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Stable Isotopes in Forensics

In October 2000, a pair of duck hunters in Utah stumbled into a murder mystery. As they walked along the south shore of the Great Salt Lake—just 100 feet from Interstate 80—they found buried in a shallow grave a plastic bag containing a white sock, an oversized t-shirt, a woven blue choker necklace, 12 bones, and a human skull. Missing was any sort of identification. Police gathered what clues they could from the remains of "Saltair Sally," named after a resort near where her remains were found, though there wasn't much to go on. The body was badly decomposed, dental records didn't reveal anything, and a widely circulated composite sketch and description of her personal effects yielded no valuable information. The case went cold.


Seven years later, there were still no leads in the case. Desperate for a break, the state medical examiner released a sample of Saltair Sally's long, blonde hair to be tested by stable isotope analysis, a new forensic technique that had been introduced in the years since Saltair Sally had been discovered.

Every molecule in our bodies—including those found in hair—are made up not just of different elements, but of different ratios of stable isotopes of those elements. Take hydrogen, for example. In its most common form, it has one proton and one electron. There are also two less common isotopes, deuterium and tritium, which have one and two neutrons, respectively, in addition to their proton and electron. Deuterium is particularly useful to forensic scientists. It is naturally occurring and very stable, meaning it is found in water in varying amounts, and, critically, those variations depend on the source of the water.

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A time of flight secondary ion mass spectrometer (TOF-SIMS) used to analyze the stable isotope composition of surfaces.
Courtesy of Pacific Northwest National Laboratory

Lesley Chesson works at Salt Lake City-based IsoForensics, the company that received the hair sample. "It was our first case, where we really got started," says Chesson, an analytical chemist. "They contacted us to see if we would analyze the hair—a long length of the hair—looking at hydrogen and oxygen to see, Was she local? Did she move into the area? If she did, where was she from prior to her death?" Such clues would give investigators valuable information as they continued to search for the victim's identity.

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To see how that's possible, let's follow a raindrop from a cloud over the Pacific Ocean to Saltair Sally's strand of hair. When those clouds rain, the heavier, oxygen-18 containing water molecules will fall out first. That typically happens near the coast. As the rain cloud moves inland, it's constantly losing molecules with oxygen-18, and its raindrops become isotopically lighter and lighter. Since our drinking water comes from rainwater, people near the coasts drink water with more oxygen-18 atoms in it than people living inland. Those atoms eventually become a part of our tissues, like hair.

If Saltair Sally had been in Salt Lake City in the weeks preceding her death, the hair closest to her scalp would reflect the isotopic signature of Salt Lake City's local water supply. If she had been in, say, Seattle instead, her hair's isotopic composition would contain more oxygen-18, giving investigators a valuable clue.

"We analyzed the hair sample," Chesson says. "It looked like within the two years leading up to her death, she had been a frequent migrant. She had moved at least three or four times, and it seemed she was making this sort of cyclic pattern. She was in a region consistent with Salt Lake City and the Intermountain West, and then she was moving someplace more towards the northwest. She did this a couple of times in the years leading up to her death."

Armed with this new information, detectives refocused their investigation on the regions Chesson and her colleagues had suggested. Still, it wouldn't be until August 2012 that they received a break in the case.

More than water

Hair isn't the only tissue scientists can analyze for clues. Fingernails are nearly identical in composition to hair, and like hair, they leave an isotopic timeline that remains unchanged until it gets cut off. In fact, every part of our bodies has potentially exploitable isotopic signatures. Though hydrogen and oxygen in hair and fingernails are often the first to be analyzed, other isotopes and body tissues can provide different types of information, says Wolfram Meier-Augenstein, a professor and principal scientist at the James Hutton Institute in Aberdeen, Scotland.

"Very rarely is one isotope alone the thing that will solve the case," he says. "It is usually a combination. Forensic science is all about context. We usually aim to build a picture not relying on one technique."

Carbon and nitrogen are two other elements commonly used by forensic scientists. Like hydrogen and oxygen, carbon and nitrogen are found throughout our bodies. But instead of chronicling the water supply, they reflect people's diets. Carbon in our bodies is largely derived from plant tissues, which get their carbon from CO2 in the atmosphere. Carbon isotopes are concentrated differently depending on which type of photosynthesis a plant uses. Wheat, rice, and sugar beets use what's known as C3 photosynthesis. Their cells take CO2 directly from the atmosphere to convert to sugar. Corn, sorghum, and sugar cane use a different photosynthetic pathway known as C4, which has an additional enzyme that concentrates CO2 before handing it off to Rubisco, which begins the carbon fixing process. The added step in C4 plants increases their carbon-13 levels relative to C3 plants. In North America, where corn is common in diets, people's tissues tend to have high carbon-13 concentrations.

"We don't find a needle in a haystack. We reduce the haystack to a manageable size."

Nitrogen isotope levels depend on whether people are omnivorous, vegetarian, vegan, or otherwise. Each step up the food chain enriches the ratio of nitrogen-15 to nitrogen-14 by 3.4‰ (per mille). That means the tissues of a person who is vegan will have less nitrogen-15 than the tissues of someone who eats mostly meat. Such clues about a victim's diet can be helpful when police are canvassing for clues.

Stable isotopes in teeth provide investigators with detailed records of where a person was living when they were formed. Unlike other tissues in the body, the isotopic composition of teeth never change.
Courtesy of Wolfram Meier-Augenstein

Unlike hair and fingernails, most tissues do not record in linear fashion where a person was or what they ate. "While the signature in hair will change relatively rapidly, the signature in other tissues, bone for instance, will change very, very slowly. The signature in teeth won't change at all," Meier-Augenstein says. Turnover in muscle tissue, which you might think would happen quickly, is a slow process. "Depending on which muscle we would probe, it would take three to six months before everything in the soft tissue department would have changed."

Chemically identical, isotopically distinct

Analyzing human tissue in cases like Saltair Sally's is an obvious application of stable isotopes, but it isn't the only one. Meier-Augenstein frequently deals with drug-related cases, including one in the United Kingdom where a man was initially arrested on the street for possessing five tablets of MDMA, or ecstasy. It's a minor offense, but the police felt he might have been hiding something.

"The police secured a search warrant and searched his place, and lo and behold, in a gym bag in a wardrobe, they found a huge plastic bag with thousands of ecstasy tablets," Meier-Augenstein recounts. The suspect denied knowledge of the stash, instead blaming it on a friend. Though the tablets looked and were chemically to similar to the five found on the suspect, police didn't feel it was enough for a solid conviction, Meier-Augenstein says. That's where stable isotopes came in. "These tablets were analyzed isotopically, and lo and behold, at a 95-plus percent confidence level, there was absolutely nothing to choose between them." Confronted with this new evidence, the suspect admitted his guilt.

Drug-related cases aren't the only type where stable isotope forensics can augment traditional chemical analysis. Today, many products are chemically identical, even if they are from separate factories. Take, for example, white paint chips found on a crime scene, Chesson says, or traces of explosives used in a terrorist attack. Evidence from the crime scene may chemically match those found in a suspect's home, but stable isotope analysis could reveal that they were made in separate factories. If that were a key piece of evidence, the isotopic analysis could be enough to keep an innocent person out of prison.

Stable isotopes can also distinguish genuine products from counterfeits. The liquor industry, which is rife with fakes and mislabeled products, has found isotopic analysis to be particularly useful. Wine may be intentionally mislabeled as a better vintage, or a cheap single-malt whiskey could be hawked as an upscale brand. By comparing a suspected counterfeit with an original sample, stable isotopes can help investigators unmask the fraud.

isotopic filters

Though stable isotopes are useful in sniffing out potential counterfeits—or potential leads in a case—they are not a means of identification, like fingerprints or DNA. "I really don't like the word identification, because that's not what we do," Meier-Augenstein says. "We try to create a picture of the person's life and life circumstance that hopefully helps jog a person's memory."

Samples being loaded into a mass spectrometer for analysis.
Courtesy of Lesley Chesson

Meier-Augenstein recommends viewing each stable isotope as a filter. In our globalized world, a John Doe could have come from anywhere. Investigators start looking for clues close to where the victim was found, but if nothing turns up, they could end up combing through every missing persons list in the world.

Stable isotopes help narrow that search. Deuterium and oxygen-18 from a victim's hair or fingernails "can exclude 80 or 90 percent of the world," Meier-Augenstein notes. A nitrogen-15 analysis of the victim's muscle tissue could reveal that he was a vegetarian, another useful clue. And heavier isotopes such as strontium leech from rocks into groundwater, eventually making their way into our bones and teeth.

Together, all of these clues narrow the field of potential matches, greatly simplifying things for detectives. "We don't find a needle in a haystack," Meier-Augenstein says. "What we do, in an ideal circumstance, is we reduce the haystack to a manageable size."

what the future holds

While stable isotopes have proven themselves useful, they aren't staples of forensic science—yet. Part of the problem is cost. Stable isotope analysis isn't cheap. Preparing samples takes lots of time and involves a host of costly chemicals. Most samples today are run through mass spectrometers, which are large, expensive, and often temperamental. Finally, interpreting the data requires an expert. These obstacles aren't insurmountable, but they're not insignificant.

To change that, Meier-Augenstein thinks a landmark case needs to hinge on evidence provided by stable isotopes. "There will be a major case, unfortunately it will involve something very unpleasant," he says. "My money would probably be on terrorism, as sad as the prospect might be, or it's a high profile murder case where isotopes provide the clincher."

Chesson is more optimistic. She feels that breakthroughs in equipment used to process stable isotopes samples will spur widespread adoption. Currently she is testing a new device that uses a laser to measure isotopic ratios. It doesn't require samples to be as carefully prepared, and it doesn't take up nearly as much space as a typical mass spectrometer. Chesson foresees a day when that technology could be further miniaturized to where it can fit on countertops or even in the trunks of detectives' cruisers.

"You could take your analyzer places that you couldn't before, which means you can analyze things that are harder to collect and get back to lab. That decreases costs, speeds up time, and opens up a whole new set of samples that you could possibly analyze," she says. "Anything that decreases cost and increases throughput will always help this type of analysis. That's why DNA analysis really exploded."

Naming saltair sally

Salt Lake Unified Police announced on August 7, 2012 that, with the help of stable isotope analysis, they had identified Saltair Sally. Her name was Nikole Bakoles. The cyclic pattern of travel between Salt Lake City and Seattle recorded in Saltair Sally's hair matched information provided in Bakoles's missing persons report, which had been filed in 2003. A DNA test confirmed the match. Bakoles, who was 20 at the time of her death, was last seen in March 2000, when she was living in a hotel in a southern suburb of Salt Lake City with her boyfriend and young child. In the preceding years, she had traveled to Seattle occasionally to visit family members, a habit which was clearly recorded in the isotopic composition of her hair. Without that information, it's likely Saltair Sally would still be a cold case.

Given where Bakoles's remains were found and the manner in which they were disposed, police suspect foul play. Stable isotopes haven't led to an arrest yet, Chesson says, "but we did help finally put a name to a person, which was really pretty cool."