Lasers for Life

Octopus, Ultra, and Astra Gemini - CLF

The Central Laser Facility (CLF) hosts an active programme of biomedical research supported by the FP7 Joint Research Activity BIOPTICHAL and its predecessor OPTBIO, in which several of CLF’s laser facilities are used according to their various strengths.

Aligning one of the Ultra laser systems.

Octopus is one of the world’s largest facilities for laser- based optical microscopy, focussed on biological and med - ical research. An extensive range of imaging techniques including multiphoton microscopy, single molecule fluo - rescence imaging in cells, and several ‘super-resolution’ mi - croscopes provide the biomedical researcher with a toolkit to investigate how biomolecular structure and dynamics in cells and tissues are responsible for the functioning of organisms in health and disease.

For example, Marisa Martin-Fernandez from the CLF is leading a project in collaboration with cancer researchers from King’s College London and a clinician from Guy’s and St Thomas’ Hospital, investigating the behaviour of a family of ‘gatekeeper’ molecules that are the targets of a number of new anti-cancer therapies. The unique combination of techniques available in Octopus enables the research team to study how the molecules transmit their signals to the cell, how active and inactive forms of the molecule are organised on the cell surface, and how mutations affect the signalling process. A collaboration with a drug de - velopment company is providing fluorescent derivatives of anti-cancer drugs so that their effect on the signalling molecules can be monitored. Ultimately, the goal of this research is to direct ‘targeted therapeutics’ by obtaining a molecular fingerprint from tumour biopsies, that can be used to ensure the patient receives the best treatment. This work is funded by the UK’s Biotechnology and Biologi - cal Sciences Research Council (BBSRC).

Octopus is also in demand from European users, with Laserlab-Europe recently funding access for groups working on advanced tracking of biological molecules in cells, plant biology, and DNA damage.

The CLF’s Ultra facility is the world’s most sensitive time-resolved vibrational spectrometer, and in a similar way to Octopus offers multiple experimental stations linked to a suite of advanced lasers. The spectroscopic techniques offered are suitable for the study of the dynamics of mol - ecules on timescales from femtoseconds to milliseconds. Ultra was partially funded by BBSRC with the specific aim of applying its unique capabilities to the investigation of biological molecules.

Laserlab-Europe has funded a number of visits to Ultra by Susan Quinn from University College Dublin. Dr Quinn is interested in the properties of DNA, and has used Ultra to investigate the photophysics of biologically relevant conformations of DNA, implicated in the mechanisms underlying photodamage. This work was recently published in Chemical Communications, in which it featured on the front cover. She has also used Ultra to investigate the inter - action of DNA with carbon nanotubes and nanoparticles.

The CLF’s Astra Gemini facility is a high power, high rep - etition rate laser with two beams, each delivering 15 joules to target in a pulse of 30 femtoseconds (i.e. a peak power of 0.5 PW), with a repetition rate of one shot every 20 sec - onds. Although the facility has been targeted at ultra-high intensity physics research, it is now finding applications in biomedical imaging. The extreme acceleration of electrons in plasma to high energies (~GeV) in a short distance (~1cm) has long been a focus of intense laser research. In recent years researchers have been looking more closely at the x-ray emission that accompanies this acceleration as the electrons wiggle in the strong transverse forces in the plasma. Because of its short pulse duration (~few fs), the single shot (peak) brightness of this source is on a par with the average brightness of synchrotron sources. The spatial coherence guaranteed by the small source size of the beam (~mm) allows the acquisition of high resolution phase contrast images. An Astra-Gemini experiment led by the Plasma Physics group at Imperial College London has recently demonstrated the capability to image medical samples with quality comparable to the state of the art with conventional techniques. Dave Clarke (CLF)


Biosensing of individual molecules with optical antennas - ICFO

Understanding how molecules interact with each other inside the cell is key to advance our knowledge in molecular and cell biology. At ICFO, an optical device has been invented with which individual biomolecules can be detected even at the high concentrations found in living cells.

Left: Artistic illustration of the antenna-in-a-box platform fabricated on
gold allowing the detection of individual DNA strands at high sample
concentrations. Right: Focussed ion beam image of an antenna-in-a-box
as used in the experiments(© Nature Nanotechnology 8, 2013, 512-516).

Together with the Fresnel Institute in Marseille we have conceived and fabricated the smallest optical device that can detect and sense individual biomolecules at concentrations that are similar to those found in the cellular context. The device, called ‘antenna-in-a-box’, consists of a tiny dimer antenna made of two gold semi-spheres and separated from each other by a gap as small as 15 nm. Light sent to this antenna is enormously amplified in the gap region, where the actual detection of the biomolecule of interest occurs. Because amplification of the light is confined to the dimensions of the gap, only molecules present in this tiny region are detected.

As an additional trick, we embed the dimer antennas inside boxes which are also of nanometric dimensions. The box screens out the unwanted contribution of millions of other surrounding molecules, reducing the background and improving the detection of individual biomolecules. When tested under different sample concentrations, this novel antenna-in-a-box device allowed for 1100-fold fluorescence brightness enhancement and detection volumes down to 58 zeptoliters (1 zL = 10-21L): the smallest observation volume in the world.

Our antenna-in-a-box could be used for ultrasensitive sensing of minute amounts of molecules, becoming an exquisite early diagnosis device for biosensing of many disease markers. It could also be used as an ultra-bright optical nanosource to lighten up molecular processes in living cells and ultimately watch how individual biomolecules interact with each other, a long awaited dream of biologists. Maria Garcia-Parajo (ICFO)


Non-invasive optical assessment of breast density as a cancer risk factor - CUSBO

Breast density is well recognised as an important and independent risk factor for breast cancer. It is currently assessed through the analysis of X-ray mammographic images and is thus generally not known until the age of fifty. Within the framework of Laserlab-Europe, the CUSBO facility at Politecnico di Milano has developed a unique system for broadband time domain diffuse optical spectroscopy in the 600-1200 nm range, which can be used to measure breast density non-invasively.

Our system allows the evaluation of the average composition of biological tissues (in terms of water, lipid and collagen content) and blood parameters (total volume of hemoglobin and oxygenation level). Information is also obtained on the structure of the tissue at the microscopic level. The optical measurement is completely non-invasive, painless and quick. It also provides an absolute operatorindependent outcome.

A first clinical system, operated at a few discrete wavelengths yet providing also spatial information, has been developed based on the knowledge obtained by the laboratory research. The system has been used in a clinical study involving more than 200 patients, showing that optically derived parameters correlate to a high degree with both qualitative and quantitative estimates of mammographic density (i.e., BIRADS categories, typically used by clinicians, and percentage density, respectively). Recently, a dedicated portable instrument has been designed within the BIOPTICHAL Joint Research Activity and built to perform point measurements over a full spectral range (600-1200 nm).

The instrumentation developed at Politecnico di Milano is unique at international level because it allows estimating the collagen content in tissue. Collagen appears to contribute fundamentally not only to breast density, but also to the origin and progression of breast cancer. Thus its estimate could provide a direct link (more direct than offered by mammographic density) with cancer risk. The impact of this pre-screening tool will be particularly significant since early diagnosis (lesion size 90% survival rate, and great improvement in overall quality of life due to less invasive treatments. Paola Taroni (CUSBO)

Left to right: x-ray mammogram and optical images at 635, 680, 785, 905, 935, 975 and 1060 nm of the left breast of a healthy subject.
Above the images, the tissue constituents that mainly determine the optical behaviour at the different wavelengths are shown.


Tissue optical studies for ultrashort pulse laser surgery - LOA

The highly nonlinear and therefore strongly localised interaction process of ultra-short laser pulses with matter enables many potential clinical applications. Researchers at the Laboratoire d’Optique Appliquée (LOA) have shown that a shift of the surgical laser wavelength would lead to a significant improvement compared to current clinical laser systems.

CAD image of the demonstrator set-up of a laser surgical device
for corneal grafting including a fibre laser unit (left, courtesy of
Institut d’Optique Graduate School), a wavefront correction
module (middle) and the beam delivery optics (right).

The first clinical ultra-short pulse laser system was commercialised with considerable commercial success in the beginning of the last decade. A number of clinical lasers are now available which provide routines for refractive and cataract surgery as well as other surgical interventions. Those systems have become increasingly widespread and produce very satisfactory results when used on transparent tissue.

However, procedures like corneal grafting need to be performed on pathological tissue, which is not perfectly transparent. In healthy cornea, the very regular arrangement of the collagen fibrils within the lamellae constituting the volume of the tissue as well as the absence of light scattering structures with micrometric dimensions are responsible for the transparency of the tissue. The transparency is lost when the regularity of the tissue structure is perturbed by pathology.

Fortunately, the light scattering processes are strongly wavelength-dependent. Our studies show the existence of a transparency window centred at about 1.65 μm even in very pathological cornea. A shift of the surgical laser wavelength from about 1 μm – which is typically used – to that window should improve the beam quality and the penetration depth considerably.

With our project partners we have developed several compact laser sources for the required wavelength range based on fibre laser technology and nonlinear optics. Our experiments on tissue show the expected results: the laser penetration depth was typically tripled and the quality of the surgical incisions was greatly improved. Interface roughness was reduced and cuts were even possible in the otherwise opaque sclera. Karsten Plamann (LOA)


Optical non-invasive lung and intestine gas monitoring in pre-term babies - LLC

Researchers at the Lund Laser Centre (LLC) are engaged in a multi-disciplinary project in which they try to measure gases inside human body cavities. The goal is to be able to continuously monitor the lung function and gas contents in the intestines of pre-term babies in a non-invasive way.

Scenario for free gas monitoring in neonatal baby lungs and intestines.
Singlemode diode lasers are used to observethe narrow molecular lines,
which are typically 10,000 times sharper than the tissue constituent
spectral features. Wavelength modulation techniques are used to
isolate the gas signals.(© J. Biomed. Opt. 18, 2013, 127005)

Assessing lung function is of prime importance for intensive care of pre-term children, since lack of surfactant in very premature children leads to the respiratory distress syndrome (RDS). Another severe problem for these small patients is necrotizing enterocolitis (NEC), affecting the intestines.

Following successful monitoring of gas contents in human paranasal sinuses using diode laser spectroscopy applied to scattering media (the so called GASMAS method), a feasibility study was first performed on pre-term baby thoracic phantoms. These were made up of animal lung tissue covered by gelatine layers with scattering particles and absorbing ink, mimicking the chest wall of a small child. Oxygen as well as water vapour could be detected in such phantoms of realistic sizes using diode laser sources around 760 and 935 nm, respectively.

Subsequently, a pilot study on three full-term babies weighing about 4 kg demonstrated the possibility of realworld gas monitoring. An ongoing study with refined equipment on several full-term healthy babies shows promising results. Measurements on the intended target patients of weight 1-2 kg are now in planning. The hope is to develop cot-side continuous optical monitoring to replace current techniques, like occasional X-ray imaging, and to help make the start in life of these small children as good as possible. Sune Svanberg (LLC)


Third harmonic generation microscopy in living tissue - LaserLaB Amsterdam

A major challenge in health and life science research is studying a single cell in its native three-dimensional environment of live tissue. Researchers from LaserLaB Amsterdam have demonstrated third harmonic generation (THG) to be an excellent tool for visualization of neuron morphology in living brain tissue.

Visualization of cell dynamical processes in life tissue is of vital importance to understand the origin and progress of diseases, from the organ and tissue down to a cellular level. To enable this, tools and methods are necessary that allow observation at the sub-microscopic level, without changing or disturbing these processes. Third harmonic generation (THG) microscopy provides non-invasive, labelfree contrast (that is, it needs no external contrast agents) of living tissue with sub-cellular resolution and intrinsic depth sectioning. The efficiency of THG depends mainly on the third-order susceptibility χ(3) of the medium and the phase-matching conditions.

Our group at LaserLaB Amsterdam has demonstrated THG to be an excellent tool for the label-free visualization of neuron morphology in living brain tissue. As lipids have a high χ(3) and the lipid content of the brain is high, THG is efficient. Neurons, blood vessels, astrocytes (the most abundant cells in the human brain) and axons were imaged in mouse ex-vivo brain slices, achieving near-video rate imaging of volumes of ~250x250x600 μm3 with <0.5 μm3 resolution.

The high-imaging speed makes THG very suitable for the study of cell dynamical processes in for example the context of neurodegenerative diseases (Alzheimer’s disease, white matter diseases) or in tissue regeneration processes. Another application is in the recognition of tumour cells in the brain during a surgical resection procedure. Tissue-conserving surgery is of extreme importance in brain cancer to minimise loss of function. The major challenge of this type of surgery is the detection of tumour margins. For this purpose, a handheld THG device is now being developed at LaserLaB Amsterdam, in collaboration with the VU Medical Center. Marloes Groot (LaserLaB Amsterdam)

Schematic representation of THG microscopy (left panel, Witte et al., PNAS 108, 2011, 5970-5975).
THG images of human brain tissue, recorded at a depth 100 μm below the surface of an ex-vivo slice
(middle and right panels). The neurons are visible as ‘black shadows’as they produce less THG intensity
than the extracellular matrix. The nucleus and nucleolus within the cells are visible. The THG signal
(green) is co-collected with 2-photon fluorescence signals (red) that mainly arise from lipofuscin particles.

Early detection of cancer | ICFO, Barcelona

Detection of HSP molecules at the surface of gold
nanosensors. © ICFO. Picture by Digivision

In a research programme led by our Spanish partner ICFO, a device is being developed which can detect molecules indicative of cancer with unprecedented sensitivity.

For the treatment of cancer, early diagnosis of the illness is crucial. Recent research shows that specific molecules, called Heat Shock Proteins (HSP), are relatively abundant at the surface of cancer cells and in the patient’s blood. Early detection of these cancer markers may therefore allow treatment of cancer patients at an earlier stage of the disease, with lower doses and less secondary effects.

In order to track a low concentration of cancer markers such as HSP70 in the blood, extremely sensitive detection devices are required. At present, such a sensitive device does not exist: diagnosis still relies mainly on macroscopic techniques, with which only tumours composed of several millions of cancer cells can be discerned.

In SPEDOC, an EU-funded research initiative led by the group of Romain Quidant at ICFO, physicists join forces with oncologists to develop a novel ultrasensitive cancer-marking sensing platform for early detection and accurate monitoring during treatment. The device will employ an optical technique called surface plasmon nano-optics to detect the presence of the HSP70 molecule in tiny volumes of blood.

Surface plasmons are electronic oscillations supported by, e.g., gold nanostructures. These plasmon oscillations are extremely sensitive to the presence of molecules near the gold surface, and can thus – combined with several other sophisticated techniques - be used to detect HSP70 proteins in blood samples.

The sensors developed in the SPEDOC programme will be implemented on an advanced microfluidics chip. On such a lab-on-a-chip, several laboratory functions are integrated on a chip of only millimetres to a few square centimetres in size. They deal with extremely small fluid volumes down to less than a picolitre – a millionth of a millionth of a litre, roughly the volume of a human cell.

Laser Science drives novel Cancer Radiotherapy from Labs to Operating Theatres | SLIC, Palaiseau

cancer therapy
Experimental set-up for laser-proton acceleration

A French-Italian team of researchers from the LASERLAB-EUROPE consortium has shown that a tabletop laser can be used to accelerate a beam of electrons suitable to be used in cancer radiotherapy.

Radiotherapy uses beams of particles to destroy tumors. Presently, millions of patients worldwide are treated each year with high-energy electron beams, directly or after their conversion into gamma-rays. The high-energy electrons are produced by specially designed accelerators driven by powerful radiofrequency generators. Usually the tumor is located deeply inside the body and, before reaching it, the electrons (or gamma-rays) release a considerable amount of energy into healthy tissues which are consequently damaged.

In order to reduce this undesired collateral effect, a novel technique known as Intra-Operatory Radiotherapy (IORT) is increasingly employed. IORT involves irradiating the patient with electrons in the operating theatre right after the tumor has been surgically removed. The idea is to destroy tumor cells that the surgery has missed. Damage to healthy tissues is then drastically reduced. Because electrons do not have to penetrate deeply, they can be fewer in number and have a lower energy, which means smaller accelerators than for ordinary radiotherapy.

However, the IORT technics presently uses linear con- ventional accelerators (the so-called LINAC) measuring typically more than two meters and over half a ton in weight. In addition, the machine must be shielded from the operating theatre and any maintenance requires the shutdown of the theatre.

A recent LASERLAB-EUROPE experiment opens new opportunities in this field. In fact, the joint CNR-CEA team has shown that most of the problems presently affecting the IORT technique can be overcome by using laser light rather than radiofrequency to produce electrons for radiotherapy. At the SLIC laboratory in CEA Saclay, France, the researchers fired ultra-short laser pulses onto a suitable target, creating a plasma oscillating at a frequency much higher than radiofrequency. In this way electrons suitable for IORT therapy of tumors are generated in a few millimeters instead of one meter or more. (A. Giulietti et al., Phys. Rev. Lett. 101 105002). This laser- based accelerator pro- duces bunches of 12 MeV electrons carrying a charge of 2nC at a repetition rate of 10 Hz : these perfor- mances are compatible with numbers required for IORT.

Besides saving on size and cost of installations, the laser-acceleration technique offers several additional advantages. Because the laser beam transport is quite easy, the laser can be located in a nearby chamber allowing insuring service and control of the system without harming the sterility of operating room. The laser beam can also be distributed at the same time in different operating theatres in order to provide more flexibility and reduce the costs. The novel equipment does not require the use of the high power radiofrequency generator and the ultra-high vacuum, as the current accelerator technique does. Finally, the radioprotection issue is expected to be considerably relaxed, because of the small size of the laser driven accelerator head, and because the radiological emission is essentially emitted in the forward direction. The only equip- ment that needs to be installed in the operation theatre is the mini-accelerator box of moderate weight and size, which is easy to be handled and to apply to the surgical wound.

A European consortium including CEA, CNR, laser and medical industries is presently considering the possibility of exploiting such a scientific breakthrough. This exciting adventure has been promoted by the LASERLAB-EUROPE Transnational Access Program.