CryoSIM: super-resolution 3D structured illumination cryogenic fluorescence microscopy for correlated ultrastructural imaging

Rapid cryopreservation of biological specimens is the gold standard for visualizing cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo-electron microscopy or cryo-soft X-ray tomography. We provide the full instructions for replicating the instrument mostly from off-the-shelf components and accessible, user-friendly, open-source Python control software. Therefore, cryoSIM democratizes the ability to detect molecules using super-resolution fluorescence imaging of cryopreserved specimens for correlation with their cellular ultrastructure.Funding: Wellcome Trust (091911/Z/11/Z, 096144/Z/11/Z, 105605/Z/14/Z, 107457/Z/15/Z, 203141/Z/16/Z, 209412/Z/17/Z); H2020Marie Skłodowska-Curie Actions (700184)

Characterisation of core histone sequences and nuclear mobility using a reproducible research approach

Chromatin is a dynamic complex that controls access to genetic information by undergoing structural reconfigurations. Understanding this dynamic can provide insights into the biological implications of chromatin organisation. We have undertaken a detailed catalogue of the human core histone genes and contributed to their annotations. Based on the reproducible research concept, we produced this catalogue with a system that is capable to generate new up to date manuscripts as a model for similar projects which can be continually improved along with genome annotations. As proof of concept, we used the same project to produce a catalogue of the current mouse histone genes. Quantitative fluorescence microscopy has been used extensively to obtain insights into the dynamics of multiple proteins in live cells. Despite many advances in model design, fluorophores, and imaging capabilities, limitations are still encountered that can lead to misinterpretation of data. By using histone proteins with extremely slow exchange rates we have tested the limitations of Fluorescence Recovery After Photobleaching (FRAP) and developed approaches to overcome some of them. Importantly, we show that movement of chromatin precludes measurements of histone dynamics on the FRAP time scale. To achieve these results we made contributions to multiple free software projects including Octave, BioPerl, and Debian. This included implementing new algorithms, refactoring code for efficiency and consistency, creating maintenance support tools, and packaging software for ease of installation by users. A core theme of this work was to create build systems capable of processing primary data from public databases or microscopy in a completely transparent way to generate complete manuscripts as implementations of the reproducible research concept. This thesis itself is an example of the approach. In these studies we have tested the limits and developed new approaches to existing methods of chromatin analysis by designing novel reagents and software for the field of chromatin dynamics

BeamDelta: simple alignment tool for optical systems

BeamDelta is a tool to help align optical systems. It greatly assists in assembling bespoke optical systems by providing a live view of the current laser beam position and a reference position. Even a simple optical setup has multiple degrees of freedom that affect the alignment of beam paths. These degrees of freedom rise exponentially with the complexity of the system. The process of aligning all the optical components for a specific system is often esoteric and poorly documented, if it is documented at all. Alignment methods used often rely on visual inspection of beams impinging on pinholes in the beam path, typically requiring an experienced operator staring at diffuse reflections for extended periods of time. This can lead to a decline in accuracy due to eye strain, flash blindness as well as symptoms such as headaches and, possibly, more serious retinal damage. Here we present BeamDelta a simple alignment tool and accompanying software interface which allows users to obtain accurate alignment as well as removing the necessity of staring at diffuse laser reflections. BeamDelta is a robust alignment tool as it doesn't require any precise alignment itself

SPEKcheck - fluorescence microscopy spectral visualisation and optimisation

<pre>SPEKcheck is a web application to visualise the efficiency of the light path in a fluorescence microscope. It can run locally and completely offline, or it can be configured to run in a site. SPEKcheck models an optical setup as a detector, a dye, an excitation source, and an arbitrary number of filters in both the excitation and emission paths. SPEKcheck then reports values for the excitation efficiency of the dye, the collection efficiency of the emitted fluorescence, and a 'brightness' score, all easy comparison between different fluorescent labels. It also displays a spectral plot of various components, and the final emitted fluorescence.</pre

The Drosophila Pericentrin-like-protein (PLP) cooperates with Cnn to maintain the integrity of the outer PCM

Centrosomes comprise a pair of centrioles surrounded by a matrix of pericentriolar material (PCM). In vertebrate cells, Pericentrin plays an important part in mitotic PCM assembly, but the Drosophila Pericentrin-like protein (PLP) appears to have a more minor role in mitotic fly cells. Here we investigate the function of PLP during the rapid mitotic cycles of the early Drosophila embryo. Unexpectedly, we find that PLP is specifically enriched in the outer-most regions of the PCM, where it largely co-localizes with the PCM scaffold protein Cnn. In the absence of PLP the outer PCM appears to be structurally weakened, and it rapidly disperses along the centrosomal microtubules (MTs). As a result, centrosomal MTs are subtly disorganized in embryos lacking PLP, although mitosis is largely unperturbed and these embryos develop and hatch at near-normal rates. Y2H analysis reveals that PLP can potentially form multiple interactions with itself and with the PCM recruiting proteins Asl, Spd-2 and Cnn. A deletion analysis suggests that PLP participates in a complex network of interactions that ultimately help to strengthen the PCM

Microscope-Cockpit: Python-based bespoke microscopy for bio-medical science [version 2; peer review: 2 approved, 2 approved with reservations]

We have developed “Microscope-Cockpit” (Cockpit), a highly adaptable open source user-friendly Python-based Graphical User Interface (GUI) environment for precision control of both simple and elaborate bespoke microscope systems. The user environment allows next-generation near instantaneous navigation of the entire slide landscape for efficient selection of specimens of interest and automated acquisition without the use of eyepieces. Cockpit uses “Python-Microscope” (Microscope) for high-performance coordinated control of a wide range of hardware devices using open source software. Microscope also controls complex hardware devices such as deformable mirrors for aberration correction and spatial light modulators for structured illumination via abstracted device models. We demonstrate the advantages of the Cockpit platform using several bespoke microscopes, including a simple widefield system and a complex system with adaptive optics and structured illumination. A key strength of Cockpit is its use of Python, which means that any microscope built with Cockpit is ready for future customisation by simply adding new libraries, for example machine learning algorithms to enable automated microscopy decision making while imaging

Microscope-Cockpit: Python-based bespoke microscopy for bio-medical science

We have developed "Microscope-Cockpit" (Cockpit), a highly adaptable open source user-friendly Python-based Graphical User Interface (GUI) environment for precision control of both simple and elaborate bespoke microscope systems. The user environment allows next-generation near instantaneous navigation of the entire slide landscape for efficient selection of specimens of interest and automated acquisition without the use of eyepieces. Cockpit uses "Python-Microscope" (Microscope) for high-performance coordinated control of a wide range of hardware devices using open source software. Microscope also controls complex hardware devices such as deformable mirrors for aberration correction and spatial light modulators for structured illumination via abstracted device models. We demonstrate the advantages of the Cockpit platform using several bespoke microscopes, including a simple widefield system and a complex system with adaptive optics and structured illumination. A key strength of Cockpit is its use of Python, which means that any microscope built with Cockpit is ready for future customisation by simply adding new libraries, for example machine learning algorithms to enable automated microscopy decision making while imaging

CryoSIM: super-resolution 3D structured illumination cryogenic fluorescence microscopy for correlated ultrastructural imaging.

Rapid cryopreservation of biological specimens is the gold standard for visualizing cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo-electron microscopy or cryo-soft X-ray tomography. We provide the full instructions for replicating the instrument mostly from off-the-shelf components and accessible, user-friendly, open-source Python control software. Therefore, cryoSIM democratizes the ability to detect molecules using super-resolution fluorescence imaging of cryopreserved specimens for correlation with their cellular ultrastructure

ome/bioformats: v7.1.0

Bio-Formats is a Java library for reading and writing data in life sciences image file formats. It is developed by the Open Microscopy Environment. Bio-Formats is released under the GNU General Public License (GPL); commercial licenses are available from Glencoe Software

ome/bioformats: v6.12.0-m1

Bio-Formats is a Java library for reading and writing data in life sciences image file formats. It is developed by the Open Microscopy Environment. Bio-Formats is released under the GNU General Public License (GPL); commercial licenses are available from Glencoe Software