In the Lasers 4 Life project, physicist Marinus Huber, a doctoral student, examines the blood samples with infrared laser light in the LEX Photonics Laser Lab at LMU Munich. After the light has passed through the sample, Huber once again analyses the spectrum of the transmitted light, which differs from that of the incident beam. This is because the molecules present in the blood have interacted with, and absorbed some of the incident light at specific wavelengths. Since the precise molecular composition of blood varies from one individual to the next, the pattern of light absorption serves as a “chemical fingerprint” of the sample donor’s metabolic state.

The next question, of course, is whether or not one can distinguish healthy individuals from people with cancer based on the laser spectroscopic analysis of blood samples. In their experiments so far, the L4L team has shown that the new laser technology is 50 times more sensitive than conventional analytical methods. In the next step, Marinus Huber will ask whether this increase in detection sensitivity is sufficient to allow him to reliably detect the presence of cancer cells.

Trustworthy medical diagnoses depend on the sensitivity, discriminatory power and reproducibility of the tests employed. Ensuring that these criteria are met is a crucial component of the Lasers4Life project (L4L). A blood test based on the application of laser spectroscopy needs to be extremely sensitive and, among other things, this means that the samples used must be prepared in a very particular way.

In the L4L team this task is in the hands of Cristina Leonardo, a chemist. She is developing a procedure which allows one to cleanly divide the soluble (non-cellular) portion of the blood sample into a protein fraction and a metabolite fraction. Both fractions are made up of organic substances that are produced in cells, and released into the circulation. Most importantly, their detailed composition is expected to differ in subtle ways, depending on whether or not cancer cells are present in the donor. The L4L team will use laser light to characterize the molecules in the two fractions, thus providing a “molecular fingerprint” for each donor. The ultimate aim is to identify the specific features of the fingerprint that reliably indicate the presence of cancer cells in the body.

We, the L4L team, would like to take this opportunity to express our heartfelt thanks for the generosity and readiness to donate on Thursday and Friday last week at the LMU Frauenklink. Now it's time to evaluate the samples.

An Important visitor made an appearance at the Center for Advanced Laser Applications and the Laboratory for Extreme Photonics last Friday. Dr. László Palkovics, Minister for Innovation and Technology was on the research campus in Garching and visited the two laser research facilities at the Ludwig-Maximilians-Universität.

While there, he received a tour of the large laser systems in the laboratories from Professor Ferenc Krausz and Dr. Andreas Döpp. The minister was particularly impressed by the enormous developments that laser technology has made in recent years and the associated opportunities for their use in medicine. Of particular interest for him, was the BIRD project and its blood analysis using laser light. Collaboration with clinics in Hungary in the framework of the project is currently being planned.

Immediately after taking the blood, the L4L team processes your donation in the biobank. Before the blood can be examined under laser light, several careful processing steps are necessary.

About half of the blood is fluid: the blood plasma. It is in this fluid that the blood cells can be found. In just one milliliter, there are around five billion red blood cells, 200 million platelets and five to ten million white blood cells. Our study assistants eliminate the solid components of the blood, so that only the liquid is retained. This happens via a kind of spin process during so-called centrifuging.

Since the blood is not immediately examined under laser light, it is first transferred to small cannulas and then stored in special refrigerators at minus 80 degrees Celsius. This slows down all biological processes to such an extent that it is possible to work with the valuable samples for up to several years.

Junior researchers at LMU Munich are also actively involved in the Lasers4Life project. One of them is Maša Bozič. As part of her Master’s project, she is using visible light to analyse blood samples, before they are examined with the newly developed near-infrared laser.

Both of these approaches use spectroscopy to characterize blood serum and blood plasma. The term ‘serum’ refers to the liquid phase obtained after whole blood has been allowed to coagulate, which therefore contains no clotting factors. Plasma retains all the normal clotting factors, but activation of the cascade of enzyme reactions that leads to blood clotting is prevented by the addition of an anticoagulant. Coagulation is normally triggered by damage to blood vessels. This in turn causes blood platelets to adhere the damaged vessel wall and ultimately leads to conversion of the protein fibrinogen into the fibrin network, which forms the mature clot and seals the wound.

Both serum and plasma are obtained from whole blood by centrifugation in the presence (plasma) or absence (serum) of an anticoagulant. The centrifugation step serves to remove the red and white blood cells (together with the clot, in the case of serum). Maša then subjects both samples to optical spectroscopy with visible light. As the beam passes through the sample, certain wavelengths of the incident light are absorbed by the substances present in the solution. The changes observed in the transmitted spectrum therefore provide information on the composition of the non-cellular fraction of the blood.

In this way, one can determine the concentration of certain proteins and lipids based on the characteristic pattern of absorption of the incident light. The method therefore allows Maša to establish the extent of day-to-day variation between samples taken from the same individual, or the range of variation between different individuals. This provides a baseline that allows one to assess whether differences in the absorption spectra lie within the normal limits of variation, or are indicative of pathological changes that reflect the presence of disease. The results also provide initial insights into the scale of the differences between the spectra obtained from cancer patients and control subjects.

Maša Bozic’s work therefore yields an essential reference for subsequent spectroscopic analyses with infrared laser light in the L4L project, as both spectroscopic methods rely on the same principle of selective absorption. However, infrared spectroscopy is far more sensitive than conventional spectroscopy with visible light. It therefore provides far more comprehensive and detailed information on the diversity of substances present in the samples, and should allow one to identify those that may be linked to the presence of malignant cells in the blood donor.

Nathalie Nagl has been awarded a doctoral scholarship by the Bischöfliche Studienförderung Cusanuswerk. She has already written her master’s thesis in Dr. Oleg Pronin’s group in the LAP team and can now continue her work as a doctoral student. Nathalie is working on a new, pulsed laser light source that emits near-infrared radiation. It uses a Cr:ZnSe crystal as a laser medium, as well as novel diodes, which are needed to pump the crystal.

The system is designed to detect specific molecules in biological samples. The molecules that researchers are interested in are often very weakly concentrated and thus difficult to find. For this reason, the laser source used must produce as little noise as possible and send out extreme strong light at very specific frequencies. Molecules each react only to a well-defined frequency of light.

Nathalie now wants to push the laser deeper into the infrared range. This could make it possible to detect an even wider range of molecules

At the beginning of October Dr. Frank Fleischmann joined the Broadband Infrared Diagnostics (BIRD) team led by Dr. Mihaela Zigman. Fleischmann began his career in biology as a botanist, but later switched to medical research. Before taking up his present position as a member of the BIRD team, he worked for a commercial provider of genetic tests, including the genotyping of cancer patients, for example.

Fleischmann’s role in the BIRD team is akin to that of an archivist. He is responsible for the cataloging and storage of blood samples. Needless to say, accurate documentation and painstaking handling of test samples are of fundamental importance in medical research. After all, its ultimate goal is to produce a therapeutic agent or procedure that will be used to treat real patients every day. Fleischmann is also in charge of the database specially developed for the Lasers4Life project, and meticulously documents everything done with each and every one of the vital samples in his care.

At the moment, the samples of blood plasma and the sera obtained from them are being stored at a temperature of −80°C. However, even this temperature is not low enough for long-term storage of such samples, as slow ice recrystallization alters their consistency, and after a certain time they have to be discarded. Fleischmann is working on an automated cooling system based on liquid nitrogen as the refrigerant, which will allow the samples to be kept at temperatures as low as −180°C. This is sufficiently cold to inhibit ice recrystallization in the liquid – and under these conditions, the constituents of the various blood fractions will remain unchanged for decades. Thus, as even more advanced methods of laser spectroscopy are developed in the future, the new system will enable the BIRD team to re-examine the samples already collected.

Imagine going to your GP for a screening test. Within minutes the test tells you whether or not you will get cancer in the near future. Would you really want to have access to such information? The fact is, it is already there in our bodies — you just have to decode it. This is the task of Dr. Kosmas Kepesidis, a physicist and data scientist who has recently joined the Broadband Infrared Diagnostics (BIRD) team at the Laboratory for Attosecond Physics. His name is fitting — ‘Kosmas’ derives from the Greek for ‘cosmos’ or ‘world’ and that is exactly what Kosmas studies: the microcosm of molecules in our blood. He does this with the help of algorithms, in other words, numbers.

The scientists on the BIRD team are developing a medical diagnostics tool to detect cancer based on the analysis of infrared light waves. These are emitted when ultrashort laser pulses excite molecules in the blood. The resulting spectra contain fingerprint-like information about the blood’s molecular make-up and thus the state of the patient’s health.

The problem is that, unlike the abstract models used in physics, biological systems are highly complex. Thousands of data points are collected and no one quite knows what to look for. Who even has the time to sift through them? Kosmas is therefore developing software which uses machine learning algorithms to carry out predictive modelling. In other words, he uses advanced computational methods to predict outcomes, such as whether a given molecular fingerprint is an indicator of early-onset cancer.

First, thousands of samples are collected from patients with and without cancer. Thus, we end up with two massive mounds of data. Eventually, the goal is to create further stacks to differentiate between distinct types of cancer. These mounds of data are then pre-processed. For instance, decisions have to be made about which patterns constitute ‘noise’ and can be ignored. Next, Kosmas performs a so-called ‘dimensionality reduction’, i.e. he ‘zooms in’ on those features of the data that are relevant. The third stage is the search for a model: which algorithm is most suited for cancer diagnostics? Kosmas hopes to use artificial ‘neural networks’, algorithms which very roughly approximate biological nervous systems in how they process high-level, as opposed to low-level information. Such algorithms work with abstract patterns rather than zeros and ones like conventional computer programs. For this to work, Kosmas needs lots of data, which are currently being collected in hospitals around the world. Finally, once a model is found, Kosmas will expose it to rigorous testing.

He hopes that his research will culminate in a simple-to-use app that enables physicians to analyse blood samples on the spot. How long it will take to develop this software is as yet uncertain. ‘There are a lot of factors involved.’ And while a screening test which, having analysed a blood sample, outputs either ‘cancerous’ or ‘non-cancerous’ is clearly sufficient for daily life at the doctor’s office, scientists of course want to know exactly which features of the molecular fingerprint are responsible for such diagnoses. But Kosmas relishes the risks and uncertainty involved in doing cutting-edge science. The tools change constantly. ‘I do not know what my work will look like in a few months’ time. I expect it to change a lot’.

Our Broadband infrared molecular fingerprinting project is expanding its network. Joint work with Hungarian clinical centers has started. We would like to welcome Dr. Gábor Csík, a specialist in managing clinical trials, who just joined the team of Mihaela Zigman. Gábor will be in charge of building up and heading the L4L network of medical collaborations in the region. Joint work with the Szeged Medical University, the Koranyi National Institute of TB and Pulmonology Budapest and the Heart and Vascular Center of Semmelweiss University in Budapest has already begun. The plan is to expand the existing clinical network for infrared molecular fingerprinting to tackle disease detection across Hungary.

Michael Trubetskov is a computer specialist. He evaluates information that researchers obtain from laser-assisted analysis of molecules in blood.

Even as a little boy growing up in the Soviet Union, Dr. Michael Trubetskov loved tinkering with metal toys. Using his Конструктор construction kits, he would often assemble components into large structures. It was here that his fascination for science was born. Trubetskov has been working at the Max Planck Institute of Quantum Optics (MPQ) since 2012. His work is still very much about creating useful tools from smaller components — his toy is now software. Trubetskov writes programs aimed at helping scientists diagnose cancer using laser light — in a non-invasive way before symptoms become apparent.

“Earlier, the components determined what I was able to build” says Trubetskov. In his office on the Garchi¬¬ng Forschungszentrum research campus, a wall of screens invite one to dive into oceans of data and every kind of programming code. Trubetskov motions to the monitors, “now I work with a toy that offers me unlimited possibilities. If I need a new component, I don’t go looking for it in my tool box anymore - I just make it myself.” Trubetskov’s job is to develop programs that prepare measured data for cancer diagnosis. “My software’s aim is to clean up unprocessed data, i.e. filter out background noise and maximize the actual information relevant to cancer diagnosis.”

As a member of the Broadband Infrared Diagnostics (BIRD) project team, Trubetskov is one link in a chain made up of physicists, mathematicians and physicians that combines laser light and cancer diagnosis. At the Ludwig-Maximilians-Universität München (LMU) and the MPQ they are working on analyzing the molecular composition of blood using infrared waves to determine a patient’s state of health. The hope is to be able to detect cancer at an early stage, when the chances of successful treatment are the highest. However, the interaction between light and molecules isn’t necessarily directly visible. The most important information is hidden deep inside measurement data and overshadowed by the “background noise” caused by instruments as well as the complex chemistry in blood. It’s Trubetskov’s job to ferret out this valuable information. With the help of his programs, scientists are able to isolate and process the relevant data before it is further analyzed by artificial intelligence.

To use a metaphor, the BIRD research group is searching for a needle in a haystack, or rather, for needles in tens of thousands of haystacks. The haystacks are the blood samples collected from cancer patients and healthy volunteers. The needles are the characteristics of blood that make cancer diagnosis possible — also called the “molecular fingerprint.” When a femtosecond light pulse (a femtosecond is a millionth of a billionth of a second) hits a blood sample, the molecules in the blood begin to vibrate. It is through this “echo” that the molecular fingerprint can be read.

The problem is that the scientists don’t exactly know which characteristics of molecular fingerprints are indicators of cancer — in other words, which needles they should be looking for. Additionally, the hay stacks are teeming with “false” needles — interfering signals generated by the instruments, which are difficult to distinguish from the characteristics they are looking for. In fact, it’s impossible to cleanly separate the original pulse from the echo, since the echo is produced and influenced by the pulse. Moreover, the short pulse laser itself is so new, that its intensity is not always constant. Its fluctuations are random and must be taken into account.

To make a comparison of the blood samples possible, Trubetskov must remove the “false” needles and suppress the interfering signals in order to isolate the desired needles. Only then is it possible to analyze the relevant characteristics. The comparison of these characteristics is subsequently carried out by so-called “neural networks”, which search the data sets for patterns.

The complicated preparation of the measured data requires a wide-ranging knowledge. Trubetskov’s training as a physicist, mathematician and computer scientist gives him the combination of theoretical and practical experience he needs. “Often, what counts is intuition,” says Trubetskov. “Sometimes you can just feel that you’re on the right track. And it’s often not possible to solve problems by just sitting at your desk.” When Trubetskov isn’t making any headway with a tricky problem, he takes a break and goes swimming and picks it up again afterwards. “Sometimes you just have to take a break and do something else — and suddenly the solution will come to you.”

Some of the biggest challenges are the constantly changing requirements. “It’s often been the case that I’ve just finished a writing program and then my colleague asks me to completely change the fundamental aspects of it,” says Trubetskov. In order to deal with these requirements, Trubetskov relies on a strategy known as “agile software development.” Instead of following fixed construction plans and designing the development of programs down to the last microscopic details, Trubetskov leaves room for change. “It’s not a linear process.” But the work is worth it. “The best feeling is when something works.” Trubetskov points to the computer tower whirring under his desk. “This isn’t much more than a clutter of silicone and cables. If we can teach this machine how to give us insights into reality and to possibly diagnose cancer, it would make me incredibly proud.” And so the boy who once tinkered with his metal toys has become a researcher who is helping to shape the science of tomorrow.

A newly installed pipetting robot allows Lasers4Life researchers to speed up the processing of blood samples prior to their characterization by means of infrared laser light.

The researchers and technicians involved in the Lasers4Life Project (L4L) recently welcomed a new member to the team. Since March, a pipetting robot has taken on the task of processing blood samples for subsequent analysis with an infrared laser. The outcome of such an analysis is a ‘molecular fingerprint’ of the metabolic products present in the sample, which are expected to differ between healthy donors and patients who are ill. Based on these differences, L4L researchers hope to develop a new analytical test for the early diagnosis of cancers.

When Dr. Frank Fleischmann enters the new Biolab in the Laboratory for Extreme Photonics, he hopes to see ‘the green light’ – “the green light that signals that the new pipetting robot is working perfectly,” he explains. Fleischmann, an expert in biobank management, supervises the processing of blood samples in the Biolab, before they are subjected to molecular analysis with a dedicated infrared laser. The samples concerned were donated by cancer patients, and by healthy individuals who serve as the control group. Laser spectrometry of these samples provides a molecular fingerprint that reveals the chemical composition of the donor’s blood plasma, which differs in characteristic ways depending on the state of the donor’s health. With the help of these data, L4L researchers hope to develop a method which allows them to identify molecules that are correlated with early stages of disease – in particular, cancers – and can therefore serve as diagnostic biomarkers.

“We now have so many samples that automation of processing makes sense,” says Fleischmann, as the instrument quietly gets on with the job. “The apparatus has been modified in accordance with the Biolab’s specifications,” he adds. For example, the instrument is equipped with a decapper, which removes and replaces the caps of the sample tubes automatically. In addition, all sample tubes are individually barcoded, which ensures that each can be traced throughout the procedure. The internal robotic arm that carries out all manipulations has a top speed of 2 m per second and deploys eight pipette tips at once. The machine has enabled the L4L team to process 2500 blood samples since the unit as delivered in April.

The robot divides the samples into smaller portions, which subsequently undergo different processing steps. For example, blood serum is required for the laser-based analysis. Serum is the supernatant liquid left behind after the insoluble fraction of clotted blood has been removed by centrifugation. The researchers also use blood plasma – the soluble fraction obtained by centrifugation of whole blood which has been collected in the presence of an anticoagulant that prevents clotting. The Biolab is located immediately adjacent to the Laser Lab, so that the blood samples can be processed by the robot and promptly subjected to laser analysis.

“Eventually the robot will be used to process blood samples for basic research on cardiovascular disease,” Fleischmann says. In that case, only donor blood plasma will be processed, since patients who are at risk for heart attacks are normally treated with coagulation inhibitors, and serum therefore cannot be obtained from them. Processing is also more complex, as the instrument will not only dispense samples into smaller aliquots, but also fractionate these into defined sets of components. For detailed laser-based investigations, researchers generally analyze three different fractions of a given sample of serum or plasma. Isolation of these fractions requires a series of steps in which specific sets of proteins are successively removed from the starting sample, and a protein-free fraction is also prepared.

The LEX Lab in which the pipetting robot is installed operates in conformity with biosafety level BIO II, because the samples processed here have not been otherwise characterized, and could potentially transmit infectious diseases. The robot provides a further safeguard against such an eventuality, thus further minimizing the risk to which researchers are exposed In the near future the instrument will have a great deal more to do, for the planned L4L study on laser-based early diagnosis of cancer will require the analysis of samples from as many as 37,000 donors. All samples are anonymised before being passed on to the research team, so that only the clinicians responsible for treating the patients are in a position to link specific samples with individual donors. Control samples are still required for the study, and healthy persons are asked to donate blood for this purpose. For further information, see: www.lasers4life.de.