Focus Documentation 1_0

1_0:2dx_image_processing

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Image Processing of 2D Crystal Images Electron crystallography of membrane proteins uses cryo-transmission electron microscopy to image frozen-hydrated/sugar embedded 2D crystals. The processing of recorded images exploits the periodic arrangement of the structures to extract the amplitudes and phases of diffraction spots in Fourier space. However, imperfections require a crystal unbending procedure to be applied to the image before evaluation in Fourier space. Here we describe the image proces…

1_0:2dx_merge2d

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

2D Merging In the previous sections we have seen how to process individual images of 2D crystals. From a single micrograph we generate a list of spots containing their amplitudes and phases in Fourier space. This section describes how to merge the projection maps from each micrograph of an non-tilted specimen in order to produce one common projection map. This is similar to creating class averages in single particle electron microscopy, where projections of particles with the same orientation a…

1_0:2dx_merge3d

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

3D Merging Theory The general goal of electron transmission imaging techniques such as Electron Crystallography, Tomography or Single Particle reconstruction is to gain a 3D computer model by integrating sets of 2D data recorded by an electron microscope. The common mathematical tool that all these techniques share is the Central Section Theorem. The theorem states that the projection along the z direction of a three dimensional volume contains the same information as the central slice in the…

1_0:2dx_refine_merge3d

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Refining Merged Map The quality of maps generated using 2D electron crystallography is limited owing to various factors including: (i) the problem of missing cone which occurs due to the fact that micrographs can be recorded until a certain tilt angle typically up to 60 degrees; (ii) the images of tilted samples have much lower quality than those of non-tilted ones; (iii) the current methods such as Lattice line fitting used to generate 3D data lack reliability. An iterative method applying kno…

1_0:2dx_test_data

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

MloK1 2D crystal image: Cryo-EM, K2 camera, counting mode, 3808 x 3808 pixels: * * * * * * * * * * * AmtB 2D crystal image: negative stain, 2k, scanned film: * MloK1 2D crystal image: cryo-EM, 4k, scanned film:

1_0:add-project

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Adding a project Projects can be conveniently added and accessed using the Project Wizard. Use this step by step guide to create a new project. Launch Focus Focus can be launched using various ways. See this page for details. Once launched, you will see Project Wizard of Focus.

1_0:download

Anonymous (anonymous@undisclosed.example.com) — 2021-09-21T16:37:02+00:00

Downloading Focus software The Focus software needs to be installed from the source code. Installation from source code

1_0:drift-correction

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Drift correction using Focus

1_0:external-tools

Anonymous (anonymous@undisclosed.example.com) — 2025-05-22T16:10:50+00:00

Setting up external software tools Focus supports a lot of image processing tasks in cryo-EM. To carry out many of these tasks, we depend on the great free software developed by the community. Some of these are essential for the functioning of Focus and others are optional. The required packages also depend on the technique you use for reconstruction. Here we list all the packages categorised as required and optional.

1_0:faq

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Frequently asked questions Interface How can I know more about a parameter? To know more information about a parameter, please hover on the legend of the parameter. It will open a tooltip revealing more information. Sometimes, there is online help also provided. In that case, a icon on the right of the field will navigate you to the appropriate link.

1_0:hardware_linux

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Hardware recommendation for Linux For a LINUX installation, we recommend UBUNTU and the following hardware For all configurations, we recommend a large 4k monitor. This can on the UBUNTU machines be directly connected to one of the GPUs. For online-processing per hour of up to 120 movies of 8k x 8k x 50 frames with MotionCor2, CTFFIND4 or gCTF and subsequent programs:

1_0:hardware_mac

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Hardware recommendation for Mac OS X computer for offline processing of 2D crystal images or movies: * MacBook Pro (Quadcore) or Mac Pro * 250 GB free space on HD * 16 GB RAM or more * OSX 10.11 or higher

1_0:import_images

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Import images and movies The import procedure involves copying raw data files into the project directory, assigning and determining a set of parameters for each image, and optionally running one or several scripts for each image during and/or after import. This process is handled by the Import Tool. The tool can be configured to automatically fetch newly recorded stacks throughout the microscopy session from a network-mounted storage location. A delay timer can be set to ensure that files are o…

1_0:install-source

Anonymous (anonymous@undisclosed.example.com) — 2025-05-17T11:45:13+00:00

Installation from source code In case a package for your system is not available or the standard package installation does not work, Focus can be installed using the source code. The compilation of Focus has some additional dependencies compared with the precompiled binary packages. Focus depends on Qt5 and FFTW to compile and additionally requires cmake and GNU compilers like

1_0:launch-focus

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Launching Focus Focus, once installed, can be launched via command line or via the desktop files in Linux and .app files in macOS. Using Command Line First, locate the installation directory. Assuming that Focus is installed in , Focus can be launched via the executable file in the bin directory of the

1_0:locating-installation

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Default installation directories The page provides the default installation paths which we use for various installation methods. Using package for macOS If you installed Focus using one of the packages for macOS, you will find Focus installed in:

1_0:main-interface

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Introduction to GUI Navigation in Focus GUI is done via the buttons that appear on the top bar. [The main window] Universal Buttons Following buttons are shown always: Project Library [Project Library Icon] Project Library consists of all the micrographs that were imported in the project. The micrographs are grouped by their name into “Image Folders”. You can select individual micrographs by clicking the checkbox in front of the image name. You can also select an entire folder by selecting…

1_0:manual_process

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Manually processing an image Once the images are imported, they are listed in the “Project Library” tab. Manual processing allows to fine tune the parameters for these images. Open image for processing The image to be processed can be opened from the

1_0:movies

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Introductory and Tutorial Movies A number of movies is available that demonstrate installation, setup, and running FOCUS. The full playlist can be found here General Downloading and Setup Compilation Processing Manual processing of one movie Basic batch processing

1_0:parallel_process

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Parallel Processing The batch-queue processor allows images to be processed in parallel in a high-throughput manner. An image along with the processing workflow (a set of scripts) defines a batch. Processing batches are added to the processor queue in the order in which they are issued and processed with the policy of ‘first come first served’, i.e., the oldest batch is processed first. Alternatively, batches can be added to the top of the processing queue if the user wants to see the result fo…

1_0:project-library

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Project Library

1_0:project_library

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Managing Images using Project Library The Project Library provides a summary of all images. These are listed in a tree structure according to the image group they belong to, with one image per horizontal line. Image statistics are shown in columns, like in an Excel sheet; users can manually choose which parameters are displayed. The Project Library also displays a set of preview images for a selected image (a selected row in the table). These appear as thumbnails on the right of the table. To e…

1_0:remote-monitoring

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Remote Monitoring using Focus

1_0:remote_collect_data

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Collection data from Focus The script “Process > Push status data” can be used to collect data such as defocus, drift and iciness. The data is collected in the local status folder and is uploaded with the other files using the upload_status script.

1_0:remote_focus_settings

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Configuring Focus for remote monitoring Focus has to be notified to collect the data. The data is first collected in a folder locally which is then pushed to server. Writing the files The settings to collect data can be changed in the Preferences window of Focus which looks like following:

1_0:remote_server

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Setting up server Focus remote monitoring works on an Apache server. An Apache server is very easy to setup and there are a lot of tutorials on the web which provides this documentation. Following link provides official documentation: Apache HTTP Documentation Once the server is setup,

1_0:remote_status_apps

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Accessing the remote monitoring via mobile apps iOS (iPhone, iPad) * Open the remote server page on Safari. * Tap the share button on the bottom (the one with up arrow) * Choose Add to Home Screen * Choose the appropriate title, or use default

1_0:user-scripts

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Adding new scripts

1_0:user-settings

Anonymous (anonymous@undisclosed.example.com) — 2020-10-22T16:28:15+00:00

Configuring user settings Settings such as software paths, microscope settings, system settings and file viewers can be set once for the user. These settings do not vary from one project to other and should be set after creating the first project. The settings manager can be invoked from the right hand side of the main navigation panel.