High Pulse Energy Supercontinuum for Combined Fluorescence and Multispectral Optoacoustic Tomography
This project will focus on the development and generation of the so-called supercontinuum lasers, which essentially are sources emitting laser light that is broadband enough to be considered white. High energy pulsed supercontinuum lasers make excellent sources for multispectral optoacoustic tomography, because, they can be spectrally filtered to provide a source which can then be tuned over the entire generated spectrum allowing probing of the response of tissue over the whole spectral range. Data obtained from probing can be used to derive functional information from cells deep in tissue which in turn can lead to new diagnostic techniques. Due to the intrinsic thermal diffusivity and propagation speed of sound in tissues, tunable light sources having long pulses with widths of few nanoseconds and in the repetition rate range of hertz to kilohertz range are generally desired. However, the pulse energy that can be emitted from current supercontinuum sources is borderline for optoacoustic applications. The main challenge is that fibers with very small cores are required to generate a wide supercontinuum but the pulse energy that a fiber can deliver is limited by the area of its core. The goal of the project is to refine new technologies developed at NKT Photonics to increase the energy of supercontinuum pulses to a level that enables optoacoustic imaging and optical coherence tomography (OCT).
Integrated Tunable Laser System for Swept Source Optical Coherence Tomography
Optical coherence tomography (OCT) is a noninvasive imaging modality that reveals structural information of tissues at millimeter depths. Current OCT systems rely on free-space optics and fiber-optical coupling which makes them bulky and hard to maintain. An integrated OCT system on a single chip would be a compact, affordable and robust alternative. The miniature size of an integrated OCT system would open the door for advancements in parallelization, multimodality and endoscopy.
In this project we use indium phosphide photonic integration technology to realize the laser and interferometer components on a single photonic integrated circuit (PIC) for the first time. Developing such a laser system requires analyzing all physical aspects of laser control, investigating laser dynamics and establishing a scan monitoring system. The design and fabrication of this device will be in done collaboration with the company Smart Photonics, based in Eindhoven. The laser system will be tested for the OCT application in collaboration with the Medical University of Vienna.
High Power Frequency Converted Tapered Diode Lasers
For multimodal bio-photonic imaging, Titanium Sapphire (Ti:Sa) lasers are excellent light sources that enable the combination of optical coherence tomography, multi-photon microscopy and other imaging modalities. Currently, frequency doubled diode-pumped solid-state (DPSS) green lasers are commonly used to pump Ti:Sa lasers because of their high output power and high beam quality. However, DPSS lasers are very expensive and have some disadvantages like large physical dimensions, limited wavelength coverage, high complexity and low efficiency which limits their widespread adoption of applications.
In this project, the design and performance of high power green lasers will be improved by exploring a number of different approaches based on nonlinear frequency conversion of high power tapered laser diodes. Different means of increasing the efficiency and output power of the frequency conversion process, for example by coherent and/or spectral combining of several tapered laser diodes, will be investigated. Frequency stabilization and external cavity power enhancement can also be used to increase nonlinear conversion efficiency. Furthermore, the project may also involve work on optical parametric oscillators pumped by tapered diode lasers. A special focus will be on ensuring low intensity noise operation, which is critical for the bio-photonic image quality. The developed laser sources integrated in Ti:Sa lasers will be used for bio-photonic imaging in collaboration with the FBI consortium partners.
High Speed Stimulated Raman Scattering in the Fingerprint Region
The aim of this project is to design and build a multimodal label-free microscope based on Stimulated Raman Scattering (SRS) and Optical Coherence Tomography (OCT). By probing the molecular vibration SRS can visualize protein, nuclear acids (DNA), cholesterol, lipids, etc. Based on the different contributions within the spectrum of the cell it is possible to discriminate healthy from cancerous tissue within living tissue label-free. Optical Coherence Tomography reveals the structure of the object.
SRS measures the transferred energy from the pump to the Stokes beam. A Ti:Sapphire laser operates at 920 nm as the pump beam and as a Stoke beam functions a photonic crystal fiber (PCF) powered by the Ti:Sapphire laser. With this approach, it is possible to operate SRS microscope in the fingerprint region, which allows simple spectroscopic identification of different molecules.  Adjusting the relative time delay between the chirped Pump pulse and the chirped Stokes pulse allows to probe not only one band but a certain region. This technique is known as spectral focusing. 
For OCT a broadband Ti:Sapphire laser centered at 800 nm will be used. The multimodal microscope will be tested on biopsies and in preclinical studies to reveal the potential in DNA imaging, cancer grading and drug trafficking.
 Freudiger, “Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy.”
 He et al., “Stimulated Raman Scattering Microscopy and Spectroscopy with a Rapid Scanning Optical Delay Line.”
Endoscopic Multimodal Biophotonic Imaging for Esophageal tracking
Presentations of esophageal cancer occur late in the disease, attributing to 400,000 annual deaths worldwide. Today white light endoscopy is a prominent technique in the detection of cancer entailing quadrat random biopsies. This protocol is laborious, plagued with missed cancers, invasive and the cost of analyzing biopsies is spiraling. Recently research has led to the rapid emerging area of early disease detection within the field of biophotonics. We present a panoramic photonic based hybrid endoscope employing multi-spectral optoacoustic tomography (MSOT) and optical coherence tomography (OCT) for esophageal tracking (ESOTRAC). OCT delivers superficial micron-scale morphological imaging and MSOT offers pathophysiological imaging and ability to penetrate deeper than OCT. ESOTRAC performs in-vivo early diagnosis and staging by replacing 2D qualitative and user dependent observations with objective, quantitative, 3D measurements of the entire esophagus wall, delivering morphological and pathophysiological cancer and pre-cancer features not available to white light endoscopy. For further information please visit: https://www.esotrac2020.eu/
Reconstruction methods for hybrid US-OCT-MSOT imaging
Antonia Longo, Doctoral Candidate at iThera Medical in Munich
The aim of this research is to develop and define standard methods to improve image quality in MSOT depending on the specific clinical requirement. The development of efficient tools for image enhancement is still a big challenge in the bioimaging field. Since image visualization plays a critical role in diagnosis and treatment disease, improving image quality in MSOT may be an essential goal for this technique in clinical applications.
Furthermore, extending the results to other imaging modalities and combining multimodal information can further support physicians in the clinical decision. For this purpose, a close collaboration with other FBI members that are currently working on hybrid US-OCT-MSOT endoscopy was started to develop inversion algorithms for MSOT endoscopy and hybrid image fusion approaches.
Development of Optical Resolution Hybrid Fluorescence Optoacoustic Endoscope
Esophageal cancer (EC), the sixth-leading cause of cancer-related deaths, carries a poor prognosis with an overall 5-year survival of less than 20%. Survival rate of EC can be significantly increased through early detection. The current gold standard of care in the detection of EC is white-light endoscopy (WLE) with random biopsies, while endoscopic ultrasound, CT and MRI as well as PET is used in the staging of EC. Since human vision, main tool in WLE (videoscopic approach), is really limited in terms of sensitivity or sub-surface tissue alterations, patients need to undergo multiple random biopsies. Therefore, EC is typically detected at an advanced stage when is treated surgically and prognosis is poor.
We plan to develop a hybrid Fluorescent and optoacoustic miniaturized imaging probe which can non-invasively provide 3-D microscopic visualization of the morphological (such as vessel diameter, connectivity and tortuosity) and patho-physiological features of the esophagus wall. Taking advantage of the strong optical absorption of endogenous agents, optical resolution multi-spectral optoacoustic microscopy (OR-MSOM) technology enables label-free quantitatively resolving inflammation, tissue blood oxygen saturation and neo-vascularity (angiogenesis). Integration of OR-MSOM with Fluorescent imaging will help us to have complementary structural and functional information which can lead us not only to reduce the number of blind-biopsies but also to detect the first stages of EC.
Multimodal, Endoscopic Biophotonic Imaging of Bladder Cancer for Point-of-Care Diagnosis
In the past two decades, bladder cancer diagnoses and bladder cancer related deaths rose by 50 %. and 30 % respectively. In any case, 2.7 million people suffering from bladder cancer history worldwide. Moreover, recurrence rates are high. Current unmet clinical needs can be addressed by optical methods. With these methods, non-invasive acquisition of morphological, molecular and functional information of tissue is possible. Providing such information in-vivo, the clinical diagnosis is made during first examination of suspicious area. Therefore, no harmful biopsy taking is necessary. Consequently, no pathology is needed, which will safe time.
Within the project a multimodal, forward-viewing endoscope, combining optical coherence tomography (OCT), Raman spectroscopy and photoacoustic tomography is developed. With such miniaturized devices the morphological (OCT), molecular (Raman) and pathophysiological (photoacoustic) information of the bladder is accessible. Cancer stage is classified by OCT, while Raman spectroscopy gives information on the cancer grade. With the multimodal endoscopic devices information of early stage cancer is detectable, due to its few microns resolution, which is not available with superficial white light endoscopy, which is the gold standard in bladder cancer diagnosis. For further information please visit: http://mib-h2020.eu/
The goal of this project is the identification of biomarkers for the early-stage diagnosis of cancerous lesions in bladder tissue. In this research project two imaging modalities, namely Two-Photon Excited Fluorescence Microscopy (TPEFM) and Optical Coherence Microscopy (OCM) are combined into one common microscopy imaging platform, capable of acquiring images from both modalities in parallel. Biomarkers for the diagnosis of cancerous lesions are to be established by evaluating distinctive features and defining a framework of measurable parameters. Superior sensitivity and specificity over single modal approaches are expected and the classification quality of cancerous lesions to results in histopathology will be compared. The research is carried out in close collaboration with clinicians.
The biomarkers can further be used in the development and use of in-vivo endoscopes for clinical diagnosis. The system will be designed such that it can be interfaced with an endoscopic TPEFM probe developed within the project mib-h2020 (see more). The probe will be characterized for performing OCM and combined imaging.
Multimodal Surgical Microscope for improved brain cancer delineation
The delineation of brain cancer during neurosurgery is of fundamental importance for complete resection of the malign tissue. Li et al. showed in a study from 2016 published in the Journal of Neurosurgery that a gross total resection can extend the median survival by more than 5 months compared to more conservative approaches. Therefore, in our project we aim to optimize the visualization of the cancer margins by incorporating additional imaging modalities like optical coherence tomography (OCT) and enhanced fluorescence detection in a commercial neurosurgical microscope. While OCT enables depth-resolved structural imaging as well as micro-angiography, enhanced fluorescence detection allows to find and analyse markers like protoporphyrin IX with increased sensitivity. Both technologies are feasible in real time and are thereby most suitable for intrasurgical use.
Multimodal image analysis for pathology detection in endoscopy
Colon cancer is one of the top leading cause of cancer death both in women and men. Early screening of the colon can prevent the development of abnormal tissue or polyps into cancer. Visual differentiation of benign and pre-malignant colonic polyps is an on-going challenge in clinical endoscopy routine. White Light Endoscopy (WLE) is the most common technique to visually assess lesions in the intestinal tract but is arguably unreliable due to hampering in polyp classification. Chromoendoscopy techniques are an alternative to enhance visual identification of gastrointestinal lesions by injecting a stain, improving differentiation between the mucosa, vessels and surface patterns in the intestinal tissue, but this requires additional chemical colorization to be injected in the body. In recent years, LED-based enhanced techniques like Blue Laser Imaging (BLI) and Linked Color Imaging (LCI) are potentially promising alternatives to avoid the use of chemical stains and to obtain similar results. In the current work, novel deep learning techniques are being applied to multi-modal gastrointestinal images to detect, classify or segment polyps to assist clinicians in the diagnosis of colon cancer.
Multimodal Endoscopy Imaging
Tumours of the skull base comprise about 12.5% of all intracranial tumours. This common and important neurosurgical problem is usually treated with endo-nasal skull base surgery, which offers a minimally-invasive approach to remove lesions at the skull base. The surgery is performed by passing an endoscope through nose and nasal cavity, thus to reach the base of the skull. Then, surgeons practice a small opening in the bone and remove the tumour from there. Unfortunately, this approach is still rudimentary in clinical practice. Navigation systems with not enough accuracy for this critical area are used and surgeons must mainly rely on what they see using the endoscope, which offers anyway a limited field of view.
We are currently working on the development of a new augmented-reality endoscope imaging technology, aimed to improve and facilitate minimally-invasive neurosurgical procedures. The system under development will allow to fuse pre-operative and intra-operative medical images of the patient, such as CTs and MRs, directly on the endoscope view. In this way, it will be possible to visualise structures beneath the surface of organs and skull bones, such as tumour lesions and blood vessels, increasing the information content of the endoscopic view and providing to the surgeon a reliable image guided surgical system.
A tunable femtosecond fiber laser source for multi-photon emission microscopy
Second harmonic or sum frequency microscopy can detect membranes and structures with a symmetry break inside cells, such as collagen fibers, tubules in neurones,… It can even be used to detect electric potential propagating along axons, and to see neurones communicate ! Two photon fluorescence occurs with numerous organic molecules. This techniques known as multi-photon microscopy can highlight many specific parts of cells and molecules without the need of markers.
To obtain enough output signal without damaging the tissues, multi-photon microscopes require ultrashort (femtosecond) lasers. Shorter pulses increase the achievable resolution. The source laser also needs to be tunable in wavelength (color) to find the resonance of different molecules or to improve penetration. Using fiber femtosecond laser we aim at producing a very short pulse tunable laser, cheaper than existing source lasers and integrate it with the microscope.
Label-free second-harmonic imaging of processes involving water at the interface of model lipid bilayer membranes
A lipid bilayer membrane is an essential part of all biological cells, the proper functioning of which determines the fate of all living things. It performs a variety of functions critical for life, providing a protective barrier for the cell, regulating the transport of basic compound and ions, signaling and stores energy in the form of lipid droplets. To function properly, the membrane hydration is critical. However, the water environment is usually neglected in studies of model biological membranes. Moreover, realistic model systems such as supported lipid bilayers or giant unilammelar vesicles are usually studied using fluorescence probes that modify not only the membrane properties and interactions but also the water environment around the bilayer. In this project, we are trying to understand the behavior of water in the vicinity of model biological lipid membranes and related interactions on a fundamental level. We use freestanding horizontal planar lipid bilayers to mimic the properties of real cell membranes and second-harmonic generation microscopy as a main experimental tool.
Multimodal Imaging of Interfacial Water Structure
Living cells on a molecular level are complex systems with a dynamic metabolic and electrical activity. These activities are related to ions influx/outflux through the ion channels in the cell’s membrane. The information is encoded in the interplay between ions and surrounding water molecules. However, the interfacial water molecules have different properties than the bulk water molecules. Therefore, to study and understand these processes, we developed a 3D nonlinear second harmonic microscope. This device is label-free, noninvasive and allows us to study the collective behaviour of water molecules at surfaces. We study the interfacial water structure on a model system of glass-water interface. Second harmonic images of glass-water interfaces are converted into surface potential maps and for every pixel along the interface an acid dissociation constant is calculated characterizing the surface chemistry.