Whole-body integration of gene expression and single-cell morphology

Animal bodies are composed of cell types with unique expression programs that implement their distinct locations, shapes, structures, and functions. Based on these properties, cell types assemble into specific tissues and organs. To systematically explore the link between cell-type-specific gene expression and morphology, we registered an expression atlas to a whole-body electron microscopy volume of the nereid Platynereis dumerilii. Automated segmentation of cells and nuclei identifies major cell classes and establishes a link between gene activation, chromatin topography, and nuclear size. Clustering of segmented cells according to gene expression reveals spatially coherent tissues. In the brain, genetically defined groups of neurons match ganglionic nuclei with coherent projections. Besides interneurons, we uncover sensory-neurosecretory cells in the nereid mushroom bodies, which thus qualify as sensory organs. They furthermore resemble the vertebrate telencephalon by molecular anatomy. We provide an integrated browser as a Fiji plugin for remote exploration of all available multimodal datasets

Lensfree super-resolution holographic microscopy using wetting films on a chip.

We investigate the use of wetting films to significantly improve the imaging performance of lensfree pixel super-resolution on-chip microscopy, achieving < 1 µm spatial resolution over a large imaging area of ~24 mm(2). Formation of an ultra-thin wetting film over the specimen effectively creates a micro-lens effect over each object, which significantly improves the signal-to-noise-ratio and therefore the resolution of our lensfree images. We validate the performance of this approach through lensfree on-chip imaging of various objects having fine morphological features (with dimensions of e.g., ≤0.5 µm) such as Escherichia coli (E. coli), human sperm, Giardia lamblia trophozoites, polystyrene micro beads as well as red blood cells. These results are especially important for the development of highly sensitive field-portable microscopic analysis tools for resource limited settings

A New Femtosecond Laser-Based 3D Tomography Technique.

Tomographic imaging has dramatically changed science, most notably in the fields of medicine and biology, by producing 3D views of structures which are too complex to understand in any other way. Current tomographic techniques require extensive time both for post-processing and data collection. Femtosecond laser based tomographic techniques have been developed in both standard atmosphere (femtosecond laser-based serial sectioning technique - FSLSS) and in vacuum (Tri-Beam System) for the fast collection (10^5 μm^3/s) of mm^3 sized 3D datasets. Both techniques use femtosecond laser pulses to selectively remove layer-by-layer areas of material with low collateral damage and a negligible heat affected zone. To the authors knowledge, femtosecond lasers have never been used to serial section and these techniques have been entirely and uniquely developed by the author and his collaborators at the University of Michigan and University of California Santa Barbara. The FSLSS was applied to measure the 3D distribution of TiN particles in a 4330 steel. Single pulse ablation morphologies and rates were measured and collected from literature. Simultaneous two-phase ablation of TiN and steel matrix was shown to occur at fluences of 0.9-2 J/cm^2. Laser scanning protocols were developed minimizing surface roughness to 0.1-0.4 μm for laser-based sectioning. The FSLSS technique was used to section and 3D reconstruct titanium nitride(TiN) containing 4330 steel. Statistical analysis of 3D TiN particle sizes, distribution parameters, and particle density were measured. A methodology was developed to use the 3D datasets to produce statistical volume elements (SVEs) for toughness modeling. Six FSLSS TiN datasets were sub-sampled into 48 SVEs for statistical analysis and toughness modeling using the Rice-Tracey and Garrison-Moody models. A two-parameter Weibull analysis was performed and variability in the toughness data agreed well with Ruggieri et al. bulk toughness measurements. The Tri-Beam system combines the benefits of laser based material removal (speed, low-damage, automated) with detectors that collect chemical, structural, and topological information. Multi-modal sectioning information was collected after many laser scanning passes demonstrating the capability of the Tri-Beam system.Ph.D.Materials Science and EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/89731/1/mechlin_1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/89731/2/mechlin_2.pd

Synaptic and peptidergic connectome of a neurosecretory centre in the annelid brain

This is the author accepted manuscript. The final version is available from eLife Sciences Publications via the DOI in this record.Neurosecretory centers in animal brains use peptidergic signaling to influence physiology and behavior. Understanding neurosecretory center function requires mapping cell types, synapses, and peptidergic networks. Here we use transmission electron microscopy and gene expression mapping to analyze the synaptic and peptidergic connectome of an entire neurosecretory center. We reconstructed 78 neurosecretory neurons and mapped their synaptic connectivity in the brain of larval Platynereis dumerilii, a marine annelid. These neurons form an anterior neurosecretory center expressing many neuropeptides, including hypothalamic peptide orthologs and their receptors. Analysis of peptide-receptor pairs in spatially mapped single-cell transcriptome data revealed sparsely connected networks linking specific neuronal subsets. We experimentally analyzed one peptide-receptor pair and found that a neuropeptide can couple neurosecretory and synaptic brain signaling. Our study uncovered extensive networks of peptidergic signaling within a neurosecretory center and its connection to the synaptic brain.The research leading to these results received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ European Research Council Grant Agreement 260821. The research was supported by a grant from the DFG - Deutsche Forschungsgemeinschaft (Reference no. JE 777/1)

A model based approach for the characterisation of radiolabelled antibodies in radioimmunotherapy

Radioimmunotherapy (RIT) utilises antibodies directed against tumour associated antigens to carry a therapeutic dose of radiation to the tumour. Using RIT, model tumours have been successfully treated and yet clinical responses have been limited by poor tumour localisation. In an attempt to overcome this, many new antibodies have been developed. Measuring the gross tumour localisation and tumour to normal tissue ratio in animal models has generally been used to assess the potential clinical use of these antibodies. However, these measurements assume all the energy from the electron emitted from the radionuclide is deposited in the source organ, and also ignore the effects of dose-rate and cell proliferation during treatment. In addition, they do not consider the effects of heterogeneous dose deposition and response within the tissues. The principal purpose of this thesis is to develop a more accurate measure of the biological effect of radiolabelled antibodies in a mouse xenograft in order to select the optimal radionuclide/antibody combination for more effective therapy in man. A structural model has been developed from mouse data to facilitate more accurate absorbed dose calculations by accounting for organ size, shape, and position relative to surrounding organs. In addition, the linear-quadratic model, conventionally used in external beam radiotherapy, has been adapted for use in RIT to account for the effects of dose-rate and proliferation during treatment. To characterise heterogeneity of dose deposition and response in tumours, images of tumour morphology and radiolabelled antibody distribution were registered. The images were obtained through digitisation of stained histological sections and storage phosphor plate technology. All data was collected using a wide range of antibodies labelled with 131I and 90Y. These models show that multivalent, tumour-specific antibodies, with intermediate clearance rates, deliver the most effective dose to xenografts. Antibody affinity and avidity facilitate the prolonged retention in radiosensitive areas of tumour where most of the dose is deposited. In addition, a significantly greater activity of 131I can be injected before causing the equivalent bone marrow toxicity. Furthermore, when antibodies are labelled with 90Y, a significant amount of the electron energy escapes the source organ and is absorbed in surrounding tissue. Nevertheless, the results clearly show that radionuclide and antibody should be matched in order to deliver optimum therapy