Sohail Ahmed

A Graph-Theoretical Approach for Tracing Filamentary Structures in Neuronal and Retinal Images

The aim of this study is about tracing filamentary structures in both neuronal and retinal images. It is often crucial to identify single neurons in neuronal networks, or separate vessel tree structures in retinal blood vessel networks, in applications such as drug screening for neurological disorders or computer-aided diagnosis of diabetic retinopathy. Both tasks are challenging as the same bottleneck issue of filament crossovers is commonly encountered, which essentially hinders the ability of existing systems to conduct large-scale drug screening or practical clinical usage. To address the filament crossovers problem, a two-step graph-theoretical approach is proposed in this paper. The first step focuses on segmenting filamentary pixels out of the background. This produces a filament segmentation map used as input for the second step, where they are further separated into disjointed filaments. Key to our approach is the idea that the problem can be reformulated as label propagation over directed graphs, such that the graph is to be partitioned into disjoint sub-graphs, or equivalently, each of the neurons (vessel trees) is separated from the rest of the neuronal (vessel) network. This enables us to make the interesting connection between the tracing problem and the digraph matrix-forest theorem in algebraic graph theory for the first time. Empirical experiments on neuronal and retinal image datasets demonstrate the superior performance of our approach over existing methods.

Quantitative imaging reveals real-time Pou5f3–Nanog complexes driving dorsoventral mesendoderm patterning in zebrafish

Formation of the three embryonic germ layers is a fundamental developmental process that initiates differentiation. How the zebrafish pluripotency factor Pou5f3 (homologous to mammalian Oct4) drives lineage commitment is unclear. Here, we introduce fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to assess the formation of Pou5f3 complexes with other transcription factors in real-time in gastrulating zebrafish embryos. We show, at single-cell resolution in vivo, that Pou5f3 complexes with Nanog to pattern mesendoderm differentiation at the blastula stage. Later, during gastrulation, Sox32 restricts Pou5f3–Nanog complexes to the ventrolateral mesendoderm by binding Pou5f3 or Nanog in prospective dorsal endoderm. In the ventrolateral endoderm, the Elabela / Aplnr pathway limits Sox32 levels, allowing the formation of Pou5f3–Nanog complexes and the activation of downstream BMP signaling. This quantitative model shows that a balance in the spatiotemporal distribution of Pou5f3–Nanog complexes, modulated by Sox32, regulates mesendoderm specification along the dorsoventral axis. DOI: http://dx.doi.org/10.7554/eLife.11475.001

Insights into the regulation of a common variant of HMGA2 associated with human height during embryonic development.

Early genetic studies in the mouse and chicken identified the HMGA oncogene as a candidate that regulates body height. Subsequent genome-wide SNP studies revealed a significant association of rs1042725 genotypes CT and CC in the 3’ UTR of HMGA2 with human height. Together, these studies indicated that HMGA2 expression levels during prenatal development might be a critical factor that contributes to the height phenotype. In the present study, we sought to gain insight into the regulation of HMGA2 during human embryonic development and provide evidence that the rs1042725 genotype is unlikely to affect HMGA2 levels in pluripotent human embryonic stem cells (hESCs). This implies that hESCs in the inner cell mass of blastocysts are most likely not involved in determining the human height phenotype associated with this SNP. By applying a computational approach and cell-based reporter assays, we then identified miR-196b as a candidate microRNA that could contribute to SNP-specific expression of HMGA2 during human prenatal development. We briefly discuss this result in the context of other known functions for miR-196b during vertebrate development.

Online 3-D Tracking of Suspension Living Cells Imaged with Phase-Contrast Microscopy

Neural stem cells/neural progenitors (NSCs/NPs) are cells that give rise to the main cell types of the nervous system: oligodendrocytes, neurons, and astrocytes. Studying NSCs/NPs with time-lapse microscopy is critical to the understanding of the biology of these cells. However, NSCs/NPs are very sensitive to phototoxic damage, and therefore, fluorescent dyes cannot be used to follow these cells. Also, since in most of NSC/NP-related experiments, a large number of cells neesd to be monitored. Consequently, the acquisition of a huge amount of images is required. An additional difficulty is related to our original suspension living, tracking objective, behavior much closer to the natural, in vivo, way of development of the cells. Indeed, unlike adherent cells, suspension cells float freely in a liquid solution, thus, making their dynamics very different from that of adherent cells. As a result, existing visual tracking algorithms that have primarily been developed to track adherent cells are no longer adequate to tackle living cells in suspension. This paper presents a novel automated 3-D visual tracking of suspension living cells for time-lapse image acquisition using phase-contrast microscopy. This new tracking method can potentially strongly impact on current 3-D video microscopy methods, paving the way for innovative analysis of NSCs/NPs and as a result, on the study of neurodegenerative diseases.

A fluorescent probe for imaging symmetric and asymmetric cell division in neurosphere formation.

We report here a novel fluorescent chemical probe which stains distinct neural stem/progenitor cells (NSPCs) by binding to acid ceramidase in mouse neurospheres. is distributed evenly or unevenly to the daughter cells during multiple mitoses enabling the live imaging of symmetric and asymmetric divisions of isolated NSPCs.

NeuronCyto II: An automatic and quantitative solution for crossover neural cells in high throughput screening

Microscopy is a fundamental technology driving new biological discoveries. Today microscopy allows a large number of images to be acquired using, for example, High Throughput Screening (HTS) and 4D imaging. It is essential to be able to interrogate these images and extract quantitative information in an automated fashion. In the context of neurobiology, it is important to automatically quantify the morphology of neurons in terms of neurite number, length, branching and complexity, etc. One major issue in quantification of neuronal morphology is the “crossover” problem where neurites cross and it is difficult to assign which neurite belongs to which cell body. In the present study, we provide a solution to the “crossover” problem, the software package NeuronCyto II. NeuronCyto II is an interactive and user‐friendly software package for automatic neurite quantification. It has a well‐designed graphical user interface (GUI) with only a few free parameters allowing users to optimize the software by themselves and extract relevant quantitative information routinely. Users are able to interact with the images and the numerical features through the Result Inspector. The processing of neurites without crossover was presented in our previous work. Our solution for the “crossover” problem is developed based on our recently published work with directed graph theory. Both methods are implemented in NeuronCyto II. The results show that our solution is able to significantly improve the reliability and accuracy of the neurons displaying “crossover.” NeuronCyto II is freely available at the website: https://sites.google.com/site/neuroncyto/, which includes user support and where software upgrades will also be placed in the future.

OpenSegSPIM: a user-friendly segmentation tool for SPIM data

UNLABELLEDnOpenSegSPIM is an open access and user friendly 3D automatic quantitative analysis tool for Single Plane Illumination Microscopy data. The software is designed to extract, in a user-friendly way, quantitative relevant information from SPIM image stacks, such as the number of nuclei or cells. It provides quantitative measurement (volume, sphericity, distance, intensity) on Light Sheet Fluorescent Microscopy images.nnnAVAILABILITY AND IMPLEMENTATIONnfreely available from http://www.opensegspim.weebly.com Source code and binaries under BSD License.nnnCONTACTnlgole@imcb.a-star.edu.sg or wmyu@imcb.a-star.edu.sg or sohail.ahmed@imb.a-star.edu.sgnnnSUPPLEMENTARY INFORMATIONnSupplementary data are available at Bioinformatics online.

Fluorescence techniques used to measure interactions between hydroxyapatite nanoparticles and epidermal growth factor receptors

The potential applications of nanomaterials in therapeutics are immense and to fully explore this potential, it is important to understand the interaction of nanoparticles with cellular components. To examine the interaction between nanoparticles and cell membrane receptors, this report describes the use of advanced fluorescence techniques to measure interactions between hydroxyapatite (HA) nanoparticles and epidermal growth factor receptors (EGFRs), as a model system. FITC‐labelled HA nanoparticles and monomeric red fluorescent protein (mRFP)‐conjugated EGFRs expressed in Chinese hamster ovary cells (CHO‐K1) were generated and their interaction measured using acceptor photobleaching‐fluorescence resonance energy transfer (AP‐FRET) and fluorescence lifetime imaging microscopy‐fluorescence resonance energy transfer (FLIM‐FRET). Results confirmed that hydroxyapatite nanoparticles not only interacted with EGFR but also attenuated downstream EGFR signalling, possibly by hindering normal dimerization of EGFR. Furthermore, the extent of signal attenuation suggested correlation with specific surface area of the nanoparticles, whereby greater specific surface area resulted in greater downstream signal attenuation. This novel demonstration establishes fluorescence techniques as a viable method to study nanoparticle interactions with proteins such as cell surface receptors. The approach described herein can be extended to study interactions between any fluorescently labelled nanoparticle‐biomolecule pair.

Neurosphere fate prediction: An analysis-synthesis approach for feature extraction

The study of stem cells is one of the current most important biomedical research field. Understanding their development could allow multiple applications in regenerative medicine. For this purpose, we need automated methods for the segmentation and the modeling of neural stem cell development process into a neurosphere colony from phase contrast microscopy. We use such methods to extract relevant structural and textural features like cell division dynamism and cell behavior patterns for biological interpretation. The combination of phase contrast imaging, high fragility and complex evolution of neural stem cells pose many challenges in image processing and image analysis. This study introduces an on-line analysis method for the modeling of neurosphere evolution during the first three days of their development. From the corresponding time-lapse sequences, we extract information from the neurosphere using a combination of fast level set and curve detection for segmenting the cells. Then, based on prior biological knowledge, we generate possible and optimal 3-dimensional configuration using registration and evolutionary optimisation algorithm.

Using dSTORM to probe the molecular architecture of filopodia

IRSp53 is a Cdc42 effector and a member of the Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain family which can induce negative membrane curvature. IRSp53 generates filopodia by coupling membrane protrusion (I-BAR domain) with actin dynamics through its SH3 domain binding partners. Dynamin 1 (Dyn1), a large GTPase associated with endocytosis, is a novel interacting partner of IRSp53 that localises to filopodia. Using rapid time-lapse TIRF microscopy we have shown that Dyn1 localized to a subcellular region just behind Mena at the leading edge, or in filopodial tip complexes when co-expressed with IRSp53. Dyn1-GFP was strongly localized in the filopodial shaft during the early phase of elongation, after which it moved rearward, suggestive of a role in early filopodia assembly. Mena and Eps8, accumulate at the tip complex in sequence and are involved in filopodial extension and retraction, respectively (Chou at al, 2014 submitted). Here we describe the use of dSTORM to investigate the molecular architecture of filopodia and in particular the size of the F-actin bundle in these structures. The data suggest that direct Stochastic Optical Reconstruction Microscopy (dSTORM) in combination with other techniques will allow the molecular architecture of