Cancers can grow in numerous places within our bodies and present tremendous threat to our health. But if one could spot cancerous growth early on, the chances to win over it would be higher. Are there ways of achieving this? The Broadband Infrared Diagnostics (BIRD) research team from the Laser Physics department of the LMU Munich revealed that infrared spectroscopic profiling can be used to pick up molecular traces that solid tumors leaves in our blood stream.

Detecting early and less aggressive cancerous lesions is paramount for having valid medical-treatment options. Besides radiographic tools to visualize tumor tissues within our body, and beyond cutting out tissue biopsies from inner organs for inspection under the microscope, modern diagnostic approaches often focus on non-invasive cancer detection: They analyze bodily fluids and try to capture macroscopically “invisible” molecular changes caused by cancer. In fact, tumors disseminate many aberrant metabolic products and signaling molecules into their surroundings. Likewise, tumors also characteristically interact with adjacent normal cells of a tissue, and later on with our immune cells and blood vessels. These interactions substantially affect the type and amount of many molecules that end up circulating in our bloodstream, even at a time when a tumor is still confined to an organ and not yet metastasizing. However, unambiguously identifying molecules that signify cancer – the holy grail for medical diagnostics and pharma – remains a challenge!

Substantial efforts have been made by the attoworld research team at the LMU Munich in paving new technological avenues for the most reliable and sensitive detection of molecules in complex liquid matrices. In this context, the BIRD research team (https://www.attoworld.de/bird) just published a paper in eLife (DOI: 10.7554/eLife.68758) where they flowed a tiny volume of a blood sample through a cuvette, shined infrared light through it, and quantified the complex changes in the light-wave patterns coming from the blood sample as a function of the identity and number of the hundreds of thousands of different molecules dissolved therein. Extracting information by using machine learning algorithms allows determining a signature that is so highly characteristic of an individual’s blood sample that the signature can be referred to as a “molecular fingerprint”. The BIRD team’s earlier work already showed that such infrared molecular fingerprints were highly reproducible over repeated blood draws from an individual (DOI: 10.1038/s41467-021-21668-5).

The size of the problem in tracing disease like cancer by infrared molecular fingerprinting now becomes immediately obvious: One has to move to the population level. Thus scientists had to analyze these fingerprints of almost two thousand individuals to be in the position to extract the difference between the average healthy fingerprint and the average disease fingerprint. How does it work in real life? In collaboration with medical doctors from LMU clinics, the BIRD team set up a matched case-control clinical study and performed comparative infrared molecular fingerprinting on samples from patients with independently diagnosed lung, prostate, breast, or bladder carcinomas. And indeed, infrared fingerprinting of blood was surprisingly robust and correctly detected the cancer state. Excitingly, infrared fingerprints could be utilized, not only for detecting cancer but also for distinguishing between different cancer types, indicating that each had triggered specific molecular alterations.

Could the approach move into the clinics one day? While still far from being a method that patients can benefit from in medical clinics, the published study substantiates the expectation that, in the future, infrared fingerprinting may become useful for aiding cancer diagnosis or even for cancer screening of low-grade cancers that would go unnoticed using current cancer tests. In particular, infrared fingerprinting may be propelled to yet another level soon. Especially when ultrafast light sources and high-precision electric-field-based metrologies from the attoworld’s research will come into play. The path can be seen, and the quest continues.

Original publication:
Marinus Huber, Kosmas V Kepesidis, Liudmila Voronina, Frank Fleischmann, Ernst Fill, Jacqueline Hermann, Ina Koch, Katrin Milger-Kneidinger, Thomas Kolben, Gerald B Schulz, Friedrich Jokisch, Jürgen Behr, Nadia Harbeck, Maximilian Reiser, Christian Stief, Ferenc Krausz, Mihaela Zigman
Infrared molecular fingerprinting of blood-based liquid biopsies for the detection of cancer
eLife research article, 26. Oktober 2021
DOI: 10.7554/eLife.68758

The Lasers4Life study team at the Klinikum Großhadern of the Ludwig-Maximilians-Universität (LMU) would like to take this opportunity to express their sincere thanks for the great willingness of interested participants to support the research project "Molecular Fingerprinting" with a blood sample. Due to an article in the magazine "Klinikum aktuell" of the Klinikum Großhadern and the corresponding article in the intranet, numerous people donated their blood.

With your blood donation you have made an important contribution to our research project!

Stay healthy,

Your Lasers4Life study team

Jacqueline Hermann, Sabine Witzens and Carola Spindler

Meet the members of the Lasers4Life Study Team @ the LMU Medical Center

We may have passed each other in the corridors of the LMU Medical Center in Großhadern at one time or another. And right here we would like to introduce ourselves!

We are members of the interdisciplinary research team Lasers4Life (L4L). The L4L team is led by Dr. Mihaela Zigman and affiliated with the LMU and the Max Planck Institute for Quantum Optics. And we are all members of the attoworld community (attoworld.de). The aim of L4L is to develop a method for the detection of cancers and other diseases at the earliest possible stage, with the aid of a unique technology based on the generation of ultrashort pulses of laser light. Such a light source can in principle be used to analyze the molecular composition of blood samples.

So can pulsed laser light tell whether or not a person has cancer? – Not yet. But this is the ultimate goal of the L4L project, in which international researchers including laser physicists, mathematicians, physicians and molecular biologists are collaborating. The goal that they all share is to develop, test and validate a procedure that can do just that.

In addition to scientists and medical experts, such an ambitious project requires a team that organizes the collection of samples with which the analytical method can be tested and standardized. – That is our task. We maintain contact with both patients and healthy blood donors, collect and document the required samples, and make them available to the researchers for analysis.

Who are we?

Jacqueline Hermann, a molecular biotechnologist, is the leader of the team. Jacqueline is our project manager, and is also responsible for liaison between the research team and the clinicians at the LMU Medical Center. In addition to these managerial tasks, she is involved in coordinating the work of the national and international research groups engaged in the project.

Jacqueline is not alone. Without the support of Sabine Witzens, her job would be impossible. – Sabine is our medical lab technician, and has years of clinical experience.

The third member of the L4L team is Carola Spindler, who provides support in the management of the project and the study data. Carola has a Master’s degree in Business Psychology, but she decided to branch out into a different field. – And her particular point of view demonstrates the advantages of transdisciplinary collaboration every day.

Where do we work?

We are based in the Department of Urology at the LMU Medical Center, which is headed by Prof. Dr. Christian Stief. Sabine and Carola are mainly involved with patients. They outline the nature of the project, and ask patients whether they would be willing to support it by donating a blood sample. They take the samples, document and process them, and enter the data into the project’s database.

Our search for sample donors

The collection of blood samples requires great care and meticulous planning. Our working day begins with the search for suitable subjects for our study. Potential donors must fulfil certain specific criteria – e.g. in relation to age and clinical history. We then explain the rationale and the aim of the clinical study.

Carola answers the inquiries and looks for suitable participants to come to LMU Klinikum.

Persons who agree to take part in the study then donate a blood sample. We draw between 5 and 10 ml of blood, which is immediately centrifuged to separate the liquid from the cellular constituents. Each fraction is then divided into 0.5-ml ‘aliquots’, little tubes that get to be stored in a freezer at −80°C, and subsequently transported to LMU’s Laboratory for Extreme Photonics (LEX) in Garching for laser analysis.

What do we do with the blood samples?

The blood samples are first processed in the laboratory at the LMU Medical Center in Großhadern.

At temperatures of −80°C, samples can be safely stored for years. Furthermore, in the laboratory at LMU, samples can be frozen in liquid nitrogen at −180°C, which ensures that they will remain unchanged for decades.

The researchers need to analyze as many physiological states as possible, so they need access to a large and highly diverse collection of blood samples. Like a person’s fingerprints, the biochemical composition of the blood is unique to each individual. To investigate these fingerprints in detail, the data obtained from laser analyses with ultrashort light pulses will be processed with the aid of methods drawn from artificial intelligence (AI). This approach enables computers to detect subtle patterns in the infrared spectral data, which in turn makes it possible to reliably connect specific features of blood chemistry with underlying disorders.

The use of liquid nitrogen for sample storage has one very important advantage. Science and technology do not stand still - thus long-term storage ensures that researchers and medical professionals can probe the same blood samples with ever more refined analytical methods and diagnostic procedures, thus gaining new insights from these samples for decades to come.

The blood samples are stored at −80°C prior to being transferred to the laser lab in Garching.

Why we are excited by this innovative research project

“It’s a very varied job, with a range of responsibilities, and it presents new challenges every day. What appeals to me most is having the opportunity to work in a forward-looking and very interesting environment,” says Carola. – And Sabine adds “I enjoy working with people, and it’s encouraging to see that a very large proportion of those we speak to are ready to take part in our study. The participants are also interested in the scientific background of the project, and put lots of questions to us.”

Would you like to know more? If so, do get in touch with us in person: We welcome everyone who wishes to help shape the future of medical probing aided by ultrafast metrology!

The Urological Clinic and Polyclinic of the LMU Hospital is looking for a Study Nurse & Physician Assistant (m/f/d) for the next possible date. The place of work is the Campus Großhadern (full-time) You will support the study management in the preparation and conduct of the study „Infrared Spectroscopy of Expressaturin and Blood for the Detection of Prostate Cancer“, which is conducted in cooperation with the Faculty of Physics of the Ludwig-Maximilians-University Munich (www.lasers4life.de).

You will be the interface between patients, investigators and clinical project management and also take over administrative tasks. Your responsibilities will include recruiting study participants, performing delegated physical examinations, collecting blood samples, processing samples in the laboratory, and recording clinical data relevant to the study.

The detailed job posting can be found at:

https://www.lmu-klinikum.de/stellenanzeigen/neue-stellenanzeige/e41f04dae0ba8ed5

Thousands of researchers are seeking ways to capture human health within a simple probe. Why? Every person is unique – in the personality as well as in terms of health. And the task of defining the healthy state at a populational level is just colossal. In the face of this, it is crucial to detect aberrations in health as soon as only possible, raising the alarm that a disease is sneaking into one’s body. Many diseases could be better coped with if we were alarmed earlier, as they only start developing. Especially cancer. This is where less invasive ways of disease detection come into play.

In a very inter-disciplinary team at the laser physics department of the LMU Munich, we believe to have made a step in this direction: We analyzed a fluid that connects all the organs – blood. The idea is familiar to anyone from medical check-ups, and it has recently been used for profiling with modern omics techniques. In our case, however, we exposed tiny amounts of blood to infrared light and captured the vibrations originating from the soluble biomolecules. In collaborative work with medical doctors from the LMU Comprehensive Pneumology Center, we set up a proof-of-principle clinical study to collect blood of individuals that were definitely known to have lung cancer, and in parallel from generally healthy, comparable individuals. We measured their blood samples with infrared spectroscopy. And finally - with quite high certainty - we succeeded to tell apart whether a person had lung cancer or not. From a mere drop of blood.

Eureka? Well, we want to go even further. While infrared fingerprints may distinguish lung cancer, they do not yet inform us about the individual blood components that make up the difference. Nevertheless, it would be good to know their identity to be able to further improve the method. This is what we demonstrated in a new study just published in Angewandte Chemie (DOI: 10.1002/anie.202103272). It is all about a combination of two techniques that are usually applied separately: mass spectrometry and infrared spectroscopy. The aim was to decode the actual chemical changes behind the previously “black box” infrared fingerprints of lung cancer. Now, with mass spectrometry of blood samples performed at the Max Planck Institute of Biochemistry in Munich, we identified a set of 12 proteins that account for the spectral signature of lung cancer, where early diagnostic markers are currently missing. All of these proteins were known since a long time. Now - like old dogs playing new tricks - the combinatorial protein signature that we defined turns a new page in diagnostic efforts. And this signature can be measured in a matter of minutes using infrared light!

So are we now one step closer to capturing disease and defining health? Certainly so. Especially as laser scientists at our department are engineering new ways of delivering ever shorter and more precise pulses of light for spectroscopic investigations. Metaphorically, this is like a magnifying lens that allows us to inspect the molecular zoo in our blood at once – in a snapshot. Finally, the results are of broader relevance for many disciplines, as one may generalize our findings to detection of other diseases that leave their traces in blood.

Yet, while we are working on new advances in capturing human health by shining light through drops of blood, we are bearing in mind the notion of Isaak Newton: “What we know is a drop, what we don’t know is an ocean.”.

Mihaela Zigman

Original article:

Angew Chem Int Ed Engl

Molecular Origin of Blood-based Infrared Spectroscopic Fingerprints

Liudmila Voronina, Cristina Leonardo, Johannes B Mueller-Reif, Philipp E Geyer, Marinus Huber, Michael Trubetskov, Kosmas V Kepesidis, Jürgen Behr, Matthias Mann, Ferenc Krausz, Mihaela Žigman

PMID: 33881784

DOI: 10.1002/anie.202103272

https://onlinelibrary.wiley.com/doi/10.1002/anie.202103272

We would like to wish a warm welcome to a new colleague: Since October 2020, the research group around Dr. Mihaela Žigman has been supported by Viola Zóka, who is our new medical laboratory assistant!

The young Hungarian fellow is a proud alumnus of the University of Pécs, where she earned her Bachelor’s degree in Chemistry. She had developed a strong passion for chemistry early on which was even more intensified while she was acquiring professional experiences at a company laboratory in Nagykanizsa (Hidrofilt Ltd. Hungary). She was performing qualitative and quantitative analysis of water samples using various analytical chemistry methods.

Shortly after Viola has heard about the newly emerging infrared molecular fingerprinting technique, which by far allows for more sophisticated analysis of human blood samples than other methods she was familiar with, she applied for a job at the Center for Molecular Fingerprinting (CMF) in Budapest. CMF has a very close collaboration with the Laser Physics Department of the Ludwig Maximilians University (LMU) in Munich, blood samples collected for clinical studies of CMF will therefore be analyzed at the LMU.

Since it is still a couple of years ahead until CMF is establishing its own analytical laser laboratories, Viola has changed her residency from Hungary to Germany to pursue a new professional career at the Garching Research Center and directly fuel the CMF research goals from there. Together with laser scientist and molecular biologists, Viola will analyze blood samples for their molecular composition using infrared spectroscopy - something that everyone is very much excited about!

The new liquid-nitrogen cooling system at the Centre for Advanced Laser Applications (CALA) fulfills all prerequisites for secure long-term storage of blood donated to the Lasers4Life project. In the Biobank, the samples are maintained under cryogenic conditions, i.e. at less than -150°C in the gaseous phase over liquid nitrogen. The 11-tier system can accommodate approximately 60,000 (0.5-ml) samples of plasma or serum. Storage and retrival of samples is carried out by an integrated robotic system. The latter steps are performed at a temperature of -100°C, so that the whole operation takes place without interruption of the refrigeration chain.

In terms of both storage and handling, the new system has marked advantages over the previous procedure, in which samples were manually stored at -80°C. Most importantly, thanks to the use of ultracold liquid nitrogen, samples remain at the set temperature even in the unlikely event of a power cut lasting for several days. They can also be stored for at least 10 years without any alteration or loss. This means that samples can always be (re-)analysed with the latest generation of lasers available, in order to extract still more information than could previously be obtained.

The BIRD research group aims to bring blood-based infrared molecular fingerprints closer to clinical, daily setting of monitoring human health, we would like to evaluate the impact of pre-analytical parameters. Towards this end we are just preparing a new Lasers4Life study where we investigate the influence of blood sampling parameters to the molecular fingerprinting.

Thus, we are turning to you all as we are in search for volunteers of all ages, to participate in our study and actively collaborate in our research. Any information from your participation will be fully anonymized.

You may come either on 30th June or on 2nd July 2020 between 8 am and 12 am/noon to the Centre for Advanced Laser Applications (CALA), Am Coulombwall 1b, 85748 Garching. The team would expect you to participate twice. Another invitation for a second donation will follow in September this year. The researchers will explain to you the study, you will fill out a short questionnaire and donate 50 ml of your blood. It will altogether take around 20 minutes of your precious time, something we would highly appreciate. Please do not eat anything on that very day prior to visiting us. We will gladly provide all the volunteers with a small snack afterwards.

All volunteers interested in participating are kindly asked to directly contact our study team via following email: jacqueline.hermann@med.uni-muenchen.de where you may ask any further questions you might have.

To ensure that samples of plasma and serum obtained in the course of the Lasers4Life (L4L) project can be stored under optimal conditions, the BIRD Group recently took delivery of an automated refrigeration system designed for use at liquid-nitrogen temperatures. The new Biobank was installed in BIRD’s own laboratory at LEX Photonics, which minimizes the interval between sample recovery and laser analysis. The consignment, which was delivered on six pallets, consisted of the necessary components and peripherals, as well as a workbench specifically designed for the handling of frozen samples in the laboratory. Deployment of the various components of the new system required the help of a crane.

Askion’s Hermetic Storage HS200S system is capable of storing approximately 60,000 samples at temperatures below -150°C. Under these ‘cryogenic’ conditions, samples can be kept for 10 or more years without qualitative deterioration.

In order to maintain the cold chain even during the storage and removal of the samples, the HS200S is equipped with a robotic arm that can perform these tasks at a temperature of approximately -110°C. The dedicated workbench mentioned above facilitates the manipulation of samples at and below these temperatures, and it also provides for the automated freezing of samples in accordance with predetermined temperature profiles.

Scientists at the Laboratory for Attosecond Physics have developed a unique laser technology for the analysis of the molecular composition of biological samples. Could a combination of laser sciences and molecular detection be cracking the limits of molecular sensing?

The combination of molecules found in body fluids such as blood plasma is unique to each individual, and the composition of this ‘brew’ can provide information on an organism’s state of health. The problem lies in learning to decipher the information it contains. Complete molecular characterization has been impossible up to now, because our instruments are not sensitive enough to identify and quantify the entire range of chemical compounds present. But this goal has now moved a step closer to realization. Researchers at Laboratory for Attoseond Physics (LAP), which is run jointly by the Max Planck Institute for Quantum Optics (MPQ) and Munich‘s Ludwig-Maximilian University (LMU), in cooperation with scientists of the King Saud University and the Hungarian Center for Molecular Fingerprinting have developed a laser-based system – the first of its kind in the world – that is capable of detecting minimal variations in the chemical make-up of biological fluids across the whole spectrum of molecular species.

At the biochemical level, organisms can be thought of as complex collections of different species of molecules. In the course of their metabolism, biological cells synthesize chemical compounds, and modify them in multifarious ways. Many of these products are released into the intercellular medium and accumulate in body fluids like the blood. One major aim of biomedical research is to understand what these immensely complex mixtures of molecules can tell us about the state of the organism concerned. All differentiated cell types contribute to this ‘soup’. But precancerous and malignant cells add their own specific molecular markers – and these provide the first indications of the presence of tumour cells in the body. So far, however, very few of these indicator molecules have been identified, and those that are known appear in minuscule amounts in biological samples. This makes them extremely difficult to detect. It is assumed that many of the most informative molecular signatures comprise combinations of compounds that belong to all the various types of molecules found in cells – proteins, sugars, fats and their diverse derivatives. In order to define them, a single analytical method that is versatile and sensitive enough to detect and measure the levels of all of them is needed.

An interdisciplinary team led by Prof. Ferenc Krausz has now built a new laser-based system that is specifically designed for this purpose. The group is based at the Laboratory for Attoseond Physics (LAP), which is run jointly by the Max Planck Institute for Quantum Optics (MPQ) and Munich‘s Ludwig-Maximilian University (LMU), and it includes physicists, biologists and data scientists. This system enables one to obtain chemical fingerprints in the form of spectra of infrared light, which reveal the molecular compositions of samples of all sorts, including samples of biological origin. The technique offers unprecedented sensitivity and can be used for all known classes of biomolecules.

The new laser spectrometer builds on technologies that were originally developed in the LAP for the production of ultrashort laser pulses, which are used to study the ultrafast dynamics of subatomic systems. The instrument, which was built by Dr. Ioachim Pupeza and his colleagues, is designed to emit trains of extremely powerful pulses of laser light that cover a broad segment of the spectrum in the infrared wavelength. Each of these pulses lasts for a few femtoseconds (in scientific notation 1 fs = 10-15s, one millionth of a billionth of a second). These extremely brief flashes of infrared light cause the bonds that link atoms together to vibrate. The effect is analogous to that of striking a tuning fork. After the passage of the pulse, the vibrating molecules emit coherent light at highly characteristic wavelengths or, equivalently, oscillation frequencies. The new technology makes it possible to capture the complete ensemble of wavelengths emitted. Since every distinct compound in the sample vibrates at a specific set of frequencies, it contributes its own well defined ‘subspectrum’ to the emission. No molecular species has anywhere to hide.

“With this laser, we can cover a wide range of infrared wavelengths – from 6 to 12 micrometers – that stimulate vibrations in molecules,” says Marinus Huber, joint first author of the study and a member of biologist Dr. Mihaela Zigman’s group, which was also involved in the experiments carried out in the LAP. “Unlike mass spectroscopy, this method provides access to all the types of molecules found in biological samples,” she explains.

Each of the ultrashort laser pulses used to excite the molecules consists of only a few oscillations of the optical field. Moreover, the spectral brightness of the pulse (i.e. its photon density) is up to twice as high as those generated by conventional synchrotrons, which have hitherto served as radiation sources for comparable approaches to molecular spectroscopy. In addition, the infrared radiation is both spatially and temporally coherent. All of these physical parameters together account for the new laser system’s extremely high sensitivity, enabling molecules present in very low concentrations to be detected and high-precision molecular fingerprints to be produced. Not only that, samples of living tissue up to 0.1 mm thick can, for the first time, be illuminated with infrared light and analyzed with unparalleled sensitivity. In initial experiments, the team at the LAP has applied the technique to leaves and other living cells, as well as blood samples.

Ioachim Pupeza and Marinus Huber are of one accord: It’s fascinating to be able to detect the signals emitted by excited molecules with such extraordinarily high sensitivity, they say. This ability to accurately measure variations in the molecular composition of body fluids opens up new possibilities in biology and medicine, and in the future the technique could find application in the early detection of disorders,” Zigman adds.

The mixture of molecules found in blood and other body fluids can provide information relating to the donor’s state of health. This implies that, if it were feasible to identify and quantify the molecules in the circulation, it would also be possible to detect signs of specific disease states. However, the precise analysis of such complex mixtures is very difficult, and requires the use of highly sensitive techniques such as infrared spectroscopy. A team at the Laboratory of Attosecond Physics, which is run jointly by the Max Planck Institute for Quantum Optics and the Ludwig-Maximilian University (LMU) in Munich, recently developed a laser specifically for this task, and initial experiments on the characterization of the molecular mélanges are now underway.

With a view to developing a diagnostic test for early-stage prostate cancer, the physicians and physicists now wish to analyse what are called ‘expressed prostate fluid’ (EPF) with infrared laser light. EPS is a combination of various prostate secretions such as pre-ejaculate, and is emitted soon after prostate stimulation. The molecular composition of EPF could yield clues as to whether or not the donor has prostate cancer. The team has already collected numerous samples from patients undergoing treatment at the LMU Medical Center in Großhadern. Now they need control samples from healthy individuals, and are therefore seeking male volunteers of all ages as sample donors, in order to assemble a reference database for comparison with the samples obtained from patients.

You can help by agreeing to provide samples of urine and EPF on at least three separate occasions at the LMU Medical Center in Großhadern (Marchioninistraße 15, 81377 München). After the provision of a conventional urine sample, the prostate is very briefly stimulated and a second sample of midstream urine is collected. All samples are then pseudonymized. Donors will receive a voucher worth 10€ after each visit.

Our clinical investigator Dr. Michael Chaloupka will be available on 11.11, 18.11, 25.11, 2.12 and 5.12 for volunteers who have made appointments, and further opportunities to provide samples will be offered regularly. Individual appointments may also be arranged at other times. Appointments may be made and further information obtained at L4L-studien@med.uni-muenchen.de or by calling 089-4400-59250.

Please help us to reliably detect prostate cancer at an early stage.

We look forward to hearing from you.

Juyeon Park comes from South Korea and is currently doing a practical in the Laboratory for Attosecond Physics. She is fascinated by the challenge of understanding the interface where physics becomes biology.

Juyeon Park wants to understand the fundamental links between physical processes and biological phenomena. Having studied Biology and Physics in her native South Korea, she is now doing a practical with the Broadband Infrared Diagnostics (BIRD) group at the Laboratory for Attosecond Physics (LAP). BIRD’s members are developing a laser-based method for the diagnosis of cancers.

Juyeon speaks with quiet intensity, and observes the world around her with bright, attentive eyes. “I would like to understand how physics can help us to obtain a better understanding of the human body,” she says. This is a goal she shares with the scientists involved in the BIRD project at the LAP. BIRD is an interdisciplinary research project which is exploring how laser technology could be employed as the basis for the non-invasive diagnosis of cancers – prior to the appearance of overt symptoms. The basic idea is the following. The laser emits infrared light, causing the diverse molecules in a blood sample to vibrate at characteristic frequencies. Because of the resulting absorption at these specific frequencies, the spectrum of the transmitted light differs from that of the initial beam. Hence, the transmitted spectrum can serve as a fingerprint that provides information about the chemical structures of the molecules found in the sample donor’s bloodstream. Since most of these molecules are derived from cellular metabolism, they essentially provide a picture of the donor’s state of health. Thus, alterations in the products of cellular metabolism owing to pre-cancerous changes in cell metabolism should also be detectable with this approach.

The physicists, biologists and data scientists on the BIRD team are now engaged in turning this idea into a practicable diagnostic tool, and Juyeon is now part of that team. One of her tasks is to measure the concentrations of known substances by means of absorption spectroscopy, using mixtures of 26 different proteins whose individual concentrations are known. By analysing the spectra of these mixtures mathematically, one can identify the spectroscopic signatures specific to their individual components. She can also use this method to establish the lowest concentration of each protein that the spectrometer can detect.

Juyeon is also examining blood samples obtained from patients with lung cancer with the aid of infrared spectroscopy. She can compare these spectra with the donor’s condition at the time when the blood sample was taken. In this way, it should be possible to detect correlations between the features of the spectra, such as the intensities of specific absorption lines, and the state of the tumour. “This is the first time that I have experienced directly how laser physics can provide insights into the complex world of biology,” she says.

Juyeon has always been fascinated by the workings of nature. She grew up in the province of Gyeonggi-do, which is located south of Seoul. As a child, she began to wonder about the variations in the local climate. “I wanted to understand why the temperatures changed from one season to the next, and why the winds varied in direction and speed,” Juyeon recalls. “It all appeared inexplicable to me, and somehow that aroused my curiosity.” But what interested her even more than the weather was the extraordinary complexity of the human body. She therefore chose to major in biology at Ewha Women‘s University in Seoul, and subsequently developed a strong interest in physics. “After all, the body is a physical entity, and physics is the fundamental basis of biology.” Furthermore, advances in physics have led to high-precision methodologies that enable us to gain a better understanding of how the body works – and this is what brought Juyeon to Garching. Her practical, which is financially supported by the Max Planck Postech/Korea Research Initiative in South Korea and the Max Planck Institute for Quantum Optics, continues until autumn 2019.

This is Juyeon’s first visit to Germany, and her life here is rather different from her daily round at home. “Garching is very quiet – at least by comparison with the noise and the bustling crowds in Seoul, in which I am regularly swallowed up on my way to university,” she explains. On the Garching Research Campus and in the meadows along the banks of the Isar, she finds it easy to concentrate on her work. “It’s so peaceful here and the air quality is very good.” She also appreciated the stillness and the broad vistas of the Bavarian Alps, which she visited during the Easter holidays. When she takes a break from research and mulling over data, Juyeon seeks tranquillity in yoga. “I’m also taking a German language course at LMU – but I find the pronunciation of German incredibly difficult,” she admits with a wry smile. Juyeon plans to continue her studies, and looks forward to delving deeper into the connections between physics and the living world.

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.

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.

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.

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’.

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.

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

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.

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.

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.

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.

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.

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.