Dipanjan Bhattacharya

Spatio-temporal plasticity in chromatin organization in mouse cell differentiation and during Drosophila embryogenesis.

Cellular differentiation and developmental programs require changing patterns of gene expression. Recent experiments have revealed that chromatin organization is highly dynamic within living cells, suggesting possible mechanisms to alter gene expression programs, yet the physical basis of this organization is unclear. In this article, we contrast the differences in the dynamic organization of nuclear architecture between undifferentiated mouse embryonic stem cells and terminally differentiated primary mouse embryonic fibroblasts. Live-cell confocal tracking of nuclear lamina evidences highly flexible nuclear architecture within embryonic stem cells as compared to primary mouse embryonic fibroblasts. These cells also exhibit significant changes in histone and heterochromatin binding proteins correlated with their distinct epigenetic signatures as quantified by immunofluorescence analysis. Further, we follow histone dynamics during the development of the Drosophila melanogaster embryo, which gives an insight into spatio-temporal evolution of chromatin plasticity in an organismal context. Core histone dynamics visualized by fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and fluorescence anisotropy within the developing embryo, revealed an intriguing transition from plastic to frozen chromatin assembly synchronous with cellular differentiation. In the embryo, core histone proteins are highly mobile before cellularization, actively exchanging with the pool in the yolk. This hyperdynamic mobility decreases as cellularization and differentiation programs set in. These findings reveal a direct correlation between the dynamic transitions in chromatin assembly with the onset of cellular differentiation and developmental programs.

Direct observation of stick-slip movements of water nanodroplets induced by an electron beam

Dynamics of the first few nanometers of water at the interface are encountered in a wide range of physical, chemical, and biological phenomena. A simple but critical question is whether interfacial forces at these nanoscale dimensions affect an externally induced movement of a water droplet on a surface. At the bulk-scale water droplets spread on a hydrophilic surface and slip on a nonwetting, hydrophobic surface. Here we report the experimental description of the electron beam-induced dynamics of nanoscale water droplets by direct imaging the translocation of 10- to 80-nm-diameter water nanodroplets by transmission electron microscopy. These nanodroplets move on a hydrophilic surface not by a smooth flow but by a series of stick-slip steps. We observe that each step is preceded by a unique characteristic deformation of the nanodroplet into a toroidal shape induced by the electron beam. We propose that this beam-induced change in shape increases the surface free energy of the nanodroplet that drives its transition from stick to slip state.

Rankl-induced osteoclastogenesis leads to loss of mineralization in a medaka osteoporosis model

Osteoclasts are macrophage-related bone resorbing cells of hematopoietic origin. Factors that regulate osteoclastogenesis are of great interest for investigating the pathology and treatment of bone diseases such as osteoporosis. In mammals, receptor activator of NF-κB ligand (Rankl) is a regulator of osteoclast formation and activation: its misexpression causes osteoclast stimulation and osteoporotic bone loss. Here, we report an osteoporotic phenotype that is induced by overexpression of Rankl in the medaka model. We generated transgenic medaka lines that express GFP under control of the cathepsin K promoter in osteoclasts starting at 12 days post-fertilization (dpf), or Rankl together with CFP under control of a bi-directional heat-shock promoter. Using long-term confocal time-lapse imaging of double and triple transgenic larvae, we monitored in vivo formation and activation of osteoclasts, as well as their interaction with osteoblasts. Upon Rankl induction, GFP-positive osteoclasts are first observed in the intervertebral regions and then quickly migrate to the surface of mineralized neural and haemal arches, as well as to the centra of the vertebral bodies. These osteoclasts are TRAP (tartrate-resistant acid phosphatase) and cathepsin K positive, mononuclear and highly mobile with dynamically extending protrusions. They are exclusively found in tight contact with mineralized matrix. Rankl-induced osteoclast formation resulted in severe degradation of the mineralized matrix in vertebral bodies and arches. In conclusion, our in vivo imaging approach confirms a conserved role of Rankl in osteoclastogenesis in teleost fish and provides new insight into the cellular interactions during bone resorption in an animal model that is useful for genetic and chemical screening.

Nanoparticle Dynamics in a Nanodroplet

We describe the dynamics of 3-10 nm gold nanoparticles encapsulated by ∼30 nm liquid nanodroplets on a flat solid substrate and find that the diffusive motion of these nanoparticles is damped due to strong interactions with the substrate. Such damped dynamics enabled us to obtain time-resolved observations of encapsulated nanoparticles coalescing into larger particles. Techniques described here serve as a platform to study chemical and physical dynamics under highly confined conditions.

Improving signal-to-noise ratio of structured light microscopy based on photon reassignment.

In this paper, we report a method for 3D visualization of a biological specimen utilizing a structured light wide-field microscopic imaging system. This method improves on existing structured light imaging modalities by reassigning fluorescence photons generated from off-focal plane excitation, improving in-focus signal strength. Utilizing a maximum likelihood approach, we identify the most likely fluorophore distribution in 3D that will produce the observed image stacks under structured and uniform illumination using an iterative maximization algorithm. Our results show the optical sectioning capability of tissue specimens while mostly preserving image stack photon count, which is usually not achievable with other existing structured light imaging methods.

Strain maps characterize the symmetry of convergence and extension patterns during Zebrafish gastrulation

During gastrulation of the zebrafish embryo, the cap of blastoderm cells organizes into the axial body plan of the embryo with left-right symmetry and head-tail, dorsal-ventral polarities. Our labs have been interested in the mechanics of early development and have investigated whether these large-scale cells movements can be described as tissue-level mechanical strain by a tectonics-based approach. The first step is to image the positions of all nuclei from mid-epiboy to early segmentation by digital sheet light microscopy (DSLM), organize the surface of the embryo into multi-cell spherical domains, construct velocity fields from the movements of these domains and extract 3D strain rate maps. Tensile/expansive and compressive strains in the axial and equatorial directions are detected during gastrulation as anterior and posterior expansion along the anterior-posterior axis and medial-lateral compression across the dorsal-ventral axis corresponding to convergence and extension. In later stages in development are represented by localized medial expansion at the onset of segmentation and anterior expansion at the onset of neurulation. Symmetric patterns of rotation are first detected in the animal hemispheres at mid-epiboly and then the vegetal hemispheres by the end of gastrulation. By analysing the temporal sequence of large scale movements, deformations across the embryo can be attributed to a combination of epiboly and dorsal convergence-extension. Significance Strain is an emergent property of tissues that originates from the mechanical coupling of cell-cell and cell-substrate interactions, individual cell shape changes, and cell level forces. By imaging the positions of nuclei from mid-epiboly to early segmentation of the zebrafish embryo we are able to calculate three types of strain maps by a plate tectonics based method. The regions of expansive and compressive axial and equatorial strain correspond to areas undergoing convergence and extension, a major step in the formation of the embryonic body plan as well as the formation of somite and head structures. The most striking signatures of strain are: 1. the bilateral symmetry of linear strain across the anterior-posterior, dorsal-ventral axis during gastrulation, 2. the complementary counter-rotational strains or curl in the animal hemisphere at mid epiboly, and 3. a divergence or saddle point in the region of the dorsal organizer, head-trunk boundary. These strains represent a general method to describe large-scale tissue-level mechanics not only of embryonic development but also tissue homeostasis and disease.

Three-dimensional Modes of Illumination and Computational Reconstruction in Light-sheet Mcroscopy

Three-dimensional spatial and temporal modulation of the illumination and optimal algorithms for reconstruction improve contrast from thick fluorescent tissue. Theoretical analysis and experimental results will be presented, with applications to developmental biology and live animal imaging.