High-Throughput Automated Multi-Target Super-resolution Imaging

Super-resolution microscopy techniques developed through the past few decades enable us to surpass the classical diffraction limit of light, and thus open new doors to investigate the formerly inaccessible world of nanometer-sized objects. Most importantly, by using super-resolution microscopy, one can visualize sub-cellular structures in the range of 10 to 200 nm. At this range, we can investigate exciting problems in biology and medicine by visualizing protein-protein interactions and spatiotemporal analysis of structures of interest on the surface or inside cells. These techniques (collectively known as nanoscopy) have a high impact on understanding and solving biological questions. This dissertation starts with a brief and general description of current super-resolution techniques and then moves toward a multi-target super-resolution imaging strategy using sequential imaging that has benefits over conventional multi-color imaging methods. Sequential microscopy takes advantage of the photo-physical properties of the most suitable dye for a particular technique to achieve the optimal and consistent resolution for each of multiple targets of imaging. For example, for dSTORM imaging, this is currently AlexaFluor647.\ Sequential dSTROM has an advantage for multi-target imaging due to having a single imaging channel which avoids dealing with differential aberration-problems between multiple emission paths unlike other multi-color imaging based methods. We show that sequential imaging method can be facilitated using automated imaging. In this dissertation, a sequential microscope is designed, calibrated, and tested on multiple structures. We show that it can automatically re-find the position of each initially registered cell and can account for sample drift through an entire experiment. The microscope has been used in multiple collaborations with other groups to investigate biological problems of interest. Two labeling strategies that facilitate sequential imaging are described.\ The first strategy is DNA-strand-displacement , which allows imaging of multiple structures in a controlled and time-efficient binding-unbinding scenario. The second strategy is imaging with the small, actin binding peptide Lifeact. Finally, future directions and suggestions are made about how we can further improve the microscope. In the Appendix I provide a guide on how to use and troubleshoot the microscope, how to measure the efficiency of the microscope, as well as how to fix and label cells for optimal imaging and how to prepare various imaging buffers

Comparing alveolar bone regeneration using Bio-Oss and autogenous bone grafts in humans: a systematic review and meta-analysis

INTRODUCTION: Bone regeneration grafts (BRG) are widely used in the treatment of osseous defects and oral surgery. The various techniques and associated success rates of bone augmentation require evaluation by systematic review and meta-analysis of eligible studies. The aim of this systematic review was to compare alveolar bone regeneration in humans using Bio-Oss and autogenous bone graft. MATERIALS AND METHODS: The computerized bibliographical databases including Pubmed, Google, ScienceDirect and Cochrane were searched for randomized and cohort studies in which autogenous grafts were compared to Bio-Oss in the treatment of periodontal defects. The inclusion criteria were human studies in English that were published 1998-2009. Exclusion criteria included non randomized observation and cohort studies, papers which provided summary statistics without the variance estimates, and studies that did not use BRG intervention alone, were excluded. The screening of eligible studies, assessment of the methodological quality of the trials and data extraction were collected by two observers independently. For comparing autogenous grafts used alone against Bio-Oss used alone 5 situations were investigated. Thirteen studies were included in the review which compared autogenous against Bio-Oss, autogenous combined with guided tissue regeneration (GTR) against GTR, Bio-Oss combined with GTR versus GTR, autogenous alone versus Open Flap Debridement (OFD), Bio-Oss versus OFD. In meta-analysis, changes in bone level (bone fill) was used as the measure. Data were analyzed using Bayesian meta-analysis by WinBUGS and Boa software. RESULTS: Only one comparison demonstrated that the difference in bone augmentation between Bio-Oss and OFD was statistically significant. CONCLUSION: There is insufficient evidence to show that Bio-Oss is superior to autogenous grafts in bone augmentation techniques however autogenous bone involves donor site surgery and thus donor site morbidity, so we can conclude that Bio-Oss is better than autogenous for alveolar regeneration. [Iranian Endodontic Journal 2009;4(4):125-30

Nasal Nosocomial Myiasis Infection Caused by Chrysomya bezziana (Diptera: Calliphoridae) Following the Septicemia: A Case Report

A 74 yr old woman from Gonabad, southern part of Khorasan Razavi Province of Iran was admitted to a Hospital of Gonabad, because of respiratory distress, exertional dyspnea and fever. Close contact with domes­tic animals, history of chronic obstructive pulmonary disease (COPD), and completely resolved pulmonary tuberculosis (TB) in remote past, were nota­ble parts of her past medical history. Due to clinical, paraclinical and radio­graphic findings and because of recent hospitalization, she was admitted to internal medicine ward with the diagnosis of health care associated pneumo­nia (HCAP). Despite the application of broad-spectrum antibiotics and ap­propriate supportive care, she had a poor response to the treatment. During the daily visit in Intensive Care Unit (ICU), numerous white larvae were de­tected in both nostrils. Further investigation of oropharynx and tracheal tube aspiration, showed no more larvae in mentioned parts. An hour later, nasal spontaneous bleeding occurred. Otorhinolaryngology consultation was per­formed and led to surgical procedure. In ENT examination, there were nu­merous larvae and massive clot formation in both inferior meatuses and distal nasal septum perforation. Thirty-seven extracted larvae were transferred to Medical Entomology lab by vial 70% ethanol and 5 live larvae for rearing. After pre­cise investigation by aid of light microscopy, the larvae were identified as Chrysomya bezziana. Due to discovered 2nd larvae stage and duration of hospitalization, this infestation was identified as nasal myiasis

Towards community-driven metadata standards for light microscopy: tiered specifications extending the OME model [preprint]

Digital light microscopy provides powerful tools for quantitatively probing the real-time dynamics of subcellular structures. While the power of modern microscopy techniques is undeniable, rigorous record-keeping and quality control are required to ensure that imaging data may be properly interpreted (quality), reproduced (reproducibility), and used to extract reliable information and scientific knowledge which can be shared for further analysis (value). Keeping notes on microscopy experiments and quality control procedures ought to be straightforward, as the microscope is a machine whose components are defined and the performance measurable. Nevertheless, to this date, no universally adopted community-driven specifications exist that delineate the required information about the microscope hardware and acquisition settings (i.e., microscopy “data provenance” metadata) and the minimally accepted calibration metrics (i.e., microscopy quality control metadata) that should be automatically recorded by both commercial microscope manufacturers and customized microscope developers. In the absence of agreed guidelines, it is inherently difficult for scientists to create comprehensive records of imaging experiments and ensure the quality of resulting image data or for manufacturers to incorporate standardized reporting and performance metrics. To add to the confusion, microscopy experiments vary greatly in aim and complexity, ranging from purely descriptive work to complex, quantitative and even sub-resolution studies that require more detailed reporting and quality control measures. To solve this problem, the 4D Nucleome Initiative (4DN) (1, 2) Imaging Standards Working Group (IWG), working in conjunction with the BioImaging North America (BINA) Quality Control and Data Management Working Group (QC-DM-WG) (3), here propose light Microscopy Metadata specifications that scale with experimental intent and with the complexity of the instrumentation and analytical requirements. They consist of a revision of the Core of the Open Microscopy Environment (OME) Data Model, which forms the basis for the widely adopted Bio-Formats library (4–6), accompanied by a suite of three extensions, each with three tiers, allowing the classification of imaging experiments into levels of increasing imaging and analytical complexity (7, 8). Hence these specifications not only provide an OME-based comprehensive set of metadata elements that should be recorded, but they also specify which subset of the full list should be recorded for a given experimental tier. In order to evaluate the extent of community interest, an extensive outreach effort was conducted to present the proposed metadata specifications to members of several core-facilities and international bioimaging initiatives including the European Light Microscopy Initiative (ELMI), Global BioImaging (GBI), and European Molecular Biology Laboratory (EMBL) - European Bioinformatics Institute (EBI). Consequently, close ties were established between our endeavour and the undertakings of the recently established QUAlity Assessment and REProducibility for Instruments and Images in Light Microscopy global community initiative (9). As a result this flexible 4DN-BINA-OME (NBO namespace) framework (7, 8) represents a turning point towards achieving community-driven Microscopy Metadata standards that will increase data fidelity, improve repeatability and reproducibility, ease future analysis and facilitate the verifiable comparison of different datasets, experimental setups, and assays, and it demonstrates the method for future extensions. Such universally accepted microscopy standards would serve a similar purpose as the Encode guidelines successfully adopted by the genomic community (10, 11). The intention of this proposal is therefore to encourage participation, critiques and contributions from the entire imaging community and all stakeholders, including research and imaging scientists, facility personnel, instrument manufacturers, software developers, standards organizations, scientific publishers, and funders

Sequential super-resolution imaging using DNA strand displacement.

Sequential labeling and imaging in fluorescence microscopy allows the imaging of multiple structures in the same cell using a single fluorophore species. In super-resolution applications, the optimal dye suited to the method can be chosen, the optical setup can be simpler and there are no chromatic aberrations between images of different structures. We describe a method based on DNA strand displacement that can be used to quickly and easily perform the labeling and removal of the fluorophores during each sequence. Site-specific tags are conjugated with unique and orthogonal single stranded DNA. Labeling for a particular structure is achieved by hybridization of antibody-bound DNA with a complimentary dye-labeled strand. After imaging, the dye is removed using toehold-mediated strand displacement, in which an invader strand competes off the dye-labeled strand than can be subsequently washed away. Labeling and removal of each DNA-species requires only a few minutes. We demonstrate the concept using sequential dSTORM super-resolution for multiplex imaging of subcellular structures

Supporting data for Sequential Super-Resolution Imaging using DNA Strand Displacement

Sequential labeling and imaging in super-resolution fluorescence microscopy allows the imaging of multiple structures in the same cell using a single fluorophore species. In this work, we describe a method based on DNA strand displacement that can be used to quickly and easily perform the labeling and removal of the fluorophores during each sequence. Labeling for a particular structure is achieved by hybridization of site-specific antibody-bound ssDNA with a complimentary dye-labeled strand. After imaging, the dye is removed using toehold-mediated strand displacement, in which an invader strand competes off the dye-labeled strand than can be subsequently washed away. We demonstrate the concept using sequential dSTORM super-resolution for multiplex imaging of subcellular structures

Mechanism of Stx17 recruitment to autophagosomes via IRGM and mammalian Atg8 proteins

Autophagy is a conserved eukaryotic process with metabolic, immune, and general homeostatic functions in mammalian cells. Mammalian autophagosomes fuse with lysosomes in a SNARE-driven process that includes syntaxin 17 (Stx17). How Stx17 translocates to autophagosomes is unknown. In this study, we show that the mechanism of Stx17 recruitment to autophagosomes in human cells entails the small guanosine triphosphatase IRGM. Stx17 directly interacts with IRGM, and efficient Stx17 recruitment to autophagosomes requires IRGM. Both IRGM and Stx17 directly interact with mammalian Atg8 proteins, thus being guided to autophagosomes. We also show that Stx17 is significant in defense against infectious agents and that Stx17–IRGM interaction is targeted by an HIV virulence factor Nef

Phosphorylation of Syntaxin 17 by TBK1 Controls Autophagy Initiation

Syntaxin 17 (Stx17) has been implicated in autophagosome-lysosome fusion. Here, we report that Stx17 functions in assembly of protein complexes during autophagy initiation. Stx17 is phosphorylated by TBK1 whereby phospho-Stx17 controls the formation of the ATG13+FIP200+ mammalian pre-autophagosomal structure (mPAS) in response to induction of autophagy. TBK1 phosphorylates Stx17 at S202. During autophagy induction, Stx17pS202 transfers from the Golgi, where its steady-state pools localize, to the ATG13+FIP200+ mPAS. Stx17pS202 was in complexes with ATG13 and FIP200, whereas its non-phosphorylatable mutant Stx17S202A was not. Stx17 or TBK1 knockouts blocked ATG13 and FIP200 puncta formation. Stx17 or TBK1 knockouts reduced the formation of ATG13 protein complexes with FIP200 and ULK1. Endogenous Stx17pS202 colocalized with LC3B following induction of autophagy. Stx17 knockout diminished LC3 response and reduced sequestration of the prototypical bulk autophagy cargo lactate dehydrogenase. We conclude that Stx17 is a TBK1 substrate and that together they orchestrate assembly of mPAS