Khaled Khairy | European Molecular Biology Laboratory (original) (raw)

Papers by Khaled Khairy

Journal of Physical Chemistry A, 2006

A number of groups have utilized molecular dynamics (MD) to calculate slow-motional electron para... more A number of groups have utilized molecular dynamics (MD) to calculate slow-motional electron paramagnetic resonance (EPR) spectra of spin labels attached to biomolecules. Nearly all such calculations have been based on some variant of the trajectory method introduced by Robinson, Slutsky and Auteri (J. Chem. Phys. 1992,96, 2609-2616). Here we present an alternative approach that is specifically adapted to the diffusion operator-based stochastic Liouville equation (SLE) formalism that is also widely used to calculate slow-motional EPR line shapes. Specifically, the method utilizes MD trajectories to derive diffusion parameters such as the rotational diffusion tensor, diffusion tilt angles, and expansion coefficients of the orienting potential, which are then used as direct inputs to the SLE line shape program. This approach leads to a considerable improvement in computational efficiency over trajectory-based methods, particularly for high frequency, high field EPR. It also provides a basis for deconvoluting the effects of local spin label motion and overall motion of the labeled molecule or domain: once the local motion has been characterized by this approach, the label diffusion parameters may be used in conjunction with line shape analysis at lower EPR frequencies to characterize global motions. The method is validated by comparison of the MD predicted line shapes to experimental high frequency (250 GHz) EPR spectra.

Journal of Magnetic Resonance, 2006

A comparison between the full Newton-type optimization NL2SNO, the Levenberg-Marquardt method wit... more A comparison between the full Newton-type optimization NL2SNO, the Levenberg-Marquardt method with the model-trust region modification, and the simplex algorithm is made in the context of the iterative fitting of EPR spectra. EPR lineshape simulations are based on the stochastic Liouville equation (SLE), with an anisotropic diffusion tensor and an anisotropic restraining potential describing the motional amplitude of the spin label. The simplex algorithm was found to be the most reliable, and an approach-incorporating both NL2SNO as well as the downhill simplex methods-is proposed as a strategy-of-choice.

Biophysical Journal, 2009

]. In this previous work, MD simulations were performed using the AMBER suite of programs, the al... more ]. In this previous work, MD simulations were performed using the AMBER suite of programs, the all-atom AMBER99 force field, the particle mesh Ewald (PME) method for treating long-range electrostatic interactions, and the SPC/E water model. In order to test the effect of the water model used in the AMBER-based molecular dynamics simulations, calculations for a small nitroxide [3-hydroxymethyl-(1-oxy-2,2,5,5-tetramethylpyrroline) or 3MeOHSL] are being performed using a variety of different water models (SPC/E, TIP3P, TIP4P). The EPR spectra calculated from these molecular dynamics simulations of 3MeOHSL will be compared to experimental data.

Microscopy Research and Technique, 2004

The atomic force microscope (AFM) is introduced as a biomolecular manipulation machine capable of... more The atomic force microscope (AFM) is introduced as a biomolecular manipulation machine capable of assembling biological molecules into well-defined molecular structures. Native collagen molecules were mechanically directed into well-defined, two-dimensional templates exhibiting patterns with feature sizes ranging from a few nanometers to several hundreds of micrometers. The resulting nanostructured collagen matrices were only ϳ3-nm thick, exhibited an extreme mechanical stability, and maintained their properties over the time range of several months. Our results directly demonstrate the plasticity of biological assemblies and provide insight into the physical mechanisms by which biological structures may be organized by cells in vivo. These nanoscopic templates may serve as platforms on non-biological surfaces to direct molecular and cellular processes.

Cellular and Molecular Bioengineering, 2008

Cells and organelles are shaped by the chemical and physical forces that bend cell membranes. The... more Cells and organelles are shaped by the chemical and physical forces that bend cell membranes. The human red blood cell (RBC) is a model system for studying how such forces determine cell morphology. It is thought that RBCs, which are typically biconcave discoids, take the shape that minimizes their membrane-bending energies, subject to the constraints of fixed area and volume. However, recently it has been hypothesized that shear elasticity arising from the membraneassociated cytoskeleton (MS) is necessary to account for shapes of real RBCs, especially ones with highly curved features such as echinocytes. In this work we tested this hypothesis by following RBC shape changes using spherical harmonic series expansions of theoretical cell surfaces and those estimated from 3D confocal microscopy images of live cells. We found (i) quantitative agreement between shapes obtained from the theoretical model including the MS and real cells, (ii) that weakening the MS, by using urea (which denatures spectrin), leads to the theoretically predicted gradual decrease in spicule number of echinocytes, (iii) that the theory predicts that the MS is essential for stabilizing the discocyte morphology against changes in lipid composition, and that without it, the shape would default to the elliptocyte (a biconcave ellipsoid), (iv) that we were able to induce RBCs to adopt the predicted elliptocyte morphology by treating healthy discocytes with urea. The latter observation is consistent with the known connection between the blood disease hereditary elliptocytosis and spectrin mutations that weaken the cell cortex. We conclude that while the discocyte, in absence of shear, is indeed a minimum energy shape, its stabilization in healthy RBCs requires the MS, and that elliptocytosis can be explained based on purely mechanical considerations.

Medical Image Analysis, 2008

Numerical deconvolution of 3D fluorescence microscopy data yields sharper images by reversing the... more Numerical deconvolution of 3D fluorescence microscopy data yields sharper images by reversing the known optical aberrations introduced during the acquisition process. When additional prior information such as the topology and smoothness of the imaged object surface is available, the deconvolution can be performed by fitting a parametric surface directly to the image data. In this work, we incorporate such additional information into the deconvolution process and focus on a parametric shape description suitable for the study of organelles, cells and tissues. Such membrane-bound closed biological surfaces are often topologically equivalent to the sphere and can be parameterized as series expansions in spherical harmonic functions (SH). Because image data are noisy and the SH-parameterization is prone to the formation of high curvatures even at low expansion orders, the parametric deconvolution problem is ill-posed and must be regularized. We use the shape bending energy as a regularizing (smoothing) function, and determine the regularization parameter graphically with the help of the L-curve method. We demonstrate the complete deconvolution scheme, including the initial image segmentation, the calculation of a good starting surface and the construction of the L-curve, using real and synthetic image data.

American Journal of Physics, 2010

We define four complex numbers representing the parameters needed to specify an elephantine shape... more We define four complex numbers representing the parameters needed to specify an elephantine shape. The real and imaginary parts of these complex numbers are the coefficients of a Fourier coordinate expansion, a powerful tool for reducing the data ...

Journal of Molecular Biology, 2005

Collagen fibres within the extracellular matrix lend tensile strength to tissues and form a funct... more Collagen fibres within the extracellular matrix lend tensile strength to tissues and form a functional scaffold for cells. Cells can move directionally along the axis of fibrous structures, in a process important in wound healing and cell migration. The precise nature of the structural cues within the collagen fibrils that can direct cell movement are not known. We have investigated the structural features of collagen that are required for directional motility of mouse dermal fibroblasts, by analysing cell movement on two-dimensional collagen surfaces. The surfaces were prepared with aligned fibrils of collagen type I, oriented in a predefined direction. These collagen-coated surfaces were generated with or without the characteristic 67 nm D-periodic banding. Quantitative analysis of cell morphodynamics showed a strong correlation of cell elongation and motional directionality with the orientation of D-periodic collagen microfibrils. Neither directed motility, nor cell body alignment, was observed on aligned collagen lacking D-periodicity, or on D-periodic collagen in the presence of peptide containing an RGD motif. The directional motility of fibroblast cells on aligned collagen type I fibrils cannot be attributed to contact guidance, but requires additional structural information. This allows us to postulate a physiological function for the 67 nm periodicity.

PLOS One, 2010

Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generati... more Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generating complexes that are anchored at the plasma membrane and that pull on microtubules growing out from the spindle poles. Although asymmetric distribution of the force generators is thought to underlie asymmetric positioning of the spindle, the number and location of the force generators has not been well defined. In particular, it has not been possible to visualize individual force generating events at the cortex. We discovered that perturbation of the acto-myosin cortex leads to the formation of long membrane invaginations that are pulled from the plasma membrane toward the spindle poles. Several lines of evidence show that the invaginations, which also occur in unperturbed embryos though at lower frequency, are pulled by the same force generators responsible for spindle positioning. Thus, the invaginations serve as a tool to localize the sites of force generation at the cortex and allow us to estimate a lower limit on the number of cortical force generators within the cell.

PLOS One, 2010

Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generati... more Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generating complexes that are anchored at the plasma membrane and that pull on microtubules growing out from the spindle poles. Although asymmetric distribution of the force generators is thought to underlie asymmetric positioning of the spindle, the number and location of the force generators has not been well defined. In particular, it has not been possible to visualize individual force generating events at the cortex. We discovered that perturbation of the acto-myosin cortex leads to the formation of long membrane invaginations that are pulled from the plasma membrane toward the spindle poles. Several lines of evidence show that the invaginations, which also occur in unperturbed embryos though at lower frequency, are pulled by the same force generators responsible for spindle positioning. Thus, the invaginations serve as a tool to localize the sites of force generation at the cortex and allow us to estimate a lower limit on the number of cortical force generators within the cell.

Biophysical Journal, 2009

three dimensional (3D) resolution. In this study, both pure and fluorescently labeled (3% by weig... more three dimensional (3D) resolution. In this study, both pure and fluorescently labeled (3% by weight) collagen gels are assembled over a range of concentrations (0.1, 0.5, 1.0, 2.0, 5.0, and 5.5 mg/mL) and at various temperatures (37, 32, 27, and 22 C). The networks are investigated using confocal reflectance microscopy (CRM) and confocal fluorescence microscopy (CFM). Comparison between CRM and CFM reveals that they are not equally sensitive to details of network structure, with CRM (CFM) displaying higher sensitivity to fibers perpendicular (parallel) to the optical axis. Furthermore, analysis of background signal in CFM images suggests the existence of small fibrillar structures that are not resolved by CRM. Despite these differences, image analyses performed on 2D slices of CFM and CRM images to quantify mesh size, number of fibers, and fiber length reveal identical trends as a function of gel concentration and gelation temperature. Fiber width approximated from both CRM and CFM is in good accord with fiber width determination using electron microscopy. Overall network structures (as quantified via mesh size, fiber number, fiber length and fiber width) are related to bulk mechanical properties measured by rheology. Finally, we demonstrate the ability to form collagen gels of varied mesh size at identical collagen concentration, all of which are compatible with cell health and 3D cell studies. Tuberculosis is a disease that infects one in three humans today. The long, expensive drug course required to cure the active form, combined with its increasing resistance to conventional antibiotic treatment, necessitates the development of a new class of TB drugs. To evaluate the efficacy of these drugs, as well as to gain increased basic knowledge of the disease's progression, we are building a novel 0.8 mm diameter bronchoscope as part of a multi-institutional initiative to develop a primate-based tuberculosis model system. In order to monitor the in vivo microenvironment of the tuberculosis granuloma, monkeys will be inoculated with transgenic pH reporting tuberculosis bacilli and GFP expression measured to quantify the local pH and other micro-environmental parameters. CT scans will be used to reveal the induced nodules/lesions and guide the bronchoscope to the granulomas. The lung tissue itself contains many 488 nm excitable endogenous fluorophores (e.g. elastin, collagen) and autofluorescence limits the level of reporter quantification. To overcome this problem we are employing a photoactivatable protein (Dronpa) as the reporter expressed by the bacteria. Using a novel pulsed UV/Blue non-laser light source, the protein's fluorescence can be modulated to distinguish reporter signal from the constant autofluorescence background and therefore produce highly quantitative measurements of changes in the granuloma microenvironment during the progression of the disease and during drug treatment. (Supported by the Gates Foundation and NIH/NIBIB P41 RR04224 to WRZ.)

Current Opinion in Genetics & Development, 2011

Light sheet-based fluorescence microscopy (LSFM) is emerging as a powerful imaging technique for ... more Light sheet-based fluorescence microscopy (LSFM) is emerging as a powerful imaging technique for the life sciences. LSFM provides an exceptionally high imaging speed, high signal-to-noise ratio, low level of photo-bleaching and good optical penetration depth. This unique combination of capabilities makes light sheet-based microscopes highly suitable for live imaging applications. There is an outstanding potential in applying this technology to the quantitative study of embryonic development. Here, we provide an overview of the different basic implementations of LSFM, review recent technical advances in the field and highlight applications in the context of embryonic development. We conclude with a discussion of promising future directions.

We address the problem of segmenting 3D microscopic volumetric intensity images of a collection o... more We address the problem of segmenting 3D microscopic volumetric intensity images of a collection of spatially correlated objects (such as fluorescently labeled nuclei in a tissue). This problem arises in the study of tissue morphogenesis where cells and cellular components are organized in accord with biological role and fate. We formulate the image model as stochastically generated based on biological priors and physics of image formation. We express the segmentation problem in terms of Bayesian inference and use data-driven Markov Chain Monte Carlo to fit the image model to data. We perform an initial step in which the intensity volume is approximated as an expansion in 4D spherical harmonics, the coefficients of which capture the general organization of objects. Since cell nuclei are membrane-bound their shapes are subject to membrane lipid bilayer bending energy, which we use to constrain individual contours. Moreover, we parameterize the nuclear contours using spherical harmonic functions, which provide a shape description with no restriction to particular symmetries. We demonstrate the utility of our approach using synthetic and real fluorescence microscopy data.

Nature Methods, 2010

Articles nAture methods | ADVANCE ONLINE PUBLICATION | recording light-microscopy images of large... more Articles nAture methods | ADVANCE ONLINE PUBLICATION | recording light-microscopy images of large, nontransparent specimens, such as developing multicellular organisms, is complicated by decreased contrast resulting from light scattering. early zebrafish development can be captured by standard light-sheet microscopy, but new imaging strategies are required to obtain high-quality data of late development or of less transparent organisms. We combined digital scanned laser light-sheet fluorescence microscopy with incoherent structuredillumination microscopy (dslm-si) and created structuredillumination patterns with continuously adjustable frequencies. our method discriminates the specimen-related scattered background from signal fluorescence, thereby removing out-offocus light and optimizing the contrast of in-focus structures. dslm-si provides rapid control of the illumination pattern, exceptional imaging quality and high imaging speeds. We performed long-term imaging of zebrafish development for 58 h and fast multiple-view imaging of early Drosophila melanogaster development. We reconstructed cell positions over time from the Drosophila dslm-si data and created a fly digital embryo.

Journal of Physical Chemistry A, 2006

A number of groups have utilized molecular dynamics (MD) to calculate slow-motional electron para... more A number of groups have utilized molecular dynamics (MD) to calculate slow-motional electron paramagnetic resonance (EPR) spectra of spin labels attached to biomolecules. Nearly all such calculations have been based on some variant of the trajectory method introduced by Robinson, Slutsky and Auteri (J. Chem. Phys. 1992,96, 2609-2616). Here we present an alternative approach that is specifically adapted to the diffusion operator-based stochastic Liouville equation (SLE) formalism that is also widely used to calculate slow-motional EPR line shapes. Specifically, the method utilizes MD trajectories to derive diffusion parameters such as the rotational diffusion tensor, diffusion tilt angles, and expansion coefficients of the orienting potential, which are then used as direct inputs to the SLE line shape program. This approach leads to a considerable improvement in computational efficiency over trajectory-based methods, particularly for high frequency, high field EPR. It also provides a basis for deconvoluting the effects of local spin label motion and overall motion of the labeled molecule or domain: once the local motion has been characterized by this approach, the label diffusion parameters may be used in conjunction with line shape analysis at lower EPR frequencies to characterize global motions. The method is validated by comparison of the MD predicted line shapes to experimental high frequency (250 GHz) EPR spectra.

Journal of Magnetic Resonance, 2006

A comparison between the full Newton-type optimization NL2SNO, the Levenberg-Marquardt method wit... more A comparison between the full Newton-type optimization NL2SNO, the Levenberg-Marquardt method with the model-trust region modification, and the simplex algorithm is made in the context of the iterative fitting of EPR spectra. EPR lineshape simulations are based on the stochastic Liouville equation (SLE), with an anisotropic diffusion tensor and an anisotropic restraining potential describing the motional amplitude of the spin label. The simplex algorithm was found to be the most reliable, and an approach-incorporating both NL2SNO as well as the downhill simplex methods-is proposed as a strategy-of-choice.

Biophysical Journal, 2009

]. In this previous work, MD simulations were performed using the AMBER suite of programs, the al... more ]. In this previous work, MD simulations were performed using the AMBER suite of programs, the all-atom AMBER99 force field, the particle mesh Ewald (PME) method for treating long-range electrostatic interactions, and the SPC/E water model. In order to test the effect of the water model used in the AMBER-based molecular dynamics simulations, calculations for a small nitroxide [3-hydroxymethyl-(1-oxy-2,2,5,5-tetramethylpyrroline) or 3MeOHSL] are being performed using a variety of different water models (SPC/E, TIP3P, TIP4P). The EPR spectra calculated from these molecular dynamics simulations of 3MeOHSL will be compared to experimental data.

Microscopy Research and Technique, 2004

The atomic force microscope (AFM) is introduced as a biomolecular manipulation machine capable of... more The atomic force microscope (AFM) is introduced as a biomolecular manipulation machine capable of assembling biological molecules into well-defined molecular structures. Native collagen molecules were mechanically directed into well-defined, two-dimensional templates exhibiting patterns with feature sizes ranging from a few nanometers to several hundreds of micrometers. The resulting nanostructured collagen matrices were only ϳ3-nm thick, exhibited an extreme mechanical stability, and maintained their properties over the time range of several months. Our results directly demonstrate the plasticity of biological assemblies and provide insight into the physical mechanisms by which biological structures may be organized by cells in vivo. These nanoscopic templates may serve as platforms on non-biological surfaces to direct molecular and cellular processes.

Cellular and Molecular Bioengineering, 2008

Cells and organelles are shaped by the chemical and physical forces that bend cell membranes. The... more Cells and organelles are shaped by the chemical and physical forces that bend cell membranes. The human red blood cell (RBC) is a model system for studying how such forces determine cell morphology. It is thought that RBCs, which are typically biconcave discoids, take the shape that minimizes their membrane-bending energies, subject to the constraints of fixed area and volume. However, recently it has been hypothesized that shear elasticity arising from the membraneassociated cytoskeleton (MS) is necessary to account for shapes of real RBCs, especially ones with highly curved features such as echinocytes. In this work we tested this hypothesis by following RBC shape changes using spherical harmonic series expansions of theoretical cell surfaces and those estimated from 3D confocal microscopy images of live cells. We found (i) quantitative agreement between shapes obtained from the theoretical model including the MS and real cells, (ii) that weakening the MS, by using urea (which denatures spectrin), leads to the theoretically predicted gradual decrease in spicule number of echinocytes, (iii) that the theory predicts that the MS is essential for stabilizing the discocyte morphology against changes in lipid composition, and that without it, the shape would default to the elliptocyte (a biconcave ellipsoid), (iv) that we were able to induce RBCs to adopt the predicted elliptocyte morphology by treating healthy discocytes with urea. The latter observation is consistent with the known connection between the blood disease hereditary elliptocytosis and spectrin mutations that weaken the cell cortex. We conclude that while the discocyte, in absence of shear, is indeed a minimum energy shape, its stabilization in healthy RBCs requires the MS, and that elliptocytosis can be explained based on purely mechanical considerations.

Medical Image Analysis, 2008

Numerical deconvolution of 3D fluorescence microscopy data yields sharper images by reversing the... more Numerical deconvolution of 3D fluorescence microscopy data yields sharper images by reversing the known optical aberrations introduced during the acquisition process. When additional prior information such as the topology and smoothness of the imaged object surface is available, the deconvolution can be performed by fitting a parametric surface directly to the image data. In this work, we incorporate such additional information into the deconvolution process and focus on a parametric shape description suitable for the study of organelles, cells and tissues. Such membrane-bound closed biological surfaces are often topologically equivalent to the sphere and can be parameterized as series expansions in spherical harmonic functions (SH). Because image data are noisy and the SH-parameterization is prone to the formation of high curvatures even at low expansion orders, the parametric deconvolution problem is ill-posed and must be regularized. We use the shape bending energy as a regularizing (smoothing) function, and determine the regularization parameter graphically with the help of the L-curve method. We demonstrate the complete deconvolution scheme, including the initial image segmentation, the calculation of a good starting surface and the construction of the L-curve, using real and synthetic image data.

American Journal of Physics, 2010

We define four complex numbers representing the parameters needed to specify an elephantine shape... more We define four complex numbers representing the parameters needed to specify an elephantine shape. The real and imaginary parts of these complex numbers are the coefficients of a Fourier coordinate expansion, a powerful tool for reducing the data ...

Journal of Molecular Biology, 2005

Collagen fibres within the extracellular matrix lend tensile strength to tissues and form a funct... more Collagen fibres within the extracellular matrix lend tensile strength to tissues and form a functional scaffold for cells. Cells can move directionally along the axis of fibrous structures, in a process important in wound healing and cell migration. The precise nature of the structural cues within the collagen fibrils that can direct cell movement are not known. We have investigated the structural features of collagen that are required for directional motility of mouse dermal fibroblasts, by analysing cell movement on two-dimensional collagen surfaces. The surfaces were prepared with aligned fibrils of collagen type I, oriented in a predefined direction. These collagen-coated surfaces were generated with or without the characteristic 67 nm D-periodic banding. Quantitative analysis of cell morphodynamics showed a strong correlation of cell elongation and motional directionality with the orientation of D-periodic collagen microfibrils. Neither directed motility, nor cell body alignment, was observed on aligned collagen lacking D-periodicity, or on D-periodic collagen in the presence of peptide containing an RGD motif. The directional motility of fibroblast cells on aligned collagen type I fibrils cannot be attributed to contact guidance, but requires additional structural information. This allows us to postulate a physiological function for the 67 nm periodicity.

PLOS One, 2010

Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generati... more Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generating complexes that are anchored at the plasma membrane and that pull on microtubules growing out from the spindle poles. Although asymmetric distribution of the force generators is thought to underlie asymmetric positioning of the spindle, the number and location of the force generators has not been well defined. In particular, it has not been possible to visualize individual force generating events at the cortex. We discovered that perturbation of the acto-myosin cortex leads to the formation of long membrane invaginations that are pulled from the plasma membrane toward the spindle poles. Several lines of evidence show that the invaginations, which also occur in unperturbed embryos though at lower frequency, are pulled by the same force generators responsible for spindle positioning. Thus, the invaginations serve as a tool to localize the sites of force generation at the cortex and allow us to estimate a lower limit on the number of cortical force generators within the cell.

PLOS One, 2010

Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generati... more Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generating complexes that are anchored at the plasma membrane and that pull on microtubules growing out from the spindle poles. Although asymmetric distribution of the force generators is thought to underlie asymmetric positioning of the spindle, the number and location of the force generators has not been well defined. In particular, it has not been possible to visualize individual force generating events at the cortex. We discovered that perturbation of the acto-myosin cortex leads to the formation of long membrane invaginations that are pulled from the plasma membrane toward the spindle poles. Several lines of evidence show that the invaginations, which also occur in unperturbed embryos though at lower frequency, are pulled by the same force generators responsible for spindle positioning. Thus, the invaginations serve as a tool to localize the sites of force generation at the cortex and allow us to estimate a lower limit on the number of cortical force generators within the cell.

Biophysical Journal, 2009

three dimensional (3D) resolution. In this study, both pure and fluorescently labeled (3% by weig... more three dimensional (3D) resolution. In this study, both pure and fluorescently labeled (3% by weight) collagen gels are assembled over a range of concentrations (0.1, 0.5, 1.0, 2.0, 5.0, and 5.5 mg/mL) and at various temperatures (37, 32, 27, and 22 C). The networks are investigated using confocal reflectance microscopy (CRM) and confocal fluorescence microscopy (CFM). Comparison between CRM and CFM reveals that they are not equally sensitive to details of network structure, with CRM (CFM) displaying higher sensitivity to fibers perpendicular (parallel) to the optical axis. Furthermore, analysis of background signal in CFM images suggests the existence of small fibrillar structures that are not resolved by CRM. Despite these differences, image analyses performed on 2D slices of CFM and CRM images to quantify mesh size, number of fibers, and fiber length reveal identical trends as a function of gel concentration and gelation temperature. Fiber width approximated from both CRM and CFM is in good accord with fiber width determination using electron microscopy. Overall network structures (as quantified via mesh size, fiber number, fiber length and fiber width) are related to bulk mechanical properties measured by rheology. Finally, we demonstrate the ability to form collagen gels of varied mesh size at identical collagen concentration, all of which are compatible with cell health and 3D cell studies. Tuberculosis is a disease that infects one in three humans today. The long, expensive drug course required to cure the active form, combined with its increasing resistance to conventional antibiotic treatment, necessitates the development of a new class of TB drugs. To evaluate the efficacy of these drugs, as well as to gain increased basic knowledge of the disease's progression, we are building a novel 0.8 mm diameter bronchoscope as part of a multi-institutional initiative to develop a primate-based tuberculosis model system. In order to monitor the in vivo microenvironment of the tuberculosis granuloma, monkeys will be inoculated with transgenic pH reporting tuberculosis bacilli and GFP expression measured to quantify the local pH and other micro-environmental parameters. CT scans will be used to reveal the induced nodules/lesions and guide the bronchoscope to the granulomas. The lung tissue itself contains many 488 nm excitable endogenous fluorophores (e.g. elastin, collagen) and autofluorescence limits the level of reporter quantification. To overcome this problem we are employing a photoactivatable protein (Dronpa) as the reporter expressed by the bacteria. Using a novel pulsed UV/Blue non-laser light source, the protein's fluorescence can be modulated to distinguish reporter signal from the constant autofluorescence background and therefore produce highly quantitative measurements of changes in the granuloma microenvironment during the progression of the disease and during drug treatment. (Supported by the Gates Foundation and NIH/NIBIB P41 RR04224 to WRZ.)

Current Opinion in Genetics & Development, 2011

Light sheet-based fluorescence microscopy (LSFM) is emerging as a powerful imaging technique for ... more Light sheet-based fluorescence microscopy (LSFM) is emerging as a powerful imaging technique for the life sciences. LSFM provides an exceptionally high imaging speed, high signal-to-noise ratio, low level of photo-bleaching and good optical penetration depth. This unique combination of capabilities makes light sheet-based microscopes highly suitable for live imaging applications. There is an outstanding potential in applying this technology to the quantitative study of embryonic development. Here, we provide an overview of the different basic implementations of LSFM, review recent technical advances in the field and highlight applications in the context of embryonic development. We conclude with a discussion of promising future directions.

We address the problem of segmenting 3D microscopic volumetric intensity images of a collection o... more We address the problem of segmenting 3D microscopic volumetric intensity images of a collection of spatially correlated objects (such as fluorescently labeled nuclei in a tissue). This problem arises in the study of tissue morphogenesis where cells and cellular components are organized in accord with biological role and fate. We formulate the image model as stochastically generated based on biological priors and physics of image formation. We express the segmentation problem in terms of Bayesian inference and use data-driven Markov Chain Monte Carlo to fit the image model to data. We perform an initial step in which the intensity volume is approximated as an expansion in 4D spherical harmonics, the coefficients of which capture the general organization of objects. Since cell nuclei are membrane-bound their shapes are subject to membrane lipid bilayer bending energy, which we use to constrain individual contours. Moreover, we parameterize the nuclear contours using spherical harmonic functions, which provide a shape description with no restriction to particular symmetries. We demonstrate the utility of our approach using synthetic and real fluorescence microscopy data.

Nature Methods, 2010

Articles nAture methods | ADVANCE ONLINE PUBLICATION | recording light-microscopy images of large... more Articles nAture methods | ADVANCE ONLINE PUBLICATION | recording light-microscopy images of large, nontransparent specimens, such as developing multicellular organisms, is complicated by decreased contrast resulting from light scattering. early zebrafish development can be captured by standard light-sheet microscopy, but new imaging strategies are required to obtain high-quality data of late development or of less transparent organisms. We combined digital scanned laser light-sheet fluorescence microscopy with incoherent structuredillumination microscopy (dslm-si) and created structuredillumination patterns with continuously adjustable frequencies. our method discriminates the specimen-related scattered background from signal fluorescence, thereby removing out-offocus light and optimizing the contrast of in-focus structures. dslm-si provides rapid control of the illumination pattern, exceptional imaging quality and high imaging speeds. We performed long-term imaging of zebrafish development for 58 h and fast multiple-view imaging of early Drosophila melanogaster development. We reconstructed cell positions over time from the Drosophila dslm-si data and created a fly digital embryo.