ICP)

Hours upon hours of ossicle sorting have almost paid off—we're finally going to analyze the samples using the ICP-MS.


I'll explain in a moment, but first: some ossicle samples from the brittle star O. californica were analyzed using hypo-spectronomy and found to transmit light between wavelengths of 530 and 590 nanometers—the spectrum of visible green light—which happens to be the color of brittle star bioluminescence. Therefore, it is very likely that the tiny bones in the arms of certain brittle stars are optimized to transmit and ideally amplify the light from their luminescent reactions. How amazing is that?! These are specialized bones in a specific luminescent system that aid in defense or mating or whatever else the brittle stars use luminesce for. Well, anyways, I was excited.

Okay. Back to the issue of ICP analysis.

ICP-MS stands for Inductively Coupled Plasma Mass Spectronomy. As another intern explained to me, it is the process of heating up argon gas into the fourth state of matter, that is, plasma. Once there is plasma in the chamber of the machine, samples (which need to be in liquid form) are sent into the chamber in a kind of mist after being nebulized. The drops vaporize near the plasma and solids break down into individual atoms after being vaporized and certain chemical elements are ionized. These ions are sorted in a special device for mass spectronomy, which can determine the presence of elements of atomic mass 7 to 250 and their proportions in the sample. In the case of the brittle star ossicles, we want to find out how much of what elements are in the bones of luminescent brittle star species compared to proportions of the same elements in non-luminescent species in order to determine if those elements (mainly metals) play any role in specialized light transmission and refraction.
During this and last week, I continued to sort more of the A. pujetana ossicles since I had less of that sample compared to my O. californica and A. squamata samples. I sorted as much as I could on Thursday and then weighed all the samples. All the weighing and digesting, etc. had to be done in order so that it would be easier to organize the samples once we got the results back from the ICP. The order I used was: Blank_1, Blank_2, Blank_3, OCL, OCT, OCV, ASL, AST, ASV, APL, APT, APV. The first two initials stand for the species name (i.e. O. californica) and the last letter stands for what kind of ossicle it is, that is, Lateral Shield, Top/Bottom Shield, or Vertebrae. 
After the ossicles were weighed, I added approximately 150 microliters of 70% Nitric Acid (or about two large drops from a disposable glass pipette) to each tube in order to dissolve the samples into a liquid. A glass pipette was used instead of the more accurate mechanical pipette because Nitric Acid is so strong it or its fumes could potentially harm the sensitive measuring device in a normal pipette and make it unusable or in need or serious repair. Once the acid was added, the ossicles sat for 12 or more hours to insure optimum digestion.

The container and tube were tared, then the sample was added. The weight (usually in milligrams) was recorded in a text file on the computer that the scale was hooked up to. [Note: in the picture it shows a tube with a cap on; in the actual weighing the cap was never put on in the scale due to the fact that if more grams are tared the less accurate the measurements of the samples will be.]

"JUSTRITE: Acids and Corrosives Storage Cabinet"

Not More than 70% Nitric Acid. It's extremely bad for your health (note the skull and crossbones), severely reactive, and extremely dangerous on contact, but worry not! it's not flammable. You need to wear goggles & shield, lab coat & apron, and proper gloves when using this acid under a vent hood.

The amazing vent hood, which, in the clean room, is always blowing on high, keeping this part of the lab at high negative pressure and sucking out any harmful or plain stinky acid or chemical smells.

 In the pictures below, acid is added to the sample of O. californica vertebral ossicles and bubbling occurs due to the reaction between the calcium carbonate in the tiny bones and the Nitric Acid, which facilitates the creation and release of Carbon dioxide gas.

The tubes, once the sample and acid are weighed, are put in a tube rack under the fume hood so that the acid can fully dissolve the ossicles into a fluid liquid to be diluted with MilliQ water after full digestion. The resultant liquid will be nebulized into the ICP-MS for analysis.

I recorded the weights on the computer and in my notebook. Notice how minuscule the sample sizes are in terms of weight (in grams).

I have yet to get the results from the ICP, but am nonetheless optimistic that they will be quite interesting and certainly worth a follow-up post!

In Search of Beetles

Today my mentor, Jenny (another intern who attends UCSD) and I went to the Natural History Museum to search their entomological archives. Basically, they have a very large collection of dead bugs. Technically, their collection consists of over 900,000 insect and arachnid specimens, which is relatively small compared to some other collections (of course, the Smithsonian has 35 million specimens). In this vast archive of thousands of organisms we were to search for iridescent or fluorescent organisms. This required searching through cabinets full of glass display cases that contained tens of dead insects pinned to foam mats.

Why?

My mentor had an upcoming funding presentation and when it comes to research science, you need to have some great PR once in a while to keep the public informed and supportive, and your funding steady. We have been working on iridescent fish scales and butterflies (the standard specimen when it comes to structural light coloration studies), but we hadn't yet looked at other land organisms. However, if we found some intriguing insects or what have you that were found in the San Diego area, it would be a good continuation of our structural coloration research and possibly serve as a contrast between marine and terrestrial iridescence.

When we arrived at the Natural History museum and taken behind-the-scenes to the entomological section, we had no idea what we were looking for. Granted, we knew what the glimmer and shine of iridescence looked like, but we were to look through hundreds, if not thousands of insects. At least the entomologists had an idea for where we should start.

The room was full of those large rolling shelving units like they have in the archives of big libraries. The metal cabinets lined the walls and only opened for one small passageway at a time before they closed their dull teeth behind you with the slow creaking sound of the old wheels. These were definitely archives--stored away behind dull gray doors that often stubbornly refused to open, unwilling to reveal the amazing specimens that were trapped in this fluorescent-lit prison, a zoo and a cemetery alike.

Inside the gray doors were wooden cases with glass on top to display the specimens, every one of which was pinned to the foam beneath. Many of them had labels that revealed (in what has to be some of the smallest legible writing in the world) the date of collection, genus/species, location of collection, and other notes. All of this information was crammed perfectly onto small tags pinned underneath the bugs.

The curator suggested we look at insects of the order Coleoptera, commonly known as beetles. They make up a lot of the collection at the Natural History Museum besides butterflies and Coleoptera actually has more organisms than any other order. So in a way, it was good to start there because there was so much to choose from, but that also meant that between the 3 of us, there were thousands and tens of thousands of beetles waiting to be seen. In the picture above there are about 45 cabinets. In each cabinet, there were anywhere from 6 to 12 display cases full of tens of beetles. And we looked through all the cabinets shown in that picture and more!

Some beetles looked plain and unassuming, but had great color contrast, which is important in the natural world. Mostly, we were looking for some beautiful examples of iridescence that was preferably not green iridescence, which is really common. We wanted more reds or purples, which are harder colors to produce and much rarer in nature.

Although these beetles don't seem to be anything special, they are an example of nature's less showy wonders.
Their coloration (which is structural [all beetle coloration is structural]) consists of a white, which deflects all wavelengths of light, and a brown, which absorbs many wavelengths of light. This contrast is probably used for camouflage or to confuse predators, but to produce two very contrasting colors simultaneously is an amazing development.

Some very cool beetles. There's a very iridescent purple-blue-teal-green beetle in the middle and a scarab that looks like it truly was preserved in gold.

These otherwise boring light brown beetles iridesce green as you can see on many of their backs. There are a few that haven't been hit by the light in the right way or at the right angle, but many iridesce a nice, light green.

From Arizona, these green beetles had amazing silver stripes down their backs. The light refracting off of them is the iridescence of the silver lines.

Striking reddish purple coloring with green trimming defines these beetles.

The common green iridescence with red highlights and ...posteriors.

An assortment of bees, in the upper left there are some smaller bees that the picture below depicts (in poor lighting).
These bees had iridescent "fuzz" on them and usually the smaller ones tended to be more colorful.
Also, in the picture above, a normal common bee that we usually see would be a size between those small bees and those giant bees.

It might be hard to see in the poor lighting of the photo, but these small bees have some strong iridescent green colors. Surprisingly enough, the none iridescent portions of some of them turned out to be fluorescent, which is certainly intriguing!

The entomological curator (left) removes an insect that my mentor (right) is renting from the Natural History Museum.
We are allowed to borrow the specimens for a year and if we need them even longer we can just contact them and extend our lease.

In the end, we ended up borrowing 13 insects for further examination in the lab. One of the most amazing things was that we borrowed a beetle that was collected in 1832! We were allowed to lease a 180 year old insect and take it back to the lab with us. Amazing, huh?

Although looking through all those beetles took us the better part of a day, ever the gluttons for punishment in the name of science, it was decided that we would probably go back in the near future to look for good damselfly or dragonfly specimens. Our only concern was being a burden to the ever helpful curator, who had the daunting task of rearranging the collection according to the ever-changing taxonomy standards that seem to be constantly re-classifying insects. It's no small task to reorganize a small entomological collection (only 900,000 bugs, remember?) The best of luck to him and to us as we delve deeper into the world of terrestrial organisms, which us marine biologists know surprisingly little about...