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Peer-Reviewed Publications in 2004PARTNER 1 FORTH1) J. Ripoll, D. Yessayan, G. Zacharakis, and V. Ntziachristos, "Experimental determination of photon propagation in highly absorbing and scattering media," J. Opt. Soc. Am. A in press (2004).
ABSTRACT: Optical imaging and tomography in tissues can facilitate the quantitative study of several important chromophores and fluorophores. Several theoretical models have been validated for diffuse photon propagation in highly scattering and low absorbing media which describe the optical appearance of tissues in the near-infrared (NIR) region. However, these models are not generally applicable to quantitative optical investigations in the visible due to the significantly higher tissue absorption in this spectral region compared to the NIR. In this paper we performed photon measurements through highly scattering and absorbing media for ratios of the absorption coefficient to the reduced scattering coefficient approximately ranging from zero to one. We examined experimentally the performance of the absorption-dependent diffusion coefficient defined by Aronson and Corngold, J. Opt. Soc. Am. A 16: 1066-1071 (1999), for quantitative estimations of photon propagation at the low and high absorption regimes. Through steady state measurements we verified that the transmitted intensity is well described by the diffusion equation by considering a modified diffusion coefficient with a non linear dependence on the absorption. This study confirms that simple analytical solutions based on the diffusion approximation are suitable even at high absorption regimes and evinces that diffusion approximation based models are valid for quantitative measurements and tomographic imaging of tissues in the visible.
2) G. M. Turner, G. Zacharakis, A. Soubret, J. Ripoll, and Vasilis Ntziachristos, "Complete angle projection diffuse optical tomography using early photons", Opt. Lett. In press (2004).
ABSTRACT: We present the first experimental images of complex shaped phantoms embedded in diffuse media using optical tomography. Imaging is based on a complete-angle projection tomographic technique utilizing transmitted early photons. Results are contrasted with measurements obtained at later gates as well as pseudo-continuous wave data. The scanning system developed employs non-contact illumination and detection technologies that allow for high spatial sampling of transmitted photons. Combining this system with complete angle illumination is found to be an important strategy toward improved imaging performance, resulting in a better posed inversion problem. The appropriateness of reconstruction algorithms similar to those employed in X-ray computed tomography are showcased and suggestions for model improvements are provided.
3) Kostas Marias, Jorge Ripoll, Heiko Meyer,Vasilis Ntziachristos and Stelios Orphanoudakis, "Image Analysis for Assessing Molecular Activity Changes in Time-Dependent Geometries", in press in IEEE. Trans. Med. Imag.
ABSTRACT: In-vivo fluorescence molecular imaging and tomography has facilitated monitoring of genomics and proteomics over time and on the same animal. A highly important issue however has been the robust registration of animals imaged at different time points to obtain accurate description of activity and location. This paper presents a method for aligning temporal data of small animals based on surface anatomical features and improving the accuracy of monitoring fluorophore distribution. The method can account for differences in the positioning and compression of small animals and can be extended to 3D as well as to other imaging modalities.
4) G. Zacharakis, J. Ripoll, R. Weissleder and V. Ntziachristos, "Fluorescent Protein Tomography scanner for small animal imaging", in press in IEEE Trans. Med. Imag.
ABSTRACT: Microscopy of fluorescent proteins has enabled unprecedented insights in visualizing gene expression in living systems. Imaging deeper into animals has been however limited due to the lack of accurate imaging methods for the visible. We present a novel system designed to perform tomographic imaging of fluorescent proteins through whole animals. The tomographic method employed a multi-angle, multi-projection illumination scheme, while detection was achieved using a highly sensitive charge-coupled device (CCD) camera with appropriate filters. Light propagation was modeled using a modified solution to the diffusion equation to account for the high absorption and high scattering of tissue at the visible wavelengths. We show that the technique can quantitatively detect fluorescence with sub millimeter spatial resolution both in phantoms and in tissues. We conclude that the method could be applied in tomographic imaging of fluorescent proteins for in vivo targeting of different diseases and abnormalities.
5) J. Ripoll and V. Ntziachristos, "Imaging turbid media from a distance: Theory and applications of Non-contact tomography", in press in Modern Physical Reviews B.
ABSTRACT: Optical tomography of turbid media has been largely limited to systems that require fixed geometries or measurements employing fibers. Non-contact optical measurements from diffuse media could facilitate the use of large detector arrays at multiple angles that are well suited for tomography applications. Such imaging strategies eliminate the need for individual fibers in contact with the highly scattering volume, the use of restricted geometries and the need for matching fluids. Here we review the different approaches and systems developed for non-contact optical measurements and concurrent registration of the three-dimensional surface information of the diffuse medium. We present the basic theoretical formulation and its experimental validation, finally applying it to the specific case of fluorescence tomography of small animals. We discuss how these new technologies can considerably simplify experimental procedures and improve our ability to visualize functional and molecular processes in-vivo. Future perspectives and work are also outlined.
6) J. Ripoll and V. Ntziachristos, "Quantitative Point-source Photoacoustic Inversion Formulas for Scattering and Absorbing Media", in press in Phys. Rev. E.
ABSTRACT: We here present a novel expression for the photo-acoustic contribution of an optical point source in a diffusive and absorbing medium. By using this measurement as a reference, we present a direct inversion formula that recovers the absorption map quantitatively, at the same time accounting for instrumental factors such as the source strength, the shape of the optical pulse and the impulse response and finite size of the transducers. We further validate this expression through accurate numerical simulations showing that the absorption map is recovered quantitatively in the presence of a rotating geometry. We finally discuss how the presented solutions for point sources within the photo-acoustic problem enable the use of concurrent fluorescence and ultrasound measurements as appropriate for a hybrid tomographic system. The proposed system could retrieve absorption information using photo-acoustic measurements, and use this data to more accurately describe the fluorescence problem and improve reconstruction fidelity.
7) G. Filippidis, C. Kouloumentas, D. Kapsokalyvas, G. Voglis, N. Tavernarakis, T. G. Papazoglou “Imaging of Caenorhabditis elegans samples and sub-cellular localization of new generation photosensitizers for Photodynamic Therapy, using non-linear microscopy ” submitted to Journal of Physics D: Applied Physics. Abstract: A compact, inexpensive and reliable set-up utilizing femtosecond pulses for excitation was developed for the Two-Photon Excitation Fluorescence (TPEF) and Second-Harmonic Generation (SHG) imaging of biological samples. We achieved high resolution imaging and mapping of Caenorhabditis elegans (C. elegans) neurons and muscular structures (pharynx), at the microscopic level by performing SHG and TPEF measurements. The identification of the endogenous structural proteins (actomyosin complexes and collagen), that are the main contributors for the observation of the high SHG signals, was managed.
PARTNER 2ULUND
1) Swartling J, Svensson J, Bengtsson D, Terike K, and Andersson-Engels S 2004 Fluorescence spectra provide information on the depth of fluorescent lesions in tissue. Applied Optics (accepted)
The measured fluorescence spectrum from a fluorophore in tissue is affected by the absorption and scattering properties of the tissue, as well as the measurement geometry. We analyze this effect with Monte Carlo simulations and by measurements on phantoms. The spectral changes can be used to estimate the depth of a fluorescent lesion embedded in the tissue by measuring the fluorescence signal in different wavelength bands. By taking the ratio between the signals at two wavelengths we show that it is possible to determine the depth of the lesion. Simulations were performed and validated by measurements on a phantom in the wavelength range 815-930 nm. The depth of a fluorescing layer could be determined with 0.6 mm accuracy down to at least a depth of 10 mm. Monte Carlo simulations were also performed for different tissue types with various composition. The results indicate that depth estimation of a lesion should be possible with 2-3 mm accuracy, with no assumptions made about the optical properties, for a wide range of tissues.
PARTNER 3UCLM .Schweiger, A. Soubret, V Ntziachristos, S.Arridge, (2004) Non-linear effects in Fluorescence Tomography, submitted to IEEE Transactions on Image Processing (special issue on Molecular Imaging) Abstract We investigate the performance of a nonlinear model-based reconstruction technique in imaging of fluorochrome concentration of optically thick objects consisting of a two-step strategy. In the first step, the distribution of inhomogeneous background absorption is reconstructed from intrinsic data obtained at the excitation wavelength, using a nonlinear iterative Gauss-Newton minimisation algorithm. The second step then recovers the distribution of fluorochrome concentration from measurements at the emission wavelength. This is a linear problem of inverting the Jacobian of the forward operator of the fluorescence light transport model. By using the absorption distribution recovered in the first step in the construction of the Jacobian, the distorting effect of inhomogeneous background absorption distributions on the recovery of the fluorescence image is eliminated. We demonstrate the performance of this reconstruction approach on experimental data obtained from phantom measurements.
PARTNER 4UKMPaper submitted for publication to the J.Inv.Radiol: C. Bremer, V. Ntziachristos, B. Weitkamp, G. Theilmeier, W. Heindel, R. Weissleder “Optical imaging of spontaneous breast tumors using protease sensing smart optical contrast agents” Abstract: Objectives: To determine if spontaneous breast cancer lesions can be detected by fluorescence reflectance imaging (FRI) and fluorescence mediated tomography (FMT) using protease-sensing optical probes. Materials and Methods: Transgenic (FVB/N-TgN (WapHRAS)69Lin YSJL)) mice that spontaneously develop carcinomas of the breast were i.v. injected with a cathepsin-sensing fluorescent probe. FRI and FMT were performed 24 hrs after contrast injection and region of interest (ROI) analysis was performed. MR images were acquired for anatomical co-registration with the FMT data. Moreover, correlative immunohistochemistry and fluorescence microscopy were performed. Results: All tumor nodules were clearly delineated by FRI showing an average signal intensity of 380 +- 106 AU. Fluorescence of the animal could be clearly localized to the tumor by FMT. In accordance with the imaging findings immunohistochemistry confirmed cathepsin-B expression of the tumors and fluorescence microscopy revealed a strong Cy 5.5. deposition in the tissue. Conclusions: FRI and FMT using smart protease sensing probes allow to detect experimental spontaneous breast cancers. Since the expression levels of various proteases correlate with patient outcome this technique may not only help to detect but also to differentiate breast cancers non-invasively.
PARTNER 5CRSA1) M. Thomas, J.-J. Greffet, R. Carminati and J.R. Arias-Gonzalez, 2004, "Single-molecule spontaneous emission close to absorbing nanostructures", Appl. Phys. Lett. vol. 85, 3863. Abstract : The spontaneous emission of a single molecule is substantially modified close to a metallic nanostructure. We study the spectral behavior of the radiative and non-radiative decay rates and of the local-field factor in the vicinity of a plasmon resonance. We show that the highest fluorescence enhancement is obtained for an emission wavelength red-shifted from the plasmon resonance, and that quenching always dominates at plasmon resonance. These results may have experimental implications in spectroscopy and monitoring of elementary light sources.
2) M. Thomas, J.-J. Greffet, R. Carminati, 2005, "Single-molecule fluorescence enhancement using metallic nanoparticles and tips", manuscript in preparation, to be submitted to Optics Express in february 2005.
3) R. Carminati, R. Elaloufi and J.-J. Greffet, 2004, "Beyond the diffusing-wave spectroscopy model for the temporal fluctuations of scattered light", Phys. Rev. Lett. vol. 92, 213903.
Abstract : We extend the theory of Diffusing-Wave Spectroscopy, using a random-walk approach and a numerical solution of the Radiative Transfer Equation. The theory is not restricted to the diffusive regime, and allows to describe the crossover between the single-scattering and the diffusive regimes which has been observed experimentally. It also predicts a lower bound of the scattered-field correlation time at long paths. This extended theory should have broad experimental applications in the field of imaging through biological tissues.
4) R. Elaloufi, R. Carminati and J.-J. Greffet, 2004, "Diffusive-to-ballistic transition in dynamic light transmission through thin scattering slabs: a radiative transfer approach", J. Opt. Soc. Am. A vol. 21, 1430.
Abstract : We study the deviation from diffusion theory which occurs in the dynamic transport of light through thin scattering slabs. Solving numerically the time-dependent. Radiative Transfer Equation, we obtain the decay time and the effective diffusion coefficient Deff. We observe a non-diffusive behavior for systems whose thickness L is smaller than 8 l*, where l* is the transport mean-free path. We introduce a simple model that yields the position of the transition between the diffusive and the non-diffusive regimes. The size dependence of Deff in the non-diffusive region is strongly affected by internal reflections. We show that the reduction of about 50% of Deff which was observed experimentally [Phys. Rev. Lett. vol. 79, 4369-4372 (1997)] can be reproduced by the radiative transfer approach. This study demonstrates that the Radiative Transfer Equation is an appropriate tool to study dynamic light transport in thin scattering systems, when coherent effects play no significant role.
PARTNER 6UAM1) G. M. Sacha, A. Verdaguer, J. Martinez, J. J. Saenz, F. Ogletree and M. Salmeron (2004). Effective tip radius in Electrostatic Force Microscopy. Applied Physics Letters (submitted) Abstract: A method to determine the effective electrostatic tip radius of arbitrarily shaped conducting tips in Atomic Force Microscopy is presented. The method is based on the finding that for conductive samples the electrostatic force can be separated into two contributions: one from a constant background that depends only on the macroscopic shape of the tip (cone or pyramid and cantilever), and another that depends only on the radius of curvature of the tip apex. Based on a simple theoretical expression derived from the Generalized Image Charge Method, we show that the tip radius can be directly determined from experimental force-distance characteristics. For irregular tip shapes, we show that the measured tip radius is the average of two principal curvatures, in agreement with tip shape images obtained by scanning electron microscopy.
2) Raquel Gomez-Medina and Juan Jose Saenz , “Strong Optical Interactions between Particles in Quasi-One-Dimensional Geometries”, Physical Review Letters 93 (2004), 243602 Abstract: A theoretical analysis of the optically induced interaction between small particles in a quasi-one-dimensional system is presented. The total reflection of light modes near a geometric resonance leads to strong radiation pressure on a single particle. The presence of the two particles splits the resonance leading to a nontrivial oscillating interaction. The existence of stable, optically bound dimers under two counterpropagating (noncorrelated) light modes is also discussed. PARTNER 7IMMBraga J, Desterro JM, Carmo-Fonseca M. 2004. Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes. Mol Biol Cell. 2004 Oct;15(10):4749-60 Abstract: Fluorescence recovery after photobleaching (FRAP) is a widely used tool for estimating mobility parameters of fluorescently tagged molecules in cells. Despite the widespread use of confocal laser scanning microscopes (CLSMs) to perform photobleaching experiments, quantitative data analysis has been limited by lack of appropriate practical models. Here, we present a new approximate FRAP model for use on any standard CLSM. The main novelty of the method is that it takes into account diffusion of highly mobile molecules during the bleach phase. In fact, we show that by the time the first postbleach image is acquired in a CLSM a significant fluorescence recovery of fast-moving molecules has already taken place. The model was tested by generating simulated FRAP recovery curves for a wide range of diffusion coefficients and immobile fractions. The method was further validated by an experimental determination of the diffusion coefficient of fluorescent dextrans and green fluorescent protein. The new FRAP method was used to compare the mobility rates of fluorescent dextrans of 20, 40, 70, and 500 kDa in aqueous solution and in the nucleus of living HeLa cells. Diffusion coefficients were lower in the nucleoplasm, particularly for higher molecular weight dextrans. This is most likely caused by a sterical hindrance effect imposed by nuclear components. Decreasing the temperature from 37 to 22 degrees C reduces the dextran diffusion rates by approximately 30% in aqueous solution but has little effect on mobility in the nucleoplasm. This suggests that spatial constraints to diffusion of dextrans inside the nucleus are insensitive to temperature. PARTNER 8ICNo publications during the first reporting period. PARTNER 9UALansbergen G, Komarova Y, Modesti M, Wyman C, Hoogenraad CC, Goodson HV, Lemaitre RP, Drechsel DN, Van Munster E, Gadella TW Jr, Grosveld F, Galjart N, Borisy GG, Akhmanova A. “Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization”. J Cell Biol. 166 (2004), 1003-1014 . Abstract: Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactin subunit p150(Glued) are structurally related proteins, which associate specifically with the ends of growing microtubules (MTs). Here, we show that down-regulation of CLIP-170 by RNA interference results in a strongly reduced accumulation of dynactin at the MT tips. The NH(2) terminus of p150(Glued) binds directly to the COOH terminus of CLIP-170 through its second metal-binding motif. p150(Glued) and LIS1, a dynein-associating protein, compete for the interaction with the CLIP-170 COOH terminus, suggesting that LIS1 can act to release dynactin from the MT tips. We also show that the NH(2)-terminal part of CLIP-170 itself associates with the CLIP-170 COOH terminus through its first metal-binding motif. By using scanning force microscopy and fluorescence resonance energy transfer-based experiments we provide evidence for an intramolecular interaction between the NH(2) and COOH termini of CLIP-170. This interaction interferes with the binding of the CLIP-170 to MTs. We propose that conformational changes in CLIP-170 are important for binding to dynactin, LIS1, and the MT tips. PARTNER 10-1MRC-NIMRNo publications during the first reporting period.
PARTNER 10-2MRC-HGUNo publications during the first reporting period.
PARTNER 11EMBL1. A paper describing the first small molecule FRET probe to monitor PLA2 activity and perform inside living cells will be submitted before March 1st. Title: ‘A small molecule FRET probe to monitor PLA2 activity in cells and organisms’. (Communication without abstract).
2. A paper describing the use of nile red-based red dyes to monitor lipid-protein interaction will be submitted within the next two months. Title: ‘Probing lipid and drug binding domains with fluorescent dyes’. Abstract: A series of 2-OH and 3-OH nile red dyes was prepared with the aim of generating water-soluble probes that could be used to probe lipid binding to proteins. Various substitutions in three positions x, y, and z, shifted wavelengths while maintaining the environmental sensitivity of nile red. In order to increase the solubility of the dyes in aqueous solutions, we attached butyric acid groups as well as other acyl groups to the 2- or 3-OH position. In addition, phenothiazine dyes, which exhibited particularly long excitation properties, were synthesized and tested for the first time. All dyes showed Stoke’s shifts of 70-100 nm and changes in excitation and emission of over 100 nm depending on the hydrophobicity of the environment. Binding studies with bovine and human serum albumin and the non-specific lipid transport protein SCP2, revealed emission changes of more than 30 nm upon binding to the protein and a five-fold increase in emission intensity. Titration of the dye-loaded proteins with various lipids or drugs replaced the dye and thereby reversed the shift in wavelength intensity. This allowed us to estimate the lipid binding affinity of the investigated proteins. For SCP2 calorimetric data verified the titration data. NMR titration of SCP2 with NRBA proved that the latter is binding to a lipid binding pocket and not to the protein surface. These results give valuable insight into lipid and drug transport by proteins outside and inside cells.
3. A concept paper, discussing novel possibilities for monitoring intracellular events in real time, simultaneously, is currently being prepared in a joint effort between the groups of Dorus Gadella (PARTNER 9UA) and Carsten Schultz (PARTNER 11EMBL). The title of this paper is 'Multiparameter analysis of intracellular signaling events', authors are :J. Goedhart, A. Schleifenbaum, T. Gadella, C. Schultz. This paper will be submitted to ChemBioChem early in January 2005. PARTNER 13ETHNo publications during the first reporting period. PARTNER 14IBCH1) Chudakov DM, Verkhusha VV, Staroverov DB, Souslova EA, Lukyanov S, Lukyanov KA Photoswitchable cyan fluorescent protein for protein tracking. Nature Biotechnology 22 (2004), 1435-1439. Abstract: In recent years diverse photolabeling techniques using green fluorescent protein (GFP)-like proteins have been reported, including photoactivatable PA-GFP, photoactivatable protein Kaede, the DsRed 'greening' technique and kindling fluorescent proteins. So far, only PA-GFP, which is monomeric and gives 100-fold fluorescence contrast, could be applied for protein tracking. Here we describe a dual-color monomeric protein, photoswitchable cyan fluorescent protein (PS-CFP). PS-CFP is capable of efficient photoconversion from cyan to green, changing both its excitation and emission spectra in response to 405-nm light irradiation. Complete photoactivation of PS-CFP results in a 1,500-fold increase in the green-to-cyan fluorescence ratio, making it the highest-contrast monomeric photoactivatable fluorescent protein described to date. We used PS-CFP as a photoswitchable tag to study trafficking of human dopamine transporter in living cells. At moderate excitation intensities, PS-CFP can be used as a pH-stable cyan label for protein tagging and fluorescence resonance energy transfer applications.
Non-refereed Journals:
2) Lukyanov KA (2004) Novel members of the Green Fluorescent Protein family. BIOforum Europe, 06/2004, 60-61. Abstract: Green Fluorescent Protein (GFP) from jellyfish Aequorea and its mutants are widely used in molecular and cellular biology as genetically encoded fluorescent tags. Several years ago a great spectral diversity of GFP-like proteins including yellow and red fluorescent proteins and purple-blue non-fluorescent chromoproteins was revealed in corals. Recent studies demonstrated that jellyfishes also contain a variety of color classes of GFP-like proteins. In particular, a yellow fluorescent protein and a non-fluorescent chromoprotein were characterized. Importantly, GFP homologs were found in copepods (crustaceans, phylum Arthropoda). This finding demonstrated a broad evolution diversity of this protein family. Novel fluorescent and chromoproteins often provide new useful spectral features that broads area of practical application of GFP-like proteins.
PARTNER 15UC3M1.) A. D. Kim and M. Moscoso, 2004, Light transport in two-layered tissues, submitted to Journal of Biomedical Optics.
Abstract: We study theoretically light backscattered by tissues using the radiative transport equation. In particular we consider a two-layered medium in which a finite slab is situated on top of a half space. We solve the one dimensional problem in which a plane wave is incident normally on the top layer and is the only source of light. The solution to this problem is obtained formally by imposing continuity between the solutions for the upper and lower layers. However, we are interested solely in probing the top layer. Assuming that the optical properties in the lower layer are known, we remove it from the problem yielding a finite slab problem by prescribing an alternate boundary condition. This boundary condition is derived using the theory of Green’s functions and is exact. Hence, one needs only to solve the transport equation in a finite slab using this alternate boundary condition. We derive an asymptotic solution for the case when the slab is optically thin. We extend these results to the three dimensional problem using Fourier transforms. These results are validated by comparisons with numerical solutions for the entire two-layered problem.
2.) O. Dorn, 2005, Shape reconstruction for an inverse radiative transfer problem arising in medical imaging, accepted for publication in Springer series 'Computational Science and Engineering', volume "numerical methods for multidimensional radiative transfer problems".
Abstract: A novel shape reconstruction method for medical imaging with near-infrared light is presented which uses adjoint fields and level sets. The propagation of photons in tissue is modeled by the time-dependent radiative transfer equation in 2D. In the shape reconstruction approach, it is assumed that the inhomogeneous background absorption parameter is known up to small random fluctuations. The background can consist of regions filled with diffusive material as well as of non-scattering regions. Inside the diffusive regions several unknown objects are imbedded with an intermediate to high contrast to the background in the absorption parameter. The goal is to reconstruct the exact shape, number and location of these obstacles from the boundary data. The method uses a level set technique for representing the unknown shapes, combined with adjoint fields for calculating shape derivatives. It is an iterative method which starts with some initial guess for the shapes, and constructs an artificial shape evolution which is designed to minimize a given cost functional during the evolution. Numerical experiments in 2D are presented which demonstrate that our method is able to reconstruct the shapes in a stable and efficient way.
3.) A. D. Kim and M. Moscoso, 2004, Beam propagation in sharply peaked forward scattering media, J. Opt. Soc. Am. A, Vol 21, pp 797-803.
Abstract: We calculate the radiance of a light beam propagating in a uniformly scattering and absorbing slab and determine the point-spread function. We do this by solving numerically the governing radiative transport equation by use of plane-wave mode expansions. When scattering is sharply peaked in the forward direction and it becomes difficult to solve the radiative transport equation, we replace it with either the Fokker-Planck or the Leakeas-Larsen equation. We also solve these equations by using plane-wave mode expansions. Numerical results show that these two equations agree with the radiative transport equation for large anisotropy factors. The agreement improves as the optical thickness increases.
4.) A D Kim and M. Moscoso, 2004, Backscattering of beams by forward-peaked scattering media, Optics Letters, Vol. 29 (1), pp. 74-76.
Abstract: For a beam impinging on a scattering medium the diffusion approximation to the radiative transport equation is not valid for analyzing the radiance near the source, especially if the medium scatters strongly with a sharp forward peak. To analyze the radiance, we use the Fokker-Planck approximation to the radiative transport equation. Numerical results show a backscattered ring appearing around the beam center. It also appears in Monte Carlo simulations of the radiative transport equation. This ring is manifested from successive near-forward scattering events, so it requires a directional description. Therefore the diffusion approximation cannot predict this ring. PARTNER 16-1 EMCR-I1) Lansbergen G, Komarova Y, Modesti M, Wyman C, Hoogenraad CC, Goodson HV, Lemaitre RP, Drechsel DN, Van Munster E, Gadella TW Jr, Grosveld F, Galjart N, Borisy GG, Akhmanova A. (2004) Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization. J Cell Biol. 166, 1003-1014. Abstract: Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactin subunit p150(Glued) are structurally related proteins, which associate specifically with the ends of growing microtubules (MTs). Here, we show that down-regulation of CLIP-170 by RNA interference results in a strongly reduced accumulation of dynactin at the MT tips. The NH(2) terminus of p150(Glued) binds directly to the COOH terminus of CLIP-170 through its second metal-binding motif. p150(Glued) and LIS1, a dynein-associating protein, compete for the interaction with the CLIP-170 COOH terminus, suggesting that LIS1 can act to release dynactin from the MT tips. We also show that the NH(2)-terminal part of CLIP-170 itself associates with the CLIP-170 COOH terminus through its first metal-binding motif. By using scanning force microscopy and fluorescence resonance energy transfer-based experiments we provide evidence for an intramolecular interaction between the NH(2) and COOH termini of CLIP-170. This interaction interferes with the binding of the CLIP-170 to MTs. We propose that conformational changes in CLIP-170 are important for binding to dynactin, LIS1, and the MT tips.
2)
Mimori-Kiyosue Y, Grigoriev I, Lansbergen G, Sasaki H, Matsui C, Severin F,
Galjart N, Grosveld F, Vorobjev I, Tsukita S, Akhmanova A.CLASP1 and CLASP2 bind
to EB1 and regulate microtubule plus-end dynamics at the cell cortex. J Cell
Biol. 2005 Jan 3;168(1):141-53.
3) van Haperen R et al., Functional expression of endothelial nitric oxide synthase fused to green fluorescent protein in transgenic mice.Am J Pathol. 2003 Oct;163(4):1677-86.
4) Cheng C, et al., The role of shear stress in atherosclerosis: action through gene expression and inflammation? Cell Biochem Biophys. 2004;41:279-94. PARTNER 16-2EMCR-II1) Beerens, N., J.H.J. Hoeijmakers, R. Kanaar, W. Vermeulen, and C. Wyman. (2004) The CSB protein actively wraps DNA. J. Biol. Chem. Online Nov. 16 M409147200. Abstract: The CSB protein is a member of the SWI2/2SNF2 family of ATP-dependent chromatin remodeling factors and is essential for transcription-coupled DNA repair. The role of CSB in this DNA repair process is unclear, but the protein was found to remodel nucleosomes, and alters DNA double helix conformation upon binding. Elucidating the nature of the change in DNA structure induced by CSB is of great interest for understanding the CSB mechanism of action. We analyzed the CSB-DNA complex by scanning force microscopy and measured a shortening of DNA contour length upon CSB binding in the presence of ATP. This DNA length reduction most likely results from DNA wrapping around the protein. Shorter DNA molecules were observed more frequently in the presence of non-hydrolysable ATP analogues. These results suggest that DNA wrapping depends on ATP-binding, whereas ATP hydrolysis results in unwrapping. We also provide evidence suggesting that CSB binds DNA as a dimer. DNA wrapping and unwrapping allows CSB to actively alter the DNA double helix conformation, which could influence nucleosomes and other protein-DNA interactions.
2) Wyman, C., R. Kanaar (2004) Homologous recombination: down to the wire. Current Biology, 14, R629-31. Abstract: Exchange of strands between homologous DNA molecules is catalyzed by evolutionarily conserved recombinases. These proteins can occur in different quaternary arrangements: rings or helical filaments. Recent results reveal that recombinase function follows from the filamentous form.
3) Essers, J., W.A. van Cappellen, A.F. Theil, E . van Drunen, N.G. Jaspers, J.H. Hoeijmakers, C. Wyman, W . Vermeulen, and R. Kanaar. (2004) Dynamics of Relative Chromosome Position during the Cell Cycle. Mol. Biol. Cell. On line Dec. 1. Abstract: The position of chromosomal neighborhoods in living cells was followed using three different methods for marking chromosomal domains occupying arbitrary locations in the nucleus; photo-bleaching of GFP-labeled histone H2B, local UV-marked DNA and photo-bleaching of fluorescently labeled DNA. All methods revealed that global chromosomal organization can be reestablished through one cell division from mother to daughters. By simultaneously monitoring cell cycle stage in the cells in which relative chromosomal domain positions were tracked, we observed that chromosomal neighborhood organization is apparently lost in the early G1 phase of the cell cycle. However, the daughter cells eventually regain the general chromosomal organization pattern of their mothers suggesting an active mechanism could be at play to reestablish chromosomal neighborhoods.
4) Wyman, C., D. Ristic and R. Kanaar. (2004) Homologous recombination-mediated double-strand break repair. DNA Repair, 3, p. 827-33. Abstract: Exchange of DNA strands between homologous DNA molecules via recombination ensures accurate genome duplication and preservation of genome integrity. Biochemical studies have provided insights into the molecular mechanisms by which homologous recombination proteins perform these essential tasks. More recent cell biological experiments are addressing the behavior of homologous recombination proteins in cells. The challenge ahead is to uncover the relationship between the individual biochemical activities of homologous recombination proteins and their coordinated action in the context of the living cell.
5) De Jager, M., K.M. Trujillo, P. Sung, K-P. Hopfner, J.P. Carney, J.A. Tainer, J.C. Connelly, D.R.F. Leach, R. Kanaar, and C. Wyman. (2004) Differential arrangements of conserved building blocks among homologs of the SMC family member and DNA repair protein Rad50/Mre11. J. of Molecular Biology, 339, 937-949. Cover illustration. Abstract: Structural maintenance of chromosomes (SMC) proteins have diverse cellular functions including chromosome segregation, condensation and DNA repair. They are grouped based on a conserved set of distinct structural motifs. All SMC proteins are predicted to have a bipartite ATPase domain that is separated by a long region predicted to form a coiled coil. Recent structural data on a variety of SMC proteins shows them to be arranged as long intramolecular coiled coils with a globular ATPase at one end. SMC proteins function in pairs as heterodimers or as homodimers often in complexes with other proteins. We expect the arrangement of the SMC protein domains in complex assemblies to have important implications for their diverse functions. We used scanning force microscopy imaging to determine the architecture of human, Saccharomyces cerevisiae, and Pyrococcus furiosus Rad50/Mre11, Escherichia coli SbcCD, and S.cerevisiae SMC1/SMC3 cohesin SMC complexes. Two distinct architectural arrangements are described, based on the way their components were connected. The eukaryotic complexes were similar to each other and differed from their prokaryotic and archaeal homologs. These similarities and differences are discussed with respect to their diverse mechanistic roles in chromosome metabolism.
PARTNER 17-1CNBNo publications during the first reporting period. PARTNER 17-2 ICMMM. Nieto-Vesperinas, P.C. Chaumet and A. Rahmani, "Near Field Photonic Forces", Philosophical Transactions of the Royal Society of London A 362, 697 (2004). Abstract: A review of recent advancements in photonic forces is presented. We discuss in detail the interaction of light and sub-wavelength particles on a substrate illuminated by total internal reflection, and we study the optical forces experienced by the particles. The effects of plasmon-mode excitations on the resulting photonic forces on metallic particles are also addressed. Moreover, we explore the possibility of using the metallic tip of a classical apertureless microscope to create optical tweezers, and thus to achieve a selective manipulation of nanoparticles. PARTNER 17-3LFSPM. Bai, C.Guerrero, S. Ioanid, E. Paz, M. Sanz and N. Garcia 2004 Measuring the speed of a surface plasmon in Phy. Rev. B 69, 115416 Abstract This paper presents experiments using an 800-nm pulsed laser and a streak camera with 2-ps resolution, which is implemented with a two-fiber differential measurements permitting a 0.2-ps resolution. With this technique, we measure the speed of the propagation of the surface plasmons. This velocity was found to be approximately (0.57±0.19)c, about 61% the value expected from simple theory (i.e., about 0.94c). The former result may clarify what is going on at the metal vacuum interface excitations, and has been measured by a direct method without the need for any theory. PARTNER 17-4 DIONo publications during the first reporting period. PARTNER 18-1KIPNo publications during the first reporting period. PARTNER 18-2UHEINo publications during the first reporting period. PARTNER 19PHOTEKNo publications during the first reporting period. PARTNER 20PKLASNo publications during the first reporting period. PARTNER 21LENSLETNo publications during the first reporting period. PARTNER 22KENTECHNo publications during the first reporting period.
Conference Proceedings
PARTNER 1FORTH1) Heiko Meyer, Anikitos Garofalakis, Giannis Zacharakis, Eleftherios N. Economou, Clio Mamalaki, Sifis Papamatheakis, Vasilis Ntziachristos and Jorge Ripoll, “A multi-projection non-contact Tomography setup for imaging arbitrary geometries”, SPIE Proc. Saratov Fall Meeting, 2004.
Abstract: Optical imaging and tomography in tissues can facilitate the quantitative study of several important chromophores and fluorophores in-vivo. Due to this fact, there has been great interest in developing imaging systems offering quantitative information on the location and concentration of chromophores and fluorescent probes. However, most imaging systems currently used in reasearch make use of fiber technology for delivery and detection, which restricts the size of the photon collecting arrays leading to insufficient spatial sampling and field of view. To enable large data sets and full 360o angular measurements, we developed a novel imaging system that enables 3D imaging of fluorescent signals in bodies of arbitrary shapes in a non-contact geometry in combination with a 3D surface reconstruction algorithm. The system is appropriate for in-vivo small animal imaging of fluorescent probes. The system consists of a rotating sample holder and a lens coupled CCD camera in combination with a fiber coupled scanning device. The accuracy of the system in obtaining the surface reconstruction was measured to be in the order of 1mm .
2) Heiko Meyer, Anikitos Garofalakis, Giannis Zacharakis, Eleftherios N. Economou, Clio Mamalaki, Vasilis Ntziachristos and Jorge Ripoll, Optical Tomography and Spectroscopy of Tissue VII, SPIE proceedings on Biomedical Optics, BiOS 2005.
Abstact: Optical imaging and tomography in tissues can facilitate the quantitative study of several important chromophores in-vivo. Due to this fact, there have been great interests in developing imaging systems offering quantitative information on the location and concentration of chromophores and fluorescent probes. Due to the simplicity of currently available optical imaging systems, which are quite well suited for basic feasibility studies, they come with significant limitations. Using fiber-based signal detection restricts the size of the photon collecting arrays which may lead to insufficient spatial matching and field of view. We developed a novel imaging system that enables 3D imaging of fluorescent signals in bodies of arbitrary shapes in a non-contact geometry in combination with a 3D surface reconstruction algorithm. The system is appropriate for in-vivo small animal imaging of fluorescent probes. The system consists of a rotating sample holder and a lens coupled CCD camera in combination with a fiber coupled scanning device. The accuracy of the system was measured to be less than 1mm, while the limit of detected cells was measured to be 50,000 cells (expressing GFP). This resolution is due to the large dataset of >106 measurements employed and the use of inversion models which gives in combination of the novel 3D data reconstruction a high capacity of the system to quantitatively reconstruct 3D fluorochrome distributions and spatial resolution. The developed system is already in use for the observation of the distribution of GFP expressing T-cells to study the function of the immune system of mice, which can then be related to the human immune system. PARTNER 2ULUND
1) Svensson J, Somesfalean G, Johansson A, and Andersson-Engels S 2004 A comparison of diagnostic fluorescence point and hyperspectral imaging spectroscopy - geometry effects, OSA Biomedical Optics, Miami, FL, USA Abstract: Fluorescence spectra have been measured at different distances from a pencil-beam excitation spot using two diagnostic instruments. These spectra have been compared to calculated theoretical data with fluorescence Monte Carlo.
2) Swartling J, Bengtsson D, Terike K, Svensson J, and Andersson-Engels S 2004 Localization of fluorophore depth in tissue fromn changes in fluorescence spectra. OSA Biomedical Optics, Miami, FL, USA Abstract: We present a novel method for determining the depth of a localized fluorophore in tissue, based on changes in the fluorescence spectrum, because of tissue absorption. Ratios of wavelength bands correlate with the depth.
PARTNER 3UCLNo conference proceedings during the first reporting period.
PARTNER 4UKMNo conference proceedings during the first reporting period.
PARTNER 5CRSA1) M. Thomas, J. Bourguignon, M. Laroche, R. Carminati and J.-J. Greffet, 2005, "Electromagnetic modes of a linear chain of metallic nanoparticles", submitted to the proceeding of CLEO/QELS Conference 2005 (Baltimore 2005).
2) R. Carminati, J.-J. Greffet and R. Elaloufi, 2005, "Beyond the difusing-wave spectroscopy model", submitted to the proceeding of CLEO/QELS Conference 2005 (Baltimore 2005).
Abstract : We extend the theory of Diffusing-Wave Spectroscopy using a random-walk approach and the Radiative Transfer Equation. The transition between the transport regimes, observed experimentally, is well reproduced, increasing its potential for biological imaging.
3) R. Pierrat, J.-J. Greffet, R. Carminati and R. Elaloufi, "Spatial coherence of a light beam in a turbid medium", submitted to the proceeding of CLEO/QELS Conference 2005 (Baltimore 2005).
Abstract: We study the optical spatial coherence in a turbid medium using the Radiative Transfer Equation. The coherence length of the diffuse light strongly depends on the transport regime. Implications for biological imaging are discussed.
PARTNER 6UAMNo conference proceedings during the first reporting period.
PARTNER 7IMMBraga J, Desterro JM, Carmo-Fonseca M. (2004) “Introducing three-dimensional information on quantitative fluorescence recovery after photobleaching experiments”, Biophys J. 86 (1): 604A-604A Part 2 Suppl. S, JAN 2004 Abstract: Fluorescence recovery after photobleaching (FRAP) is widely used tool for estimating mobility parameters of fluorescently tagged proteins. To date many quantitative analysis of FRAP experiments rely on a model proposed by Axelrod in 1976. However, some of the assumptions of this model are inappropriate when FRAP is performed with a confocal laser scanning microscope (CLSM) resulting in an incorrect estimation of diffusion coefficients. We present a new method which accounts for the three-dimensional profile of the bleaching laser. We tested this model by solving numerically a modified diffusion equation which accounts for the diffusion during bleach generating simulated FRAP recovery curves for wide a range of diffusion coefficients and immobile fractions. Further validation of the method was done by carrying an experimental determination of the diffusion coefficient of fluorescent dextrans of 20, 40, 70 and 500 KDa. Dextrans were analyzed in an aqueous solution and microinjected HeLa cell nucleus at either 37?C or 22?C. The new method yielded diffusion coefficient values in close agreement with predictions from the Stokes-Einstein equation. The data suggests that the viscosity of the nucleoplasm is approximately 8 times higher than the viscosity of water for molecules of the same size, which is higher than previously reported values.
PARTNER 8ICNo publications during the first reporting period. PARTNER 9UA1) Gadella, Jr., T.W.J., E. van Munster, P. Dhonukshe, M. Adjobo-Hermans, J. Vermeer, G.-J. Kremers and J. Goedhart (2004) “Imaging cytoskeleton and signalling dynamics in live cells with GFP-based 4D-microscopy, FRET and FLIM”. 13th European Microscopy Congress, Antwerp, Belgium (Abstract and Invited Plenary Speaker) August 2004. Abstract: By introducing chimeric proteins containing green fluorescent protein spectral variants fused to signalling molecules into live cells and analyzing the properties of the fluorescence light emitted by these molecules with new micro(spectro)scopic techniques, it is possible to monitor molecular conformation, -complex formation, and signalling dynamics with high spatiotemporal resolution. The micro(spectro)scopic techniques include multichannel confocal microscopy, spinning-disk confocal microscopy, fluorescence spectral imaging microscopy (FSPIM), and fluorescence lifetime imaging microscopy (FLIM). FSPIM and FLIM were used for analyzing fluorescence resonance energy transfer (FRET) between (or within) fusion proteins containing spectral variants of GFP, enabling monitoring of both protein conformational changes and protein-protein interactions at the subcellular level in living cells. Using FRET-FLIM and FRET-SPIM enabled us to monitor heterodimerization of CFP- and YFP-tagged MADS-box transcription factors in nuclei of single living Petunia protoplasts. FRET-FLIM/SPIM thereby offers an in planta alternative for the yeast-two-hybrid-screen for protein-protein interactions. With confocal 4D-imaging we were able to monitor dynamic instability events in living plant cells for the first time and we were able to show that microtubules are attached to the membrane by a novel mechanism involving phospholipase D forming a covalent intermediate with membane lipids. Lipid-protein interactions and lipid signaling events were monitored by using specific lipid-binding domains fused to GFP. Following this approach, signalling-induced appearance and disappearance of specific (phospho)lipids (e.g. phosphatidylinositol(4,5)-bisphosphate) could be monitored directly by confocal microscopy with high spatial and temporal resolution in single living cells, see next page figure 1. This routinely enables us to study spatially resolved hormone-stimulated phospholipid-signalling.
2) Gadella jr., T.WJ. (2004) Imaging signalling across the plasma membrane in single living cells using multimode fluorescence microscopy. Royal Microscopical Society Cell Biology Meeting 2004 on new Frontiers in Cancer Cell Imaging, Amsterdam (Abstract and invited speaker), March 2004 Abstract: The green fluorescent protein revolution in cell biology enables the study of the dynamics of signaling reactions in real time in single cells. We use fluorescent biosensors that are designed to specifically visualize lipid-signaling in single cells. Using different protein domains that highly specifically bind different phospholipids molecules and fusing those to spectral variants of GFP, enabled us to visualize the degradation of phosphatidylinositol(4,5)bisphospate (PtdIns(4,5)P2), the production of diacylglycerol (DAG) and calcium release by inositol-trisphosphate (IP3) in one living cell simultaneously. In order to further understand the importance of localized signaling processes, caged derivatives of phospholipids have been synthesized. Upon UV-induced photolysis, it can be shown that these phospholipids can serve as second messengers in their own right.
PARTNER 10-1MRC-NIMRNo conference proceedings during the first reporting period.
PARTNER 10-2MRC-HGUNo conference proceedings during the first reporting period. PARTNER 11EMBLImprovements and applications of the PKC reporter KCP-1 A. Schleifenbaum, A. Gasch, G. Stier, M. Sattler, C. Schultz ASCB 44th annual meeting, Washington DC, USA, Dec. 2004. Abstract 721. PARTNER 13ETHNo conference proceedings during the first reporting period. PARTNER 14IBCHNo conference proceedings during the first reporting period.
PARTNER 15UC3MNo conference proceedings during the first reporting period.
PARTNER 16-1 EMCR-INo conference proceedings during the first reporting period.
PARTNER 16-2EMCR-IINo conference proceedings during the first reporting period. PARTNER 17-1CNBNo conference proceedings during the first reporting period. PARTNER 17-2 ICMMNo conference proceedings during the first reporting period. PARTNER 17-3LFSPNo conference proceedings during the first reporting period. PARTNER 17-4 DIONo conference proceedings during the first reporting period. PARTNER 18-1KIPNo conference proceedings during the first reporting period. PARTNER 18-2UHEINo conference proceedings during the first reporting period. PARTNER 19PHOTEKSeptember 2004, Alexandria USA. SPIE Conference of High Speed Photography and Photonics Title: Picosecond Time Response Characteristics of Microchannel Plate PMT Detectors Authors: J. Milnes and J. Howorth. Ref: 5580-89.
Abstract: The output pulse width in the time response of photo-multiplier tubes (PMT) is much faster in micro-channel plate (MCP) models compared to more standard dynode chain PMTs due to a vastly reduced variation in the path length of the electrons through the amplifying system. Typically the pulse widths can be in the region of 200ps compared to the nanosecond domain occupied by the best conventional PMTs. Photek manufacture PMTs with one, two or three MCPs depending on the gain required, and also use the same structure without any MCPs to work as simple photodiodes. We demonstrate the variation of output pulse characteristics due to the number and design of MCPs in a range of PMT models and illustrate the importance of having a properly designed 50 ohm transmission line to deliver the pulse from the detector. PARTNER 20PKLASNo conference proceedings during the first reporting period. PARTNER 21LENSLETNo conference proceedings during the first reporting period. PARTNER 22KENTECHNo conference proceedings during the first reporting period. |
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