Cryoelectron microscope building
214628149
Simulation Based Methods for Model Building and Refinement in Cryoelectron Microscopy
Advances in cryo electron microscopy (cryo EM) have revolutionized the structural investigation of large macromolecular assemblies. In this review, we first provide a broad overview of modeling methods used for flexible fitting of molecular models into cryo EM density maps. We give special attention to approaches rooted in molecular simulations atomistic molecular dynamics and Monte Carlo. Concise descriptions of the methods are given along with discussion of their advantages, limitations and most popular alternatives. We also describe recent extensions of the widely used molecular dynamics flexible fitting (MDFF) method and discuss how different model building techniques could be incorporated into new hybrid modeling schemes and simulation workflows. Finally, we provide two illustrative examples of model building and refinement strategies employing MDFF, cascade MDFF and RosettaCM. These examples come from recent cryo EM studies that elucidated transcription preinitiation complexes and shed light on the functional roles of these assemblies in gene expression and gene regulation.
J. Chem. Inf. Model.
Thomas Dodd Chunli Yan Ivaylo Ivanov
139858095
Build Your Own Microscope: Step By Step Guide for Building a Prism Based TIRF Microscope
Prism based total internal reflection fluorescence (pTIRF) microscopy is one of the most widely used techniques for the single molecule analysis of a vast range of samples including biomolecules, nanostructures, and cells, to name a few. It allows for excitation of surface bound molecules/particles/quantum dots via evanescent field of a confined region of space, which is beneficial not only for single molecule detection but also for analysis of single molecule dynamics and for acquiring kinetics data. However, there is neither a commercial microscope available for purchase nor a detailed guide dedicated for building this microscope. Thus far, pTIRF microscopes are custom built with the use of a commercially available inverted microscope, which requires high level of expertise in selecting and handling sophisticated instrument parts. To directly address this technology gap, here we describe a step by step guide on how to build and characterize a pTIRF microscope for in vitro single molecule imaging, nanostructure analysis and other life sciences research.
Methods and protocols
Dalton R Gibbs Anisa Kaur Anoja Megalathan Kumar Sapkota Soma Dhakal
10664783
Building a high throughput microscope simulator using the Apache Kafka streaming framework
Today microscopy imaging is a widely used and powerful method for investigating biological processes. The microscopes can produce large amounts of data in a short time. It is therefore impossible t
Lovisa Lugnegard
4970652
Model Building and Refinement of a Natively Glycosylated HIV 1 Env Protein by High Resolution Cryoelectron Microscopy.
Secretory and membrane proteins from mammalian cells undergo post translational modifications, including N linked glycosylation, which can result in a large number of possible glycoforms. This sample heterogeneity can be problematic for structural studies, particularly X ray crystallography. Thus, crystal structures of heavily glycosylated proteins such as the HIV 1 Env viral spike protein have been determined by removing the majority of glycans. This step is most frequently carried out using Endoglycosidase H (EndoH) and requires that all expressed glycans be in the high mannose form, which is often not the native glycoform. With significantly improved technologies in single particle cryoelectron microscopy, we demonstrate that it is now possible to refine and build natively glycosylated HIV 1 Env structures in solution to 4.36 A resolution. At this resolution we can now analyze the complete epitope of a broadly neutralizing antibody (bnAb) PGT128, in the context of the trimer expressed with native glycans.
Structure
Jeong Hyun Lee Natalia de Val Dmitry Lyumkis Andrew B Ward
24078272
Building a tissue based molecular diagnostic system in heart transplant rejection: The heart Molecular Microscope Diagnostic (MMDx) System.
BACKGROUND The emergence of molecular systems offers opportunities for improving the assessment of rejection in heart transplant biopsy specimens. The present study developed a microarray based system for assessing heart transplant endomyocardial biopsy (EMB) specimens. METHODS We analyzed 331 protocol or for cause EMB specimens from 221 subjects in 3 centers (Edmonton, Bologna, and Paris) Unsupervised principal component analysis (PCA) and archetype analysis used rejection associated transcripts (RATs) shown in kidney transplants to be associated with antibody mediated rejection (ABMR) or T cell mediated rejection (TCMR) or both. To compare EMB specimens to kidney biopsy specimens, rejection status in both was simplified to TCMR, ABMR, or no rejection. RESULTS The pattern of RAT expression was similar in EMB and kidney specimens, permitting use of RATs to assign scores and group "cluster" membership to each EMB, independent of histology. Three clusters emerged in EMB specimens, similar to kidney specimens: TCMR, ABMR, and no rejection. This permitted each EMB specimen to be given 3 scores and assigned to 1 cluster by its highest score. There was significant agreement between molecular phenotype archetype scores or clusters and both histologic diagnoses and donor specific antibody. Area under curve estimates for predicting histologic TCMR, ABMR, and no rejection by molecular assessment were lower in EMB specimens than in kidney specimens, reflecting more uncertainty in EMB specimens, particularly in histologic diagnosis of TCMR. CONCLUSIONS Rejection associated transcripts can be used to estimate the probability of TCMR and ABMR in heart transplant specimens, providing a new dimension to improve the accuracy of diagnoses and an independent system for recalibrating the histology guidelines.
The Journal of heart and lung transplantation the official publication of the International Society for Heart Transplantation
Philip F Halloran Luciano Potena Jean-Paul Duong Van Huyen Patrick Bruneval Ornella Leone Daniel Kim Xavier Jouven Jeff Reeve Alexandre Loupy
91104743
Building a light sheet microscope to study the early development of the polyclad flatworm Maritigrella crozieri (Hyman, 1939)
The Lophotrochozoa is an evolutionary interesting clade comprising many and mostly marine phyla. Despite their diverse morphology, these animals often have a biphasic life cycle in form of a free swimming, ciliated larval stage, the trochophore, and seemingly retained a highly conserved developmental pattern called spiral cleavage. This opens the door for comparative studies to better understand the shared mechanisms of the spiralian developmental program, its deviations and the evolution of lophotrochozoan body plans. Here I studied the early development and larva formation in the polyclad flatworm Maritigrella crozieri and compared it with other members of this evolutionarily diverse group of animals. In order to conduct this research, I first built a light sheet microscope (OpenSPIM) that would allow me to follow the development of the polyclad embryo from the zygote into the larval stage and to acquire sophisticated 3d reconstructions of fixed embryonic stages. I then used this and other techniques to characterise in detail the early development of M. crozieri and its conserved spiral cleavage pattern. Precise volume measurements of 3d reconstructed early blastomeres and the investigations of associated cleavage patterns indicate that this polyclad worm may not follow a strictly equal spiral cleavage type, as was previously thought. I investigated the cleavage pattern and fate of micromere 4d, which in M. crozieri gives rise to mesoderm and generates bilaterally symmetric embryos at a cellular level. A first cell lineage analysis of this organism involved long term live imaging recordings and point to a conserved fate of blastomeres that, like in other spiral cleavers, give rise to ectodermally derived structures, specifically the larva's locomotion system (ciliary band) and a sensory organ at the apical point (apical organ) These findings strengthen the idea that these structures may be homologous to those found in other trochophore larvae. This work increased the current knowledge of the early development of the polyclad flatworm M. crozieri, which facilitates evolutionary comparisons of the development of different flatworms and lophotrochozoans more broadly and can contribute to addressing the homology of marine larvae.
Johannes Girstmair
103873048
Building with Ions in the Helium Ion Microscope
In situ direct writing by electron and ion beam from solutions opens a pathway for resistless fabrication of nanostructures at high throughput. This method utilizes the electron beam (e beam) or ion beam (ibeam) as an ionizing source that decomposes solvent (specifically water here) molecules to generate solvated electrons and other radicals in a complex cascade of reaction pathways [1,2] In water, radiolytically generated aqueous electrons (eaq act as a reducing agent when interacting with organic and inorganic compounds, this may be harnessed to guide local deposition of the material directly from the precursor. Since electron and ion beam microscopes generally operate in high vacuum, (except variable pressure scanning electron microscope) it is necessary to utilize cells to encapsulate the liquid precursor in order to prevent evaporation [3,4] These liquid cells are composed of silicon microchips and silicon nitride membranes [3,5] In situ liquid cell electron microscopy, has been used to investigate the mechanisms of electron beam induced nucleation, growth, and coalescence of nanoparticles in a variety of precursors at various concentrations [6 11] To date, most in situ liquid cell studies have been performed using a scanning electron microscope (SEM) or a (scanning) transmission electron microscope (S)TEM [12]
Microscopy and Microanalysis
Olga S Ovchinnikova
205824538
Building cell models and simulations from microscope images.
The use of fluorescence microscopy has undergone a major revolution over the past twenty years, both with the development of dramatic new technologies and with the widespread adoption of image analysis and machine learning methods. Many open source software tools provide the ability to use these methods in a wide range of studies, and many molecular and cellular phenotypes can now be automatically distinguished. This article presents the next major challenge in microscopy automation, the creation of accurate models of cell organization directly from images, and reviews the progress that has been made towards this challenge.
Methods
Robert F Murphy
59036473
Design and building of modern two photon laser scanning microscope for using in neuroscience
One of the main visualization method in neuroscience is two photon microscopy. However, scientists need to upgrade their microscopy system so regular because they are interested to get more specific data. Also this is due to the fact that this technique allows to obtain 3D images of tissues due to laser focus change, that is possible due to substantially greater penetration depth on the main wavelength into biological tissues. Self developed microscopy system allow to modify the construction of microscope in not complicated manner depending on specialized experimental models and scientific tasks. This work is small attempt to help researchers who are interested to build their own two photon laser scanning microscope for using in neuroscience. The work was performed with financial support of the government represented by the Ministry of Education and Science of the Russian Federation, the unique identifier of the project is RFMEFI57814X0079, the agreement on granting a subsidy No. 14.578.21.0079 dated 28.11.2014.
Maxim Doronin Alexander Popov
15704565
Rethinking the Microscope Building a Microscopic Microscope
icroscopes are ubiquitous in biology and clinical analysis laboratories. They are the workhorses of bioscience research and our surrogate eyes into the wondrous world of microbes and cells. Yet, since the development of the microscope beginning about 1590 by two Dutch spectacle makers, Hans and Zaccharias Janssen, then by Galileo and Antonie van Leeuwenhoek, the basic microscope design has undergone very few fundamental changes over the intervening centuries. In almost all microscopes you can expect to fi nd precise and expensive optical lenses and plenty of space for the light to travel and reshape itself the long distance between the objective lens and the eyepiece lens isn't there for aesthetic reasons! The antiquated nature of the conventional microscope design stands out even more in the context of the ongoing lab on a chip research and development, in which laboratory instruments are being systematically miniaturized into chip size devices. While a wide range of bioanalysis methods have been successfully miniaturized and implemented in a lab on a chip format, there has not been a commercially viable approach to miniaturizing microscopy until recently. The diffi culty is twofold. First, there isn't a cheap and effi cient way to create small and precise optical lenses on chips easily. Second, the space requirements of conventional microscopy confl ict with the size constraints of chip based devices. These diffi culties aside, we can ponder " what if? " situations for miniature microscopes. An on chip microscope implementation method can dramatically change the way we use microscopes. The application range of an on chip microscope is wide and will be discussed below at this junction, we would like to point out that a typical bioscience laboratory contains less than ten microscopes (size and cost are both factors in this) We invite the reader to consider the enhanced effi ciency if the number of microscopes per laboratory is to increase by a factor of ten or a hundred. To implement a cost effective and commercially viable on chip microscope, it was necessary to break with tradition, abandon the old microscope design, and rethink the whole imaging problem from the ground up. Fortunately, optical technology has come a long way since the 16th century, and we now have access to a broader range of devices than existed even 50 years ago! To motivate this redesign process, let us enumerate the functions that the microscope must perform. There are three Do you see fl oaters drifting in your eyes when you look
By Yang Demetri Psaltis