The sequence-dependence of biomolecular interactions involving nucleic acids and proteins is essential for numerous processes inside the cell. Insights into the underlying molecular mechanisms have... Show moreThe sequence-dependence of biomolecular interactions involving nucleic acids and proteins is essential for numerous processes inside the cell. Insights into the underlying molecular mechanisms have been obtained using various biochemical and biophysical methods on two different levels — bulk and single-molecule. These have complemented each other as single-molecule studies excel in observing multi-state dynamic interactions, but perform only at low throughput; while bulk studies can probe many different sequences simultaneously, but providing limited kinetic information. To unite the strengths of both levels, we developed high-throughput Single-molecule Parallel Analysis for Rapid eXploration of Sequence space (SPARXS), that allows the study of molecular structure, kinetics and interactions for thousands of different sequences simultaneously at the single-molecule level. We, for the first time, combine single-molecule fluorescence with next-generation Illumina sequencing. As a proof of principle we apply SPARXS to study the sequence-dependent kinetics of the four-way DNA Holliday junction, occurring among others during homologous recombination. Using SPARXS we observe the dynamic behavior of 120,000 Holliday junction molecules covering 3750 distinct core sequences, a result unattainable with previous techniques. Overall, the mechanistic insights obtained using SPARXS will give an entirely new view on the relationship between sequence, structure and function. Show less
Pancreatic cancer is an aggressive cancer associated with a poor prognosis as a large proportion of these patients have locally advanced disease or metastases at the time of diagnosis. Currently... Show morePancreatic cancer is an aggressive cancer associated with a poor prognosis as a large proportion of these patients have locally advanced disease or metastases at the time of diagnosis. Currently onlypatients with localized pancreatic tumors can undergo surgery, which is the cornerstone in thetreatment of pancreatic cancer, whether or not preceded by neoadjuvant (chemo/radio)therapy. To establish the diagnosis, imaging techniques are used, tumor tissue must be obtained, and certain tumor markers can be determined in the blood. During surgery, the tumor must be removed in its entirety to prevent early recurrence. This thesis describes the potential role of tumor markers, which can be determined in the blood, and several experimental optical imaging techniques applied to pancreatic cancer. Show less
Near-infrared (NIR) fluorescence imaging with indocyanine green (ICG) is a promising imaging technique for the assessment of tissue perfusion. This thesis describes the quest for valid and reliable... Show moreNear-infrared (NIR) fluorescence imaging with indocyanine green (ICG) is a promising imaging technique for the assessment of tissue perfusion. This thesis describes the quest for valid and reliable quantitative assessment of tissue perfusion using this technique, predominantly in patients with lower extremity arterial disease. Two systematic reviews were performed, describing the experience with ICG NIR fluorescence imaging within various surgical fields. In three original studies, perfusion patterns were described in various groups, including lower extremity arterial disease, healthy controls and in patients undergoing free flap reconstructive breast surgery. By applying normalization to the quantitative assessment, an increased validity and reliability was seen. To describe potential clinical applications, the use of ICG NIR fluorescence imaging was described for two indications. In patients undergoing unilateral revascularization, quantitative assessment showed an increase of inflow parameters, whilst parameters in the untreated side remained unchanged. In a cohort of patients undergoing amputation surgery, ICG NIR fluorescence imaging was able to predict postoperative skin necrosis in all four cases. Future use of ICG NIR fluorescence imaging should focus on improving validity and reliability of quantitative perfusion assessment. Show less
Intraoperative visualization of tumors could be enhanced by the use of near-infrared (nir) fluorescent contrast agents. The fluorescence imaging field first focused on clinically available contrast... Show moreIntraoperative visualization of tumors could be enhanced by the use of near-infrared (nir) fluorescent contrast agents. The fluorescence imaging field first focused on clinically available contrast agents that do not specifically bind to tumor cells. Nowadays the focus is shifting more towards novel contrast agents that target receptors overexpressed on tumor cells. Part one of this thesis illustrates the road towards the use of tumor-targeted fluorescent agents, by highlighting the disadvantages of the use of non-targeted fluorescent contrast agents and demonstrating how to select biomarkers for tumor targeted fluorescence imaging. In part two of this thesis the clinical translation of tumor-targeted agents is exemplified by describing the introduction of three different agents for several indications (ovarian cancer, breast cancer and pancreatic cancer) in four clinical trials. Part three focusses on the future perspectives of image-guided fluorescence surgery. The road toward clinical application and implementation, associated challenges, and the possible actions to overcome them are summarized in this part. Show less
This thesis is a collection of experimental attempts to enhance photoluminescence of fluorescent molecules and quantum dots with single gold nanorods (GNRs) and relevant applications. Special... Show moreThis thesis is a collection of experimental attempts to enhance photoluminescence of fluorescent molecules and quantum dots with single gold nanorods (GNRs) and relevant applications. Special attention is focused on the interactions between single emitters and GNRs. The idea is to increase the emission of weak emitters by the excellent optical properties of GNRs so that weak light emitters will then be bright enough to be detected and studied individually. We can thus generalize single-molecule fluorescence spectroscopy to weakly emitting species which are currently undetectable by conventional single-molecule spectroscopy. The research is important for extending the scope of single-molecule spectroscopy, which is a powerful technique for understanding the dynamic behaviors at the nanometer scale in biological systems and other materials. Show less
This thesis consists of two parts addressing novel imaging technologies to improve the treatment of cancer patients. In part I, the additional value of real time image guidance during surgery is... Show moreThis thesis consists of two parts addressing novel imaging technologies to improve the treatment of cancer patients. In part I, the additional value of real time image guidance during surgery is discussed and the research described in this part of the thesis showed that imaging performed during surgery can be of great value. Nevertheless, the success rate is highly dependent on the choice of imaging modality and biomarker to be targeted. In part II, a necrosis avid probe was successfully evaluated as novel method for early neoadjuvant treatment response monitoring. Show less
The breakthrough of immunotherapy for cancer has introduced promising new options, but nonetheless only a minority of cancer patients show significant clinical benefit. This situation has inspired... Show moreThe breakthrough of immunotherapy for cancer has introduced promising new options, but nonetheless only a minority of cancer patients show significant clinical benefit. This situation has inspired two avenues of research to find solutions to this problem: mechanistic studies to decipher the working mechanisms of immunotherapies and to investigate why many patients do not respond, and studies developing combination treatments to achieve clinical benefit in situations where immunotherapy alone is not sufficient. This thesis explores both these avenues by investigating applications of visible light in immunotherapy of cancer in pre-clinical models. We developed optical imaging platforms for visualization of immune cells and immunotherapies, which can shed light on the immunological events after administration of immunotherapy. In addition, we investigated novel therapies based on the combination of tumor ablation by Photodynamic Therapy and different types of immunotherapy. Our findings may prove useful in understanding success and failure of immunotherapy, and provide new combination treatment options when the efficacy of monotherapy is insufficient. Show less
Single-molecule fluorescence was invented in the 1990s and has quickly developed into an indispensable technique in the biomedical sciences and condensed-matter research. It has revolutionized... Show moreSingle-molecule fluorescence was invented in the 1990s and has quickly developed into an indispensable technique in the biomedical sciences and condensed-matter research. It has revolutionized the fields of molecular biology, imaging (super-resolution), and catalysis, to name a few. In this thesis, we will apply fluorescence enhancement by single gold nanorods to extend single-molecule studies to chromophores with low fluorescence quantum yields and to high concentrations of probe molecules. Following single-molecule trajectories, we will explore variations in the electron-transfer rates of the metalloprotein azurin both from molecule to molecule and for the same molecule as a function of time. Evidence for conformational substates will be discussed based on dynamic heterogeneity. Show less
Surgery is considered the golden standard to treat the primary tumour and regional spread of many different cancer types. In addition to the pathological evaluation of surgical margins, the... Show moreSurgery is considered the golden standard to treat the primary tumour and regional spread of many different cancer types. In addition to the pathological evaluation of surgical margins, the evaluation of lymph nodes is needed to stage the metastatic dissemination of the disease [1,2]. Both these aspects are critical in accomplishing radical excision and are, as such, providing true prognosis. However, the quest for complete cure should be in balance with the desire for minimally invasive surgery [3–5]. Hereby it can be assumed that surgical side effects may negatively influence the patient’s quality of life. Advanced image guided surgery technologies can be used to improve the surgical resection and to minimize the invasive nature of the procedure [6]. In this thesis both these technologies are discussed. Hereby we focussed on the locoregional assessment of the lymphatic tumour spread via sentinel lymph node procedures [7–11]. In addition technologies that support nerve-sparing surgery have been pursued. To realize these ambitious goals, a combination of imaging modalities has been used, ranging from fluorescence to nuclear imaging and hybrid combinations of the same. Show less
Intraoperative imaging using near-infrared (NIR) fluorescence is a fast developing imaging modality as it provides real-time visual information during surgery (Chapter 1). The ability to detect... Show moreIntraoperative imaging using near-infrared (NIR) fluorescence is a fast developing imaging modality as it provides real-time visual information during surgery (Chapter 1). The ability to detect lymph nodes and tumours that need to be resected can assist the surgeon to improve surgery by reducing time of the procedure, reducing iatrogenic damage, and improve the number of radical resections. This thesis focuses on the introduction of NIR fluorescence imaging into the clinic. Part 1 of this thesis describes the optimization of NIR fluorescence imaging for sentinel lymph node (SLN) biopsy using the clinically available NIR tracer Indocyanine green (ICG) in various cancer types. Moreover, these studies show both the limitations as the clinical benefit of NIR fluorescence for SLN biopsy. Part 2 describes the use of NIR light for tumour detection. Tissue absorption and scattering in the NIR light spectrum was used for neoadjunvant treatment response monitoring in breast cancer patients. Moreover, NIR fluorescence imaging using NIR contrast agents was used for the intraoperative detection of otherwise difficult to localize liver metastases of colorectal cancer. Show less
The work described in this thesis shows how intraoperative lesion identification can be improved via the introduction of (hybrid) tracers and optimised (hybrid) imaging modalities capable... Show more The work described in this thesis shows how intraoperative lesion identification can be improved via the introduction of (hybrid) tracers and optimised (hybrid) imaging modalities capable of detecting this tracers. In part one, the reader is introduced to the concept of fluorescence image-guided surgery and the evolution thereof. Furthermore the hybrid approach for image-guided surgical guidance is presented. Part two describes the clinical evaluation of the hybrid approach using the hybrid tracer indocyanine green-technetium 99m-nanocolloid. To further refine the hybrid approach for surgical guidance, part three of this thesis describes the development and evaluation of different types of (hybrid) imaging modalities. Show less
Super-Resolution Microscopy is an optical fluorescence technique. In this thesis we focus on single molecule super-resolution, where the position of single molecules is determined. Typically these... Show moreSuper-Resolution Microscopy is an optical fluorescence technique. In this thesis we focus on single molecule super-resolution, where the position of single molecules is determined. Typically these molecules can be localized with a 10 to 30nm precision. This technique is applied in four different studies. To determine the spatial distribution of Ras-protein in live cells, Ripley's analysis is used on localization data to quantify size an diffusion parameters of nanodomains. Particle image correlation spectroscopy (PICS) is a second order spatial distribution analysis to determine diffusion properties of single molecule populations. A mathematical framework to correct the data when applied on 3D diffusion is presented in the second chapter. In the fourth chapter the uptake of alpha-synuclein aggregates by the cells is observed using super-resolution microscopy. Their partial degradation was followed and showed the importance of the lysosome-dependent mechanism for protecting cells from exposure to potentially toxic a-synuclein. In the fifth chapter we correlate the number of vinculin proteins in a focal adhesion protein complex, to the local force generated by the cell via this complex. A method was developed to determine the local stoichiometry of molecules by their correlated distances as obtained from SMLM. Show less
Fluorescence bias in in signals from individual SNP arrays can be calibrated using linear models. Given the data, the system of equations is very large, so a specialized symbolic algorithm was... Show moreFluorescence bias in in signals from individual SNP arrays can be calibrated using linear models. Given the data, the system of equations is very large, so a specialized symbolic algorithm was developed. These models are also used to illustrate that genomic waves do not exist, but are merely an artifact of commonly used methods. Furthermore, a new semi-parametric, single array, approach to SNP genotyping is introduced and shown to be both effective and efficient. A refined algorithm for copy number estimation, using a zero-exponent norm is proposed, which performs well, as is illustrated by thorough comparisons with other methods. Indications that the signal calibration can improve (genotyping) results from lower quality samples are also discussed. A software suite that implements the above is described and illustrated. Show less
The investigation of electron-transfer (ET) processes, as well as redox reactions is important to understand a whole series of biochemical processes. Single-molecule techniques are a precious tool... Show moreThe investigation of electron-transfer (ET) processes, as well as redox reactions is important to understand a whole series of biochemical processes. Single-molecule techniques are a precious tool whose diffusion and technical evolution made them available for the study of biologically relevant reactions. In this thesis an approach to the study of electron-transfer and redox reaction by means of single-molecule techniques is presented. Two proteins were investigated: azurin, from Pseudomonas aeruginosa, which contains a type-1 copper center, and the blue nitrite reductase (bNiR), from Alcaligenes xylosoxidans, which contains a type-1 and a type-2 copper center. By means of a resonant energy transfer (FRET) __ based approach it was possible to use a fluorescent label attached to the surface of the proteins to obtain information about their redox state. By using fluorescence correlation spectroscopy in solution it was possible to study ET processes between azurin and the label and between two covalently linked azurin monomers. Nitrite reductase was immobilized in an agarose matrix and single enzyme molecules were investigated by using scanning confocal microscopy: new details about the internal ET between the two copper centers of bNiR were revealed and new light was shed on the catalytic cycle of the enzyme. Show less
This thesis consists of two parts. In part I, we have demonstrated that preoperatively administrated systemic (neoadjuvant) therapy is a feasible treatment strategy in early stage breast cancer to... Show moreThis thesis consists of two parts. In part I, we have demonstrated that preoperatively administrated systemic (neoadjuvant) therapy is a feasible treatment strategy in early stage breast cancer to achieve improved surgical options and to assess tumor response. We also demonstrated that overexpression of the breast cancer stem cell marker aldehyde dehydrogenase-1 in early stage breast cancer patients is inversely associated with age and is of prognostic importance. In part II, we have demonstrated proof-of-principle of intraoperative tumor detection and image-guided tumor resection by using the novel technique of near-infrared fluorescence imaging. We have performed two clinical trials to optimize the use of indocyanine green as a near-infrared fluorescence lymphatic tracer for the sentinel lymph node procedure in breast cancer patients. Show less
A human consists of billions of cells. All these cells need to know in which organ they are located and what their position inside the organ is. One way to obtain this information is via morphogens... Show moreA human consists of billions of cells. All these cells need to know in which organ they are located and what their position inside the organ is. One way to obtain this information is via morphogens, small particles providing positional information. We quantitatively studied the transport of the morphogen Decapentaplegic (Dpp) in the __wing imaginal disc__ (the precursor of the wing) of fruit fly larvae. Certain cells in this disc produce Dpp, while others receive it and determine their position according to the Dpp concentration. To study Dpp transport we first developed a microscope able to follow single molecules in three dimensions in living tissue with high spatial and temporal accuracy. With this microscope we then studied the subcellular processes governing intracellular Dpp transport. We determined how long Dpp resides in different types of endosomes (a cellular compartment involved in transport). We also found that the movement of endosomes is too small to facilitate Dpp transport. Furthermore we found differences in the in- and outflow of Dpp in endosomes. This work is one of the first to quantitatively study intracellular morphogen transport. It provides new insights into growth and development of organisms. Show less
This thesis centers around a novel fluorescence based method that allows to monitor the activity of redox enzymes and of electron (ET) or oxygen transfer proteins. It takes advantage of the fact... Show moreThis thesis centers around a novel fluorescence based method that allows to monitor the activity of redox enzymes and of electron (ET) or oxygen transfer proteins. It takes advantage of the fact that the absorption spectrum of the protein__s active site varies upon changing its redox state. This change can be translated into a change in the fluorescence intensity of a label that is covalently linked to the protein on the basis of F_rster Resonance Energy Transfer (FRET). With our method we could show that different redox proteins and enzymes can be studied down to the single molecule level. This exciting finding opens the door to the study of various redox enzymes and to monitor specific substances such as for example nitrite. Depending on the function of the enzyme under investigation a wide range of substrates can be monitored. Another example is the development of an oxygen sensor by employing proteins that are capable of binding oxygen. The findings presented in this thesis might be significant for applications in oxygen sensing and, more generally, in the fast growing field of biosensing Show less
