Researchers from various backgrounds have joined in the Vital Imaging Unit [VIU] CARIM/BME because they share a common interest in microscopy techniques applied to visualize processes that take place in cells and tissues, on a molecular level. Over the years, the VIU has acquired an impressive number of microscopy set-ups: several intravital video microscopes [equipped for bright field and fluorescence], a Confocal Laser Scanning Microscope [CLSM], a digital microscope with off-line image restoration, a Fluorescence Imaging and Micro photometric System [FIMS, acquired in 1994 with support from NWO], and recently a Two-Photon excitation Lifetime Scanning Microscopy [TPLSM, 415 k support from NWO with contributions of the research institutes CARIM, NUTRIM, GROW, and EURON, and the Department BME of the Technische Universiteit Eindhoven]. The TPLSM consists of an upright and an inverted microscope allowing for experiments under optimal conditions in vivo as well as in vitro and is equipped with a Fluorescence Lifetime Imaging [FLI] module. The availability of all these vital imaging facilities within one building is unique.
The coordination, stimulation and development of both research with and education in techniques for the imaging of vital tissues.
CARIM Cardiovascular Research Institute Maastricht
BME Department of Biomedical Engineering
GROW
NUTRIM
EURON
Prof. Dr. Frans.C.S. Ramaekers (Dept. of Molecular Cell Biology, UM)
Prof. Dr. Dick.W. Slaaf (Dept. of Biophysics, UM; Vice-dean BME, TUE)
Dr. Marc.A.M.J. van Zandvoort (Dept. of Biophysics, UM)
Dr. Johan.W. Heemskerk (Dept. of Human Biology and Biochemistry, UM)
Prof. Dr. Luc. Snoeckx (Dept. of Physiology, UM; Dept. of Materials Technology, TUE)
Dr. Mirjam.G.A. oude Egbrink (Dept. of Physiology, UM)
The forum meets once in two months to coordinate the activities of the Vital Imaging Unit CARIM/BME. If you have questions concerning vital imaging, contact one the above mentioned persons. They will either answer your question themselves or direct you to the appropriate persons.
Name: Prof. Dr. Frans C.S. Ramaekers, Dept. Molecular Cell Biology,
F.C.S.Ramaekers@MCB.unimaas.nl, Tel: +31-43-3881349
Position: Professor of Molecular Cell Biology and head of the Department of Molecular Cell Biology.
Main Field of Research: From June 1989, Prof. Ramaekers is appointed professor of Molecular Cell Biology at the Maastricht University. The major research topics concern oncology and cardiovascular cell biology. At the moment he is president of the Dutch Society for Cell Biology, board member of the Dutch Society for Oncology, Dutch representative of the European Tissue Culture Society, member of the Society for Experimental Biology, representative of the SMBWO for the education of Experimental Pathobiologists and member of the editorial board of several international journals. He is the scientific director of MuBioResearch B.V.
Major Techniques: Confocal Microscopy, Cell cultures, Cytometry
Name: Prof. Dr. Dick W. Slaaf, Dept. of Biophysics, D.W.Slaaf@BF.unimaas.nl, Tel. +31-43-3881657.
Dept. of Biomedical Engineering (Eindhoven University of Technology), Tel. +31-40-2475171
Position: Professor of Physics of the Microcirculation; Vice-dean Biomedical Engineering; Research at CARIM, Main theme Vascular Biology (III); Sub theme Mechanics of Vascular Occlusion; Program Microvascular aspects of vascular occlusion.
Main field of research: The main research topics of Dick Slaaf [Physicist by training] are studies on the microcirculation with emphasis on regulation of perfusion and interactions between blood cells and the vessel wall. He has specialized in methodological developments such as microscopy techniques and measurement procedures. He represents the vital imaging unit in the Master program Biomedical Imaging and Bioinformatics of the Biomedical Engineering program of the TUE.
Major Techniques: Intravital (fluorescence) microscopy. Two-photon excitation microscopy (in vivo). Lifetime Imaging.
Name: Dr. Marc A.M.J. van Zandvoort, Dept. of Biophysics, M.A.M.J.vanZandvoort@BF.unimaas.nl, Tel: +31-43-3881661
Position: Research at CARIM, Main theme Vascular Biology (III); Sub theme Mechanisms of Vascular occlusion; Program Microvascular aspects of vascular occlusion.
Main field of operation: Within the above mentioned research program, Marc is mainly concerned with the visualization of interactions between endothelial cells of the vessel wall and blood cells, both in small and larger vessels. Such interactions involve Calcium or Nitric Oxide responses in blood cells and vascular cells. For the visualization of these responses he applies new microscopy techniques, such as two photon excitation and fluorescence lifetime contrast. The technical developments needed for vital imaging and probe characterization are main issues. Besides the research within this field, Marc cooperates with several groups (UM and TUE) in other programs in order to reveal new
Possibilities of the two photon lifetime technique. Examples are the imaging of atherosclerotic plaques in intact large vessels and the imaging of cellular apoptosis using lifetime contrast. There is also an intense cooperation with the Microscopy Division (Dr. Hans Gerritsen) of the Debije Institute at the University of Utrecht for the development of new techniques.
Major Techniques: Two photon excitation microscopy. (Confocal) fluorescence microscopy. Lifetime Imaging. Fluorescence spectroscopy.
Name: Dr. Wim Engels, Dept. of Biophysics, W.Engels@BF.unimaas.nl, Tel. +31-43-3881662
Position: technical and optical specialist at the department of Biophysics for CARIM
Main field of operation: Dr. Wim Engels is the technical expert who supports the TPLSM system and experiments on it. He furthermore supports the work on various Fluorescence microscopes and spectrophotometers. With his background in research in internal medicine, he has a broad experience in various biochemical and biomedical techniques. He furthermore has a thorough background in computers and image processing techniques.
Major Techniques: Two photon excitation microscopy. Fluorescence microscopy. Lifetime Imaging. Fluorescence spectroscopy. FIMS. Various biochemical techniques. Image processing.
Name: Dr. Johan Heemskerk, Dept. of Biochemistry, jwm.heemskerk@bioch.unimaas.nl, Tel. +31-43-3881671
Position: Research at CARIM, Main theme Thrombosis and Haemostasis (I). Areas: Signal Transduction, Cell Biology.
Main field of operation: Dr. Heemskerk is assistant professor of Human Biology and Biochemistry. Dr. Heemskerk is now working as a senior staff member at the Departments of Human Biology and Biochemistry, where he studies signal transduction and cell-cell communication in blood cells in relation to haemostasis, thrombosis and nutrition. He is the author or co-author of numerous articles and books in the field of biomembranes, haemostasis, and signal transduction. Dr. Heemskerk is a board member of the Vital Imaging Section of the Netherlands Society for Biophysics and Biomedical Technology, and of the Congress International Advisory Board of the International Society on Thrombosis and Haemostasis. He chairs the Unit of Fluorescence Imaging and Micro photometry at the Maastricht University. He is also an (elected) member of the Royal Netherlands Society for Chemistry and Molecular Biology (KNCV), the Netherlands Society for Cell Biology (NCVB), and the Biochemical Society (UK).
Major Techniques: (Confocal) fluorescence microscopy. FIMS. Fluorescence spectroscopy.
Name: Prof. Dr. Luc Snoeckx, Dept. of Physiology (UM), L.Snoeckx@fys.unimaas.nl, Tel. +31-43-3881203
Department of Materials Technology (TUE), L.H.E.H.Snoeckx@tue.nl. Tel. +31-40-2473787
Position: Research at the CARIM, main theme Cardiac Failure (II), sub-theme Cardiac mechanical dysfunction; Program Cell mechanics. Research at the BME: Main theme Soft tissue engineering and biomechanics; Program: Cell Mechanics
Main field of operation: At the two universities, Dr. Snoeckx is involved in the exploration of the function of individual living cells and their adaptation to events in their environment, such as cardiac failure, atherosclerosis and pressure sores. For this purpose, such techniques as atomic force microscopy and confocal microscopy are combined with vital imaging of cells via fluorescent labeling of intracellular structures. Changes in the intracellular environment are followed in time. These techniques allow the 3D-reconstruction of moving intracellular structures and the evaluation of changes in local stiffness, measured at the surface of cells. Besides the research in this field, Dr. Snoeckx is highly interested in the involvement of students in scientific research during their education. To this end, he coordinated the elective Research Trajectory in Medicine course for medical students at the Maastricht University. Recently, students from other directions (for instance Technical Physics, Movement Sciences) can collaborate with medical students in joint research efforts.
Major Techniques: Atomic Force Microscopy; Confocal Microscopy; culturing of individual cells; gene transfer into living cells; fluorescent tagging of molecules.
Name: Dr. Mirjam G.A. oude Egbrink, Dept. of Physiology (UM), M.oudeEgbrink@fys.unimaas.nl, Tel. +31-43-3881082/1200
Position: Research at CARIM, Main theme Vascular Biology (III), Sub theme Mechanisms of Vascular Occlusion, Program Microvascular Aspects of Vascular Occlusion.
Main field of operation: Dr. Mirjam oude Egbrink is associate professor in Physiology and principal investigator of the CARIM program Microsvascular Aspects of Vascular Occlusion. Her research focuses on the role of activated blood platelets in vascular disease (e.g., thromboembolism, atherosclerosis and ischemia/reperfusion) in vivo. In these in vivo studies, conventional methods like intravital (fluorescence) microscopy are combined with new techniques like real time fluorescence imaging of intracellular ion-changes and two-photon excitation microscopy.
Major Techniques: Intravital (fluorescence) microscopy; Real-time fluorescence imaging of intracellular ion-changes in vivo (FIMS); two-photon excitation microscopy in vivo (together with Marc van Zandvoort and Dick Slaaf).
Principle: Fluorescence microscopy uses the property of molecules that upon excitation with light, they fluoresce at a longer wavelength. These properties may be inherent to the structures under observation [auto fluorescence], or pertinent to the labels [probes, fluorochromes] added to the structures to be observed. Fluorescence microscopy is classically performed with single photon excitation. This type of fluorescence microscopy often has the drawback that probes require excitation with UV-light that may damage vital structures, and results in a limited penetration depth due to scattering of the light and out-of-focus bleaching. When research has to be performed in thicker tissues, such as mouse aorta, penetration depth has to be improved. This can be accomplished by using two-photon excitation with about double the wavelength, thereby reducing scattering. Since in this method fluorescence only occurs when 2 photons hit the same molecule almost simultaneously [i.e., within about 10-18 seconds], fluorescence only occurs in a very thin section about the focal plane. Therefore, images are actually optical sections of the preparation with characteristic resolutions similar to those of confocal microscopy. Since fluorescence only occurs in this thin optical section, bleaching does not occur outside this section. Since in TPLSM pulsed laser illumination is used, this set-up can also be used for fluorescence lifetime imaging [FLI]. The characteristic lifetime of many fluorochromes alters with changes in the immediate environment [e.g., levels of Ca2+, H1, proteins, etc.] independently of probe concentration. Quantitative FLI can thus provide new information on the intracellular state and add an extra [molecular] dimension to the three-dimensional imaging studies in vivo.
Field of Research:
A. Intercellular communication of blood cells and vessel wall.
TPLSM will be applied for in vivo and ex vivo monitoring of interaction processes that take place between blood cells [platelets, leukocytes], blood plasma [coagulation factors] and cells of the vessel wall [endothelium, smooth muscle cells]. In vivo, using mesenteric venules and arterioles from rabbits, rats and (knock-out) mice, TPLSM will allow us to study the processes of inflammation, haemostasis and thrombosis at a resolution level that has not been reached before. In addition, larger vessels with thick vessel walls, e.g., undergoing atherosclerosis, can now be visualized using TPLSM, while FLI provides new possibilities of quantification and contrast. In collaboration with the image processing group of Prof. B. ter Haar-Romeny of BME of the TU/e, software will be developed to compensate for cell and tissue movement upon recording of the images.
B. Vital imaging of (de) differentiation and apoptosis in the cardiovascular system.
Because of the low cytotoxicity and high penetration depth of TPLSM, this technique will be used to evaluate the process of dedifferentiation in the hibernating myocardium. In addition, the reversibility of this process, redifferentiation, will be studied after mechanical or hormonal stimulation. Another important application is monitoring of atherosclerotic plaques. Effects of variable blood flow [oxygen] on dedifferentiation and apoptosis of vessel wall cells in vivo is the next topic. TPLSM allows for observation of cells at a deeper level in the rather thick tissue samples. Many [home-made] vital fluorescent markers are available to mark stress, early apoptosis and differentiation stage. The feasibility studies foresee a particular advantage of using FLI in this area of application.
C. Cell and tissue mechanics.
In the studies into the effects of deformation forces on cell and tissue function, TPLSM/FLI will be used to increase the three-dimensional [dynamic] resolution of current recordings using conventional confocal LSM, FIMS and DIC-microscopy. In [skeletal] muscle samples, the monitored effects of force are the integrity of myofibrils and the organization of intracellular structural components [sarcomers and sarcoplasmic reticulum]. Adenoviral transfection technology will be used to express green fluorescent protein [GFP]-tagged structural proteins in the biopsies [experiments performed both in TU/e and UM]. In heart muscle preparations, usually isolated from model animals where ischemia or abnormal loading has been applied, TPLSM/FLI, will be used to monitor the effects of these interventions on functional and structural changes. TPLSM will add the required penetration depth, while FLI may yield increased contrast and discrimination for tissue auto fluorescence.Location: CARIM, Department of Biophysics, University of Maastricht, UNS50, room 3.347. Auxiliary chemical / biological laboratory and computer / image processing facilities available in room 3.348. Dr. Wim Engels is the technical expert who supports the system and experiments.
Contact persons: If you are interested in carrying out projects on the TPLSM, please contact Dr. Marc A.M.J. van Zandvoort and Dr. Wim Engels for information. There is (limited) space for new projects.
Principle: In vascular diseases, such as atherosclerosis and (arterial) thrombosis, interactions between blood cells and the vessel wall play an important role. Investigations into the involvement of the endothelium and of different blood cells (and their characteristics) in such diseases are performed all over the word. Most of these studies are performed in vitro, using isolated and/or cultured cells. Because it is known by now that the culturing of cells changes their phenotype, and that simple isolation of cells can influence their reaction pattern, it is important to study blood cell-vessel wall interaction in vivo as well, with the cells in their natural environment. In addition, an in vivo approach is important because of the proven heterogeneity in endothelial functioning along the vascular tree. Such apparent heterogeneity in endothelial phenotypes, that appears to be due to effects of specific micro environmental stimuli, underlies most probably the fact that many human vascular diseases are restricted to specific vessel types: this should be taken into account in research in this field. For in vivo studies of blood cell-vessel wall interactions in small vessels intravital microscopy is applied. An intravital microscope is suited to visualize tissues of living organisms (from chicken embryos and mice to rabbits and men) by either bright field or fluorescence microscopy. It provides a magnified image via an objective lens I a conventional microscope configuration. The illumination of the tissue can be accomplished by either trans- or epi-illumination. Low light level cameras are used to record the images for off-line analysis. In the vessels under study parameters like blood flow velocity and diameter can be measured, using optic-electronic devices. Image analysis software can be used during analysis of the images; in some cases, special image analysis programs have to be developed to suit specific research questions.
Fields of research:
A. The activation pathway of platelets involved in thromboembolism.
In the thromboembolic reaction that occurs at a site of a vascular lesion, interactions between blood platelets and the damaged vessel wall play an important role. These interactions can be divided into two parts. Immediately after the induction of vessel wall injury, platelets come into contact with exposed sub endothelial layers, become heavily activated and form a thrombus. This stationary thrombus that consists of tightly packed platelets that have lost their discoid shape and most of their secretory vesicles will plug the hole produced by puncture and cover the sub endothelial layers. It will not change its shape during the following hours, possibly due to the presence of fibrin. Subsequently, circulating platelets will adhere to the stationary thrombus, without loosing their shape and without a pronounced release reaction; they will form a loosely packed platelet mass. From time to time these newly formed parts will embolize. These emboli may occlude downstream micro vessels, but disaggregate within minutes (unpublished observations), resulting in restoration of normal tissue perfusion. This process of embolization of loose platelet aggregates resembles the clinical situation in which a shower of emboli is produced from carotid atherosclerotic plaques without immediate detrimental clinical consequences, unless the embolization persists for hours. Knowledge of the level of activation of the platelets involved in this embolization process, and of the mediators and receptors involved, will help to expose the underlying pathway that leads to this reaction pattern and may contribute to the development of new therapeutic, antithrombotic agents against long-lasting embolization. Because it is well-known that the cytosolic free calcium concentration ([Ca2+]I) is a crucial second messenger in stimulus-response coupling in platelets, we have chosen to focus on changes in [Ca2+]I in platelets involved in a thromboembolic reaction, as a measure of their level of activation. To enable in vivo measurements of [Ca2+]-changes in individual platelets intravital microscopy is applied in combination with the FIMS technique (see below). Recent experiments have validated this technique and proven that it can be used to investigate the role of different agonists and antagonists, and their receptors, during thrombus formation and embolization.
B. Embryonic development of the vascular tree.
Intravital microscopy is also used to investigate the development of the vascular tree during embryogenesis. Important topics in this respect are the central role of local hemodynamic conditions, control of growth and differentiation of individual vessels, and pattern formation in embryonic vascular networks. The model that is used for investigation of these topics is the chicken embryo.
Location: Laboratory for Microcirculation, Department of Physiology, Maastricht University, Universiteitssingel 50, Maastricht; room 3.159; tel. 043-3881210.
Contact persons: Dr. Mirjam oude Egbrink and Prof. Dick Slaaf.
Principle: Cells and tissues are continuously exposed to environmental changes in the form of nutrients, hormones, nerve stimulation, mechanical deformation and stress factors. The demand to respond to these changes in a coordinated way requires intimate communication of the cells both with each other and the environment. We have acquired a fluorescence imaging and micro photometric system (FIMS) to increase the limited knowledge of the language that cells of the cardiovascular system are using to talk to each other and their immediate surroundings. This acquisition was possible thanks to an investment grant from the Netherlands Foundation for Scientific Research and support form the research institutes CARIM and NUTRIM in Maastricht and CARMA (VU) in Amsterdam. After several adaptations, the equipment has been designed to monitor multiple (fluorescence) properties of cells embedded in a complex milieu at simultaneously a high temporal and high spatial resolution. Basically, the system exists of an inverted fluorescence microscope and incubation chambers or living cells or tissues, to which various instrumentation is connected for automated sample illumination and recording of vital images.
Field of Research:
Vital fluorescence imaging in the study of cell-environment interaction: the coagulation-promoting effect of blood plateletsA. Vital imaging of multiple properties of individual cells.
During the past years, the FIMS has successfully been employed to elucidate interactions between smooth muscle cells, endothelial cells, platelets and the coagulation system. Because of the possibility to record quasi-simultaneously more than one property on the level of individual cells, firm relations of functional and structural characteristics can be made. For instance, the contraction of intact blood vessels has been measured as a function of the Ca2+ fluxes in single vascular smooth muscle cells, following exogenous or neurotic stimulation of the vessel. In other studies with cultured smooth muscle or endothelial cells, considerably heterogeneity between individual cells is recorded in both the morphological and signaling responses, but also clusters or patches of cells are identified showing almost identical responses to an array of agonists. These experiments thus revealed an unexpected variation in the degree of cell-cell communication. Another example of a project, where FIMS was indispensable in the assignment of specific responses to subgroups of cells in a heterogeneous population, was a study carried out with pluripotent stem cells that were stimulated to differentiate towards the megakaryocyte lineage. By using combination of fluorescent antibodies and monitoring both morphological and functional responses for ranges of individual cells, it was possible to relate specific cellular reactions to the degree of maturation. Unfortunately, space restrictions prevent further discussion of this work. One special application concerns the experimentation used to unravel the mechanisms underlying the procoagulant response of activated platelets.
B. The platelet procoagulant response.
Almost twenty years ago, researchers from Maastricht and elsewhere discovered that the phospholipids in the plasma membrane of platelets and other blood cells are arranged in a typical asymmetrical way. In particular, anionic (signaling) phospholipids such as phosphoinositides and phosphatidylserine (PS) were found to be located almost exclusively in the inner membrane leaflet. In platelets, this asymmetry was rapidly abolished by ATP depletion or stimulation with Ca2+ ionophores. It was soon realized that this loss of lipid asymmetry (flip-flop) and the consequent surface exposure of especially PS transformed the platelets from coagulation-inactive to highly coagulation-supporting entities. Indeed, PS-coating lipid surfaces act as a site of cleavage and activation of the coagulation factors, prothrombin and factor X. Initially, the significance of this finding remained unclear because most platelet agonists appeared to be in essence unable to mimic this effect of Ca2+ ionophores. Even combinations of strong agonists, like thrombin plus collagen, evoked PS exposure in only a small subtraction of the platelets. Interest in this platelet reaction increased, however, when a patient with serious bleeding problems was identified (Mrs. Scott+), whose platelets were found to be deficient in PS exposure, thus emphasizing the importance for the in vivo situation. Recent studies agree with this, because abnormalities in the procoagulant response have also been traced in platelet-containing plasma from patients with other, more common platelet defects. While progress has been made in identifying the phospholipid transporters involved in the loss of lipid asymmetry, the physiological activators of the platelet response and the underlying signaling mechanisms have remained unclear for a long time.
C. Receptors and signal transduction involved in the procoagulant response.
Application of FIMS has led to unexpected advancement in this area, because it allowed continuous observation of individual platelets with respect to several characteristics. A first step forward was made with a simple experiment, where platelets were monitored that came into contact with a surface covered with collagen fibers. The recorded phase-contrast images showed that the platelets firstly spread along these fibers and then suddenly changed in morphology from a structure with pseudo pods to one of balloons and attached membrane blebs. Subsequent fluorescence imaging studies showed that this distinctive morphological change was always accompanied by exposure of PS (detected with fluorescent-labeled annexin V), and was preceded by a strong increase in intracellular Ca2+ concentration (detected with a Ca2+ probe). Since collagen surfaces were much more potent in inducing the bleb formation and PS exposure that other tested platelet-adhesive surfaces, the question rose whether this collagen-specific effect was receptor-mediated. When testing various collagen-like structures, it appeared that immobilized peptides that had previously been identified as ligands of the glycoprotein VI (GpVI) receptors were quite potent in inducing both PS exposure and the preceding Ca2+ response. This markedly contrasted to effect of platelet integrin Ib 3 receptor interaction with a sticky fibrinogen surface where even after stimulation with the strong agonist thrombin, only little PS exposure was observed. Since ligands of GpVI receptor were very weak in inducing PS exposure if added to platelets in suspension, in spite of their highly platelet-aggregating effect, a next question was whether platelet adhesion is needed to prime for the above-described GpVI-dependent procoagulant response. When fibrinogen-spread platelets were stimulated with GpVI ligands, but not with thrombin, this caused a prolonged, high Ca2+ signal in practically all cells, followed by PS exposure and again formation of balloons and plasma membrane blebs. These and other findings suggest that GpVI is a receptor linked to the procoagulant response, provided that the platelets are in contact with an adhesive surface. Although the structure of GpVI is yet unknown, the literature provides good evidence that it is an important signaling receptor for collagen in aggregating platelets. It elicits signal-transducing pathways that are reminiscent to those evoked by the immune receptors of lymphocytes, leading to activation of the protein tyrosine kinases Syk, Btk and Lyn, and of phospholipase C-gamma. Studies to the signaling events in collagen-adherent platelets indicate that, her, the same pathways are elicited, but that also other intracellular processes are active probably mediated by Ca2+-dependent tyrosine kinases, phosphatases and proteases.
D. Relation to arterial thrombosis.
Using FIMS, the same morphological (blebbing) and functional (increase in Ca2+ and exposure of PS) platelet responses have been observed, when whole blood was perused over a collagen-coated surface, which highly suggests that this response is indeed of physiological importance. A next goal will be, using FIMS in combination with intravital microscopy, to monitor these responses also in damaged, thrombogenic capillaries form living animals that are injected with fluorescent platelets or probes. Current pilot studies are very promising. That the platelet procoagulant response may indeed be important in arterial thrombosis, appeared from another recent study, where we found that both Ca2+ and procoagulant responses were significantly higher in platelets that were obtained from young stroke patients in comparison to the platelets from a control group of healthy volunteers.
Location: CARIM, Department of Human Biology and Biochemistry, University of Maastricht, UNS50, Room 3.349.
Contact person: Dr. Johan Heemskerk
Principle:
Fields of Research:
Vital imaging of structural proteins.The advent of the GFP technology has largely enhanced the possiblities of imaging molecules in living cells. When the (cDNA) sequence of a gene is known, fusion proteins can be generated encompassing the protein of interest (in our case structural proteins such as intermediate filament proteins and actin) and green fluorescent protein. This latter molecule, isolated from the deep sea jellyfish Aequorea victoria (see picture) is capable of emitting a bright green fluorescence when excited with the appropriate wavelength (488 nm)
the jelly fish
the
molecule
The location, behaviour and changes of fusion proteins can be examined by fluorescence microscopy. Because of the superior resolution and the relative low level of phototoxicity one- and two-photon confocal laser scanner microscopy is the choice for imaging of living cells.
Applying modern molecular biological and imaging techniques we want to elucidate the role of structural proteins in relation to a number of diseases
Deformation and damage of cytoskeletal proteins as a result of repetitive mechanical stress
The role of mutated nuclear lamins in the development of cardiomyopathies and muscle distrophies
Ad. 1 Deformation of cytoskeletal proteins
Decubitis is the result of long-term immobilisation of patients. The main cause is still unknown. In cooperation with the cell mechanics department the effects of (repetitive) mechanical stress on structural proteins will be investigated using confocal microscopy. Relative displacements of cytoskeletal proteins, such as (GFP-) vimentin can be monitored ad quantified in time (see movie1)
Also other cytoskeletal protens such as actin can be visualised in GFP-actin transfected cells
Ad 2. Nuclear lamins
Lamins are structural nuclear proteins belonging to the intermediate filaments. Lamins are present in the nucleus of all eukaryotic cells and form not only a rim-like strcuture on the inner layer of the nuclear membrane, but form also deep intranuclear tubules and a veil-like nuclear network
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Localisation of lamins in the nucleus visualised after image restoration. Broers et al. J. Cell Sci. (1999) view article here BroersJCS99.pdf |
View mitosis movie (lamc.avi) View interphase movie (intrphmv.avi)
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Reassembly of lamin proteins during mitosis can be examined in living cells |
Moreover, it is possible to examine 3-dimensional movement of the lamine in time. For 3d-viewing of this movie, red/green stereo glasses are needed. |
For other movies of mitosis and apoptosis follow this link http://137.120.28.214/mcb/cytoskeleton.html#GFP
Recently it has been discovered that A-type lamin mutations are involved in the development of several inheretable diseaseses including certain types cardiomyopathies. Until now it is completely unclear how a nuclear protein can cause severe abnormalities in muscle tissues. Using vital imaging techniques on normal cells and cells containing mutated lamins we investigate structural and functional changes as a result of lamin mutations at the cellular level. Techniques include molecular mutagenesis techniques, the generation of (stable) transfectants, imaging by confocal microscopy, investigations on structural stablility using fluorescence recovery after photobleaching (FRAP), fluorescence loss of intensitiy after photobleaching (FLIP) , and image restoration (for an example see colour picture above).
Students interested in performing research can contact Jos Broers (e-mail jos.broers@molcelb.unimaas.nl) tel 043-3881366 or Frans Ramaekers f.ramaekers@molcelb.unimaas.nl.tel. 043-3881351
A brief description of potential research topics can be found here (keuzestageBM2002)
Location:Department of Molecular and Cellular Biology, University of Maastricht, Fifth Floor of UNS50
Contact person: Prof. Frans Ramaekers and Dr. Jos Broers
Principle and fields of research: Living material is permanently adapting to changes in the environment in a very dynamic way. Because mechanical forces can be sensed by living cells, the architecture of the cytoskeleton is permanently adapting to new situations through synthesis of cytoskeletal proteins. A typical example is the axial cytoskeleton in endothelial cells, which line the internal blood vessel wall. This cytoskeleton consists of filamentous (F-) actin and is localized immediately under the cell membrane. Upon enhanced blood flow which imposes a higher shear force on the endothelial cells, adaptation in the axial cytoskeleton leads to an increased cellular stiffness, thereby enhancing the resistance to cellular deformation.
Various techniques have been developed to quantify changes in cellular stiffness such as cell poking, scanning acoustic microscopy, micropipette aspiration, optical tweezers and magnetic bead microrheometry. However, the spatial resolution of these techniques is limited such that high-resolution information of distribution of cellular mechanical properties can not been obtained. In contrast the atomic force microscope (AFM) not only enables visualization of the surface morphology of living cells but also calculation of spatial distribution of the stiffness of the cell surface. As such, it provides essential information on the stiffness characteristics of the cytoskeletal fibers, immediately underlying the cell membrane. AFM measurements allow the calculation of the Young's modulus, i.e., a measure of the local stiffness, by applying the Hertz' formula.
The Atomic Force Microscope
The figure below presents the surface morphology of a living cardiomyoblast, registered with an AFM. The F-actin containing stress fibers underneath the cell membrane are clearly visible, as well as regional differences in height of the cell.
Location: Measurements on living cells using the AFM is possible in the BME-TU/e department.
Contact person: Information on this research domain can be obtained from Prof. Dr. Luc Snoeckx or Ms. Maria Stekelenburg (tel. 0402473075; e-mail: m.stekelenburg@tue.nl)
(1.1) Portfolio of the first year Imaging and image-formation techniques
(1.5) Lecture on Endoscopy and Microscopy by Prof. Dr. D.W. Slaaf
(1.6) Lab on Practical aspects of microscopy by Prof. Dr. D.W. Slaaf
(2.1) Coordination of block Leve de Cell by. Prof. Dr. L. Snoeckx. Prof. Ramaekers is member of the planning committee
(2.7) Coordination of OTG trajectory by Prof. Dr. L. Snoekcx. For short project see the website of the Faculty of Medicine, onderwijspleinkeuzeonderwijs-beschrijving onderwijsinstituten-programmas.
(1.4) Participation in the block with a focus on Microscopy. Prof. Dr. Dick Slaaf.
(1.1) Lecture and lab on physical aspects of microscopy by Dr. Marc van Zandvoort
(2.6) Participation in the block with a lecture on cell biology by Prof. Dr. F. Ramaekers
(3.2) Participation in the block, focused on image formation.
(3.2) Elective block Signal transduction, coordinated by Dr. J. Heemskerk.
Participation of Dr. Marc van Zandvoort and Dr. Wim Engels (one project)
(4.1) Start of short projects carried out under auspices of the VIU
(4.2) Elective course Cell Mechanics Dr. Carlijn Bouten and Prof. Dr. Luc Snoeckx
(4.3) Elective course New developments in microscopy on living samples Dr. Marc van Zandvoort and Dr. Jos Broers. For more information on this course, go to the following websites:
(4) Experimental projects on Biomedical applications of TPLSM Dr. Marc van Zandvoort and Prof. Dr. Dick W. Slaaf(5) Final Project on Biomedical applications of TPLSM Dr. Marc van Zandvoort and Prof. Dr. Dick W. Slaaf
8.1.1: Lecture by Prof. F. Ramaekers on general contents: (PowerPoint presentation)
8.1.2: Lecture by Prof. F. Ramaekers on the Cytoskeleton: (AVI movies (45 MB), PowerPoint presentation)
8.1.3: Lecture Dr. J. Heemskerk on FIMS: (PowerPoint presentation)
8.1.4: Lecture by Prof. L. Snoeckx on Atomic Force Microcopy: (PowerPoint presentation)
8.1.5: Lecture by Prof. B. Rutten on Microscopic Stereology: (PowerPoint presentation)
8.1.6: Lecture by Dr. J. Broers on Confocal Microcopy: (PowerPoint presentation2, AVI movie1, AVI movie2)
8.1.7: Lecture by Dr. M. Janson on Micromanipulation techniques: Optical Tweezers and force measurements. Force measurements and light microscopy: (PowerPoint presentation)
8.1.8: Lecture by Dr. M.A.M.J. van Zandvoort on Two Photon Lifetime Microscopy: (link not available at the moment)
8.1.9: Lecture by Prof. D. Slaaf on Optical Microscopy: (link not available at the moment)
