2004 Summary

Project Objectives

The main scientific and technological objective of this project is the development, assessment and application of tomographic and microscopic technologies combined with novel fluorescent probes that will allow in vivo imaging of important biological processes in subcellular, cellular, organ, embryo and whole animal levels. To achieve this integrating and unifying view they have combined multidisciplinary research and technology expertise in Biology, Bioorganic Chemistry, Engineering, Theoretical Physics and Biomedical Optics.

In order for this interdisciplinary large-scale project to be accomplished, it has been divided into three main subprojects: The probe development subproject, which objectives are to develop and improve new fluorescent proteins, fluorophores and biosensors; an embryo and whole animal imaging subproject, devoted to developing experimental techniques and software to improve quantitation and resolution imaging at the embryo/whole animal level; and finally the cellular and subcellular imaging subproject devoted to developing and improving new microscopy techniques for imaging the cell in-vivo from the micrometer to the nanoscale. Within both imaging subprojects appropriate biological questions are posed in order to prove and validate the quality and level of improvement in the novel imaging techniques.

Sp I. Probe Development

In relation to novel probe development, four main lines of work have been established, namely new fluorescent proteins, chemical fluorophores, Biosensors and tags for nanoscale imaging.

Sp I. Workpackage 1 – Fluorescent Proteins

Progress on new fluorescent proteins was made by PARTNER 14IBCH, that has developed a monomeric dual-color photoswitchable cyan fluorescent protein (PS-CFP) and its improved variant, PS-CFP2. P14 has found that PS-CFPs are capable of efficient photoconversion from cyan to green form, changing both its excitation and emission spectra in response to 405-nm light irradiation. Complete photoactivation of PS-CFP2 results in a 2,000-fold increase in the green-to-cyan fluorescence ratio, making it the highest-contrast monomeric photoactivatable fluorescent protein described to date. PS-CFP was used as a photoswitchable tag to study trafficking of human dopamine transporter in living cells. Generally, PS-CFP and PC-CFP2 can be used widely for protein, organelle and cell tracking in living systems. At moderate excitation intensities, PS-CFPs represent as a pH-stable cyan label for protein tagging and fluorescence resonance energy transfer applications. In particular, efficient FRET was demonstrated between PS-CFP (donor) and yellow FP phiYFP (acceptor). An increase in the donor-to-acceptor emission ratio after their separation reached 6.85-fold, which is better than that for any other GFP-like protein pairs reported. On the other hand, and also in relation to Fluorescent proteins, PARTNER 10-1MRC-NIMR generated cultured cells expressing CFP or YFP/HMG-box binding protein-1 (HBP1) fusion proteins. These can be used to carry out FRET experiments and will allow the study of interactions of HBP1 with other HBP1 molecules or other FP/chromatin binding protein fusion molecules.

Sp I. Workpackages 2 & 3 – Chemical Fluorophores and Biosensors

PARTNER 11EMBL have synthesised two series of novel, long wavelength red fluorescent dyes. These dyes are unique in that they are uncharged and therefore membrane-permeant, where as all of the existing far red fluorescent dyes contain charges. Far red fluorescent dyes are particularly attractive since they avoid the haemoglobin absorbance and are thus very useful for imaging large tissue volumes in-vivo, increasing the optical penetration depth by orders of magnitude. New dyes have been used very successfully to study protein-lipid interactions, which could have application for high throughput screening of test ligands. A new 2-photon excitable caged compound was generated by PARTNER 9UA. This compound is based on coumarin that can be coupled to alcohols and free amino groups. In the near future, this cage will be coupled to signalling lipids, providing excellent tools for studying spatiotemporal lipid signalling in living cells.

Sp I. Workpackage 3 – Tags for Nanoscale Imaging

Finally, progress on development of tags for nanoscale imaging achieved during this first reporting period have been made by PARTNER 13ETH who has investigated the potential of gold nanoparticles as small as 5 nm as label for optical microscopy. The optical detection of these particles has been demonstrated and will be applied to biological samples. Additionally, two methods to tag proteins with nanoobjects have been developed as pilots by PARTNER 16-2EMCR-II, who has successfully attached gold particles (1.4 nm diameter) to purified proteins and DNA.

Sp II. Animal Imaging

Within the Animal Imaging subproject, four main lines of work have been established: Instrumentation for whole animal imaging, Instrumentation of embryo and organ imaging, Theory and experiments and Biological applications.

Sp II. Workpackages 1 & 2 – Instrumentation for Whole Animal and Embryo Imaging

Towards fulfilling their tasks PARTNER 1FORTH has developed the first full 360o tomographer for 3D in-vivo imaging of fluorescent proteins in whole animals. This prototype has been tested both with phantoms and in-vivo results, showing for the first time in-vivo imaging of GFP-tagged T-cells in the thymus, that were provided by PARTNER 10-1MRC-NIMR. PARTNER 2ULUND’s objectives have been to investigate whether fluorescence emission spectroscopy could add any information regarding depth of a fluorescence inclusion in tissue. They have been investigating if the depth resolution could be more robustly obtained by adding spectroscopic information to tomography in animal imaging. The results are promising, both from modelling and experimental work. This work is closely related to FMT developed by PARTNER 1FORTH as it could provide a more robust data analysis. PARTNER 10-2MRC-HGU designed and constructed a robust device which combines two existing technologies: OPT imaging (previously invented in that group) and organ culture techniques. Many issues have been tackled in order to achive this: A "life-support" chamber has been designed that is compatible with the OPT technology, involving the design of temperature control, maintainance of high humidity, and gas supply. A new series of adjusters has been constructed allowing accurate positioning of the specimen while in the chamber. New software has been written and tested to control the new device. They now have successfuly surpassed the previous state-of-the-art, which was the ability to perform optical projection tomography on fixed specimens. Much of this development work has been done in close collaboration with PARTNER 17-4DIO who are developing new fluorescent labelling approaches necessary to maximise the potential of this new technology. The new combined technology will provide unprecedented insight into the growth and development of an intact mouse organ in 4D.

PARTNER 10-1MRC-NIMR have generated mice that express YFP, CFP, GFP and dsRed in the whole haemopoietic system or only on lymphoid cells and they intend to expand the collection of FP mice by generating mice expressing kindling protein KFP1 in collaboration with PARTNER 14IBCH, who have also developed a dual-color photoswitchable cyan fluorescent proteins (PS-CFP and PS-CFP2) capable of photoconversion from cyan to green state in response to 405-nm light irradiation. Complete photoactivation of PS-CFP2 results in a 2,000-fold increase in the green-to-cyan fluorescence ratio. Potentially, PS-CFP2 can be used for precise photolabeling and tracking of selected cells in living embryos and animals. In probe generation PARTNER 11EMBL prepared recently a functional FRET probe to measure protein kinase C activity in living cells that gives a readout of phosphorylation levels which includes the equilibrium of kinase and phosphatase activity. Up to now, the probe is equipped with green and yellow fluorescent proteins. They are now working on replacing these fluorophores with longer wavelength dyes suitable for imaging in tissue. Further progress towards probe generation was made by PARTNER 4UKM who synthesized a tumor selective optical contrast agent designing two different fluorochromes modified by short peptides which exhibit high binding affinity to endothelial markers of tumor neovasculature. The binding affinity of both probes could be shown in vitro using different cell assays. Fluorescence reflectance imaging (FRI) experiments showed a strong retention of the targeted probes in different cancer models. It is planned to apply these targeted probes for future multi-wavelengths FMT studies.

Sp II. Workpackage 3 – Theory and Experiments

Advances on theoretical issues were made by the following partners: PARTNER 15UC3M have worked on two fundamental issues arising in the mathematical and computational treatment of small animal imaging. The first is the accurate description of pulse propagation in tissue based on the radiative transfer equation (RTE). Here, they have investigated new boundary and interface conditions for the RTE, and they have investigated alternative approximate models for light propagation in tissue which are more accurate than the diffusion equation close to interfaces and sources. The second main theme of investigation was the derivation of a rigorous mathematical model for the inverse RTE problem. Here they also have advanced a novel nonlinear shape reconstruction scheme for the inverse problem based on level sets which will be used during the second year of the project in their collaboration with PARTNER 3UCL. PARTNER 17-3LFSP developed improved imaging methods for detection of phantoms in highly scattering tissue like media, and they have been trying a few different schematics of time-resolved NIR imaging method, in transillumination and reflection schemes. One of the reflection scheme is proved to be more efficient with better resolution, has the potential of further developing to be a non-invasive diagnostic tool for breast cancer detection. PARTNER 5CRSA have performed a theoretical and numerical study of light propagation in scattering media, with absorption or gain (excited fluorescent molecules). The main results are: (1) a characterization of the transition from the ballistic to the diffusive regime using the Radiative Transfer Equation (RTE), (2) a quantitative characterization of the diffusive regime in both absorbing and amplifying media, (3) the development of a model coupling rate equations and the RTE to study the dynamic transport (pulse illumination) and the intensity fluctuations due to the motion of the scatterers. An expected end result from (2) is the development of an easy-to-use software to calculate the diffusion coefficient and the extrapolation distance for absorbing and amplifying media, which could be used within the Consortium. An expected end result from (3) is a numerical study of the potential for biomedical imaging of an extended Diffusing-Wave Spectroscopy technique. During the first year of the project PARTNER 3UCL have developed methods for the accurate description of light propogation in optical media based on the Radiative Transfer Equation. This method is designed to solve the reflection and transmission of light in optically thin media including obliquely incident light and refractive index mismatched surfaces. Secondly they developed numerical codes for solving the forward and inverse problems in fluorescence optical tomography, based on the diffusion equation. PARTNER 6UAM aimed to establish theoretical models to study light propagation in systems with small length scales and developed a numerical tool to analyse multiple scattering effects in systems of thousand scatters. The emission from a single molecule in such complex environments can also be calculated. PARTNER 21LENSLET focuses on optical processing and electro-optical systems. Within the consortium Lenslet is working on the applicability of ultra-fast image reconstruction that may elevate the tomography field to new grounds. Implementing complex algorithms on an optical processing core requires innovative algorithmic work and state of the art technology. As for now, they have initial algorithmic success and have made suitable modifications to the optical core design and concepts.

Sp II. Workpackage 4 – Biological Applications

Progress on small animal imaging in biological systems was made by the following partners: PARTNER 10-1 NIMR-MRC have analysed fluorescent cells within embryos expressing GFP under the control of CD2 regulatory sequences and intend to identify the function of these cells and their contribution in the formation of primordial structures that lead to the adult lymphoid organs. This will contribute in our knowledge on the development of the immune system during embryonic development. In order to develop tools for the improvement of the FMT tomographer PARTNER 1FORTH have generated mice that express dsRed in T cells. In the future these mice will be used for in vivo imaging of T cells and will allow the study of immune function. PARTNER 16-1EMCR-I have generated transgenic mice expressing GFP fusion proteins that are expressed in several different cell types or tissues such as, a) a mouse model using the Ly-6A gene expressing in hematopoietic stem cells, b) a mouse model expressing GFP in all endothelial cells using the eNOS gene to image atherosclerotic plaques, c) a mouse model expressing GFP-Clip 170 expressed in many cell types including the brain, and d) a mouse model expressing GFP -CTCF which is expressed in most cells to allow imaging of spermatogenesis. PARTNER 17-4DIO established the conditions for imaging living cells within a complex organ grown in culture and have assayed a system allowing visible-light and fluorescent imaging of living specimens, coupled with computer control of specimen position and rotation. This system has been applied to study the behaviour of ectodermal cells during mouse limb growth. Ectodermal limb cells were randomly labelled using DiI and their distribution and movements were recorded for up to 16 hours of mouse limb bud growth in vitro. This is the first time that mouse limb growth has been recorded and the first time that labelled cell behaviour has been time-lapse filmed during this process. During this period they have also developed transgenic models that will allow more accurate cell labelling and mesenchymal cell monitoring. PARTNER 18-2UHEI have generated transgenic mice targeting the tTA activator to the forebrain (using CaMKII plasmid and BAC) and raphe nucleus (using 5-HTT and TPH2 PACs). The expression pattern of the transgenes is being validated. Those mice will be used to express reporter genes in defined brain regions. In addition they have developed a reporter for CREB activity in vivo with higher signal to noise ratio compared to CRE reporters. This reporter is being tested and optimized in cell culture. Finally, PARTNER 8IC, even if not formally assigned tasks within SPII, has produced work that is relevant to this subproject. During this first 12 months PARTNER 8IC worked on the development of new wide-field time-gated imaging techniques that can be applicable to fluorescence imaging through turbid media. This work is intended to evolve to time-gated OPT. Their work on this task has begun with the development of suitable fluorescent optical phantoms in clear and turbid media of different scattering properties. These experimental data is currently being analysed by PARTNER 3UCL and their first images are expected before 2005. The combination of time-gated imaging and either diffuse optical tomography (DOT) or optical projection tomography (OPT) will provide improved localisation of fluorescent heterogeneities in diffuse optical media. PARTNER 17-1CNB developed a novel imaging technique : optical position tomography (OPT) allowing the study of the internal structure of entire organs or embryos. In collaboration with PARTNER 10-2MRC-HGU and PARTNER 17-4DIO, they are adapting OPT to the study of the internal structure of secondary lymphoid organs, such as spleen and lymph nodes. PARTNER 22KENTECH’s objectives at the beginning of the reporting period were to prototype an agile delay control system together with an improved high speed camera in order to gain: i) faster data acquisition by means of reducing the time taken to scan the decay signature, ii) improved spatial and temporal resolution. They have designed and built the critical elements of a prototype high speed delay system, which will allow the rapid and automated time domain control of a camera trigger signal. The most important element of the camera system is the image intensifier and they have been working with an image intensifier manufacturer to improve the temporal and spatial resolution. They have built a prototype high rate camera which takes advantage of a prototype improved intensifier.

Sp III. Cellular and Sub-cellular Imaging

In the subcellular and cellular renges, the main objectives are the development and improvement of existing microscopic technologies for the production of multidimensional high-resolution microscopy devices for in vivo cellular and sub-cellular imaging. Cell imaging contains four workpackages that aim to:

- Improve and develop microscopic approaches for imaging at the cellular level.

- Improve and develop instrumentation for cellular imaging at the nano-scale level

- Provide knowledge, methods and techniques complementary for the development of novel high-level resolution micro-devices for sub-cellullar imaging and understanding of photobleaching results.

- Use the above mentioned techniques for FRET imaging in vivo and apply photobleaching techniques (FRAP, FLIP) to characterize kinetics of macromolecule function.

Sp III. Workpackage 1 – Instrumentation for Fluorescence Microscopy Imaging

In this workpackage considerable progress has been made by PARTNER 1FORTH that has achieved multiphoton confocal microscopy. To achieve multimodal fluorescence imaging microscopy a wide-field microscope capable of measuring fluorescence lifetimes, fluorescence spectra and fluorescence spectra was upgraded by PARTNER 9UA in collaboration with PARTNER 20PKLAS as to perform z-sectioning using a spinning disk confocal unit. PARTNER 8IC has worked on the development and optimization of several novel microscope systems for in-vivo imaging of tissue, cells and sub-cellular structures. In partnership with PARTNER 20PKLAS, they have developed a FLIM microscope system with the spinning Nipkow disc to provide real-time optical sectioning. They have combined this with high-speed wide-field time domain FLIM using new technology from PARTNER 22KENTECH that can provide FLIM images at up to video rate. They are working to integrate the same high speed FLIM technology from Kentech with a multibeam, multiphoton microscope and have good first results. They have also developed a new compact, electronically tunable laser source and applied it to excitation spectroscopy and FLIM in confocal and wide-field microscopes.

FLIM images of a fixed unstained 10 microns section of human pancreas obtained in a wide-field time-domain FLIM system with two different excitation bands. Integrated fluorescence intensity, false colour FLIM image and intensity merged FLIM image respectively obtained with Pex = 1.4 mW, lex = 440-450 nm and lem > 470 nm. Same field of view but with Pex = 2.4 mW, lex = 520-530 nm and lem > 570 nm. Areas marked in red in the first image are A small artery, I islet of Langerhans and C connective tissue (mainly collagen).

PARTNER 22 KENTECH’s objectives at the beginning of the reporting period were to prototype an agile delay control system together with an improved high speed camera in order to gain faster data acquisition by means of reducing the time taken to scan the decay signature, and improved spatial and temporal resolution. They have designed and built the critical elements of a prototype high speed delay system, which will allow the rapid and automated time domain control of a camera trigger signal. The most important element of the camera system is the image intensifier and they have been working with an image intensifier manufacturer to improve the temporal and spatial resolution. A prototype high rate camera has been built which takes advantage of a prototype improved intensifier.

In order to achieve Spatially Modulated Illumination (SMI) -Microscope two counter propagating laserbeams have been brought to constructive interference by PARTNER 18-1KIP, establishing a standing wavefield (U. Spoeri et al, Journal of Applied Physics (2004); B. Albrecht et al, Applied Optics (2002)). In combination with high precision axial displacement this technique of analysing biological objects allows size measurements in molecular dimensions of some ten nanometers (up to now only for fixed cells). By the extension of this microscope for the realisation of 'in vivo' measurements, i.e. analysis of the living cell with a vertical setup of the SMI, the advantages of the SMI-Microscope can be extended to vital biological systems. The pre-setup is completed since November 2004 and the translation stages, including a piezo with a positioning accuracy < 10nm for the most relevant z-direction, are mounted. After passing the beamsplitter the two laserbeams with a wavelength of 488nm will be focussed into the two water immersion objectives through which a standing wavefield will be established in the object space. The detection light - separated by a dichroic mirror and a blocking-filter which transmitts wavelengths > 500nm - will be focussed onto the CCD-Chip of the camera. After the completion of these preparations in the coming months, the assignment of that system to 'in vivo' measurements will be realised as the next step. A novel low phototoxic confocal microscope (CLEM: controlled light exposure microscope) was built by PARTNER 9 UA displaying a 7-fold reduced phototoxicity as compared to a conventional confocal microscope. The new CLEM microscope principle was patented and will be commercialized in 2005 using exclusive licensing to a confocal microscopy manufacturer. Finally PARTNER 17-1CNB has adapted Intravital Microscopy (IVM) for a better detection of fluorescent signals and are currently establishing MP-IVM in Madrid.

Sp III. Workpackage 2 – Instrumentation for Nanoscale Imaging

This workpackage is focused on instrumentation for nanoscale imaging and PARTNER 9UA has made progress for implementing total internal reflection (TIR) into a wide-field fluorescence lifetime imaging system. They anticipate a fully automated TIR-FLIM system will be ready mid 2005. For the Spatially Modulated Illumination (SMI) -Microscope two counter propagating laserbeams are brought to constructive interference, establishing a standing wavefield by PARTNER 18-1KIP. In combination with high precision axial displacement this technique of analysing biological objects allows size measurements in molecular dimensions of some ten nanometers (up to now only for fixed cells). By the extension of this microscope for the realisation of 'in vivo' measurements, i.e. analysis of the living cell with a vertical setup of the SMI, the advantages of the SMI-Microscope can be extended to vital biological systems. The pre-setup is completed since November 2004 and the translation stages, including a piezo with a positioning accuracy < 10nm for the most relevant z-direction, are mounted. After passing the beamsplitter the two laserbeams with a wavelength of 488nm will be focussed into the two water immersion objectives through which a standing wavefield will be established in the object space. The detection light - separated by a dichroic mirror and a blocking-filter which transmitts wavelengths > 500nm - will be focussed onto the CCD-Chip of the camera. After the completion of these preparations in the coming months, the assignment of that system to 'in vivo' measurements will be realised as the next step. Based on the frame of the Cell imaging of Molecular Imaging, PARTNER 17-3 LFSP has developed and tested a modified tapping mode SNOM, in order to distinguish the high localized evanescent field and their enhancement in comparison with propagating ones. The surface plasmon (SP) imaged by this tapping mode SNOM, is proven to be a good method to image the surface defect and its optical properties. Using time-resolved technique, they have also measured the propagation speed of the SP in the near field. An imaging device for the time-dependent spectral and near field optical information is in development.

Sp III. Workpackage 3 – Theory and Experiments

This workpackage is aimed at developing novel methods and techniques for subcellular and nanoscale imaging and all tasks have been addressed. PARTNER 1FORTH has developed novel theories for high resolution sub-cellular imaging and established novel approaches for fluorescence tomography at the microscopic level. The PARTNER 5CSRA group has performed a theoretical and numerical study of the single-molecule fluorescence enhancement by metallic nano-objects such as tips or nanoparticles. The main results are: (1) the fluorescence enhancement level strongly depends on the orientation of the transition dipole with respect to the object, (2) plasmon resonances can be used to enhance the signal, but the emission wavelength should not coincide with the plasmon resonance wavelength, (3) when using tips of scanning near-field microscopes, the tip quality substantially influences the enhancement. These results should help understanding the experiments, and qualitatively guide future works. An end result is a quantitative calculation of the expected enhancement factor in a model biological environment, using a 3D numerical calculation.

PARTNER 7IMM developed a new FRAP formula to better quantify the effective diffusion coefficient and the immobile fraction was successfully derived. The group has also developed new FLIP methodology to characterize the kinetics of a mixed population of molecules diffusing at different rates within the cell. A preliminary version of the software is already available and is undergoing extensive testing. PARTNER P17-2ICMM has developed simulation procedures, based both on finite element methods and Finite Difference Time Domain (FDTD) calculations, to enhance the signal detected in the near field fom a cell modell in the presence of both dielectric (silica) and metallic (gold) nanoparticles through the excitation of their morphologic dependent resonances. In addition they are currently investigating the binding photonic force between the excited molecule and the nanoparticle. Thus, sensitive Raman spectroscopy inside living cells allow for monitoring small chemical changes in the cell which could be the precursors of larger morphological changes, visible by conventional microscopy.

PARTNER 6UAM worked on the problem of modelling of atomic force microscopy (AFM). A new numerical tool was developed to calculate electrostatic fields and forces generated by the metallic tip of an AFM on anisotropic samples. PARTNER 13ETH worked in improving the sensitivity of particle localization along the axis of an optical microscope by using a standing wave illumination. They are also interested in exploiting the interaction between a dye molecule and gold nanoparticle to learn about their local environments. PARTNER 15UC3M is mainly involved in the 'inverse problem in sub-cellular imaging' (Wk3 Task6). During the first year they have done preparatory work for addressing this task. For this purpose, the group has discussed the general inverse problem arising in cell imaging with other partners during the kick off meeting in Crete. In addition, Oliver Dorn has visited the research lab of partner P5 (Ecole Centrale Paris) on Sept 24 in order to discuss possible future collaborations on the task of addressing the 'inverse' as well as the 'direct problem' ('modelling') of sub-cellular imaging. Another visit of Oliver Dorn at the lab of P5 is provisionally planned for March 2005 for continuing these discussions. PARTNER 3UCL has been active in discussions with P5 and P15 to complete the task of 'inverse problem in sub-cellular imaging', while PARTNER 20PKLAS has attended a number of meetings and discussions and collaborative actions have been undertaken.

Lastly PARTNER 10-1MRC-NIMR have generated mice expressing a fusion protein HP1-beta/GFP, and cells expressing a fusion protein HBP1/CFP, HBP1/YFP or HBP1/GFP. They also generated mice transgenic for GFP, CFP, YFP, dsRed under the control of CD2 and vav regulatory elements. These tasks can be used to help understand photobleaching and to study the inverse problem in subcellular imaging. They intend to generate more tools using novel fluorescence proteins.

Sp III. Workpackage 4 – Biological Applications

The different biological groups in the area of biological applications have addressed all of the tasks proposed. PARTNER 9UA has made substantial progress for measuring signaling dynamics at the plasma membrane. Several optimized fluorescent biosensors have been constructed and/or introduced into single cells. Upon stimulation of the cells several signaling events can be visualized at the level of the plasma membrane. They have a strong collaboration with PARTNER 11EMBL and PARTNER 14IBCH for the generation of molecular probes for visualizing signaling events such as translocation, phosphorylation, interaction or conformational change. They also collaborated with PARTNER 16-1EMCR-I resulting in a publication. Their developments in instrumentation (SPIII, WK1 and WK2) are applied in this workpackage. PARTNER 10-1MRC-NIMR as described under WP3 have generated mice expressing a fusion protein HP1-beta/GFP, and cells expressing a fusion protein HBP1/CFP, HBP1/YFP or HBP1/GFP. They also generated mice transgenic for GFP, CFP, YFP, dsRed under the control of CD2 and vav regulatory elements. These tasks can be used to help understand photobleaching and to study the inverse problem in subcellular imaging. They intend to generate more tools using novel fluorescence proteins. PARTNER 16-1EMCR-I has studied a variety of processes at the cellullar level using fluorescently labelled proteins. The microtubular network important for the transduction of signals to and from the nucleus was visualised in collaboration with PARTNER 9UA using a variety of fusion proteins that bind to the microtubular network such as CLIP 115 and 170, EB1 and BicD. This has resulted in the imaging of the dynamics of the microtubular network in a variety of cells including for the first time neurons. CTCF, a nuclear protein thought to to be involved in long range interaction between regulatory regions in the nucleus was also labelled. Bleaching and recovery experiments in combination with biochemical experiments have shown that CTCF appears to have a chromatin organising role with most of the CTCF bound to chromatin making most CTCF immobile. Most importantly this protein is involved in long range interactions. The group is in the process of visualising the chromatin dynamics of the beta globin locus by providing binding sites for fluorescent proteins at different positions in the locus and using FISH covering large chromatin regions (with PARTNER 18-1KIP). Lastly they have initiated a collaboration with a postdoc from PARTNER 16-1EMCR-I working in the lab of PARTNER 13ETH to measure real distances in chromatin. PARTNER 17-1CNB have used intravital microscopy (IVM) and multi-photon microscopy (MP-IVM) two imaging techniques which allow the detection of leukocyte-endothelium interactions and interstitial leukocyte migration, respectively. They have adapted IVM for a better detection of fluorescent signals and are currently establishing MP-IVM in Madrid. P16-2 EMCR-II is well on track to achieve its deliverables by the delivery date. They have completed a description of the in vitro activity of CSB with important implications for its mechanism of chromatin remodeling. This work has been accepted for publication in the Journal of Biochemistry. The group has engineered and produced a version of Rad54 protein with a specific amino acid sequence tag that can be biotinylated. They are now testing the efficiency of labeling this protein with streptavidin conjugated to gold particles. This gold labeled protein will allow us to study the interaction of Rad54 with nucleosomal DNA and describe conformational changes in these chromatin mimics induced by Rad54 using scanning force microscopy. The group is also generating reagents and developing protocols to produce nucleosomal arrays for structural analysis by scanning force microscopy analysis. They have succeeded in producing histone proteins in high yield in bacteria. After purification these histones will be reconstituted into nucleosomal arrays

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