Computers & Software

There is a wide variety of software packages available to LMCB staff for image processing and analysis. Some software is free and can be installed on any computer including personal laptops but other software must be paid for and is either only installed on certain computers or is available under a network licence scheme where any computer able to connect to the licence server can run the software. The information below deals only with software used for scientific image processing and analysis. I have not included photo-editing software such as Adobe Photoshop and Gimp, or vector graphics software such as Adobe Illustrator and Inkscape, although there is a certain amount of overlap between the functions - especially when it comes to preparing images for publications and posters.

The LMCB light microscopy and electron microscopy facilities have an image processing suite currently consisting of 6 computers located in room 2.19A in the Medawar Building. There are a number of different image analysis software packages installed on the machines. The list below provides further information about the specifications of the computers and the software installed. Computers must be booked using the and training can be provided for anyone unfamiliar with the software. I have only included commercially available software in the list but the computers will also have Fiji or ImageJ installed.

Image Processing Software

Image J

Licence: FREE

Operating Systems: Windows, Mac OS, Linux

Key features: Multi-platform. Routine processing, analysis and measurement of multi-channel and colour 2D and 3D stacks, time-series

ImageJ is probably the most commonly used software for scientific image processing. It is free and runs on all platforms, which means anyone can run it anywhere. It has a set of basic image processing tools that will allow you to carry out such procedures as merging colour channels, editing stacks and making movies. You can threshold images and stacks and segment and measure features. You can then export the resulting data in a variety of formats (e.g TIFF, AVI, CSV). Much of the power of ImageJ comes through the plugins written by the ImageJ community. ImageJ has a built-in plugin language and editor if you know Java but if you want to create processing and analysis routines based on existing code it has a macro language and a macro recorder for linking together pre-existing tasks in a sequence. ImageJ is the basis of the Fiji analysis suite, μmanager device control software and OpenSPIM light sheet software.

ImageJ does not open all manufacturers' proprietary image file formats 'out of the box' but if you install the BioFormats Importer plugin this will read a huge and ever expanding range of formats. Specific analysis procedures tend to need specific plugins, which means one person's ImageJ installation can be very different to another's. This makes troubleshooting difficult, which is why I tend to remommend Fiji for most purposes. Although ImageJ opens and processes 3D and 4D data very well it's tools for 3D rendering and visualisation have not been as easy to use as those in other software when I have tried them.

ImageJ homepage at NIH

ImageJ online tutorials

Fiji

Licence: FREE

Operating Systems: Windows, Mac OS, Linux

Key features: Multi-platform. Routine processing, analysis and measurement of multi-channel and colour 2D and 3D stacks, time-series. Updater, plugins include BioFormats Importer

Fiji is an extended version of ImageJ with two major advantages. Firstly, it has an updater that allows you to keep versions constant, making troubleshooting at lot easier. Secondly, it comes bundled with pre-installed plugins (including the aforementioned BioFormats Importer) so many analysis features are already in there and you don't need to install them yourself. If you do need to install plugins this is just as easy as it is for ImageJ. I currently recommend Fiji as the software that all staff members should use for basic image processing procedures.

Fiji comes bundled with plugins but these seem to be heavily weighted towards certain applications that aren't necessarily cell biological. This probably simply reflects who is writing software for it. It is therefore likely that from time to time you will need to instal a plugin. This may be fine but some plugins may have dependencies (e.g. Java 3D) that aren't necessarily installed with Fiji.

Fiji homepage

Fiji online tutorials

Volocity

Licence: 500 licences for the Volocity core program on the LMCB network. 3 licences each for the Restoration, Visualization and Quantitation modules

Operating Systems: Windows and Mac OS

Key features: 3D surface rendering, volume rendering and ray tracing. Identify and measure 3D objects and object properties. Deconvolution.

Volocity natively analyses data sets in 3D rather than as stacks of 2D slices. This means that measurements automatically refer to 3D objects rather than single slices and are given in 3D units (e.g. cubic microns). Volocity consists of a core programs that allows you to open your data, carry out some basic processing and export in a variety of formats. If you want to carry out advanced processing in Volocity you need to use one of the analysis modules: Restoration, Visualization and Quantitation. Restoration is a deconvolution package of most use with high quality confocal data. Visualization is a 3D reconstruction and rendering package with volume and surface rendering tools and a ray tracer. Quantitation is a measurement package that can be used to apply a 'pipeline' of analysis steps to process data and extract measurements. Basically this means thresholding and segmentation, binary style procedures like erosion and dilation, object identification, filtering by object features, measurement and colocalisation, etc.

I find that the Restoration module is not great at deconvolving data with lower signal to noise ratios, so I tend to use Huygens and AutoQuant for deconvolution. It is difficult to set up an analysis pipeline using the Quantitation but all such tools are difficult and require a lot of trial and error, so this isn't unusual. The analysis and charts tools in the Quantitation module are not intuitive at all so most of the time I export data as a spreadsheet for analysis in other packages (e.g. Excel). The Tracking tools for measuring objects moving over time seem to be a lot more flexible and user friendly in Imaris.

How to get a copy of Volocity to use on your computer (LMCB network only)

Volocity homepage

Volocity online tutorials

Volocity Registration Correction

Volocity Acquisition Quick Start

Imaris

Licence: Single licence installed on Beast analysis computer

Operating Systems: Windows (but can also run on Linux)

Key features: 3D surface and volume rendering. Identify and measure 3D objects and object properties.

Imaris is a 3D rendering and quantification software package. 3D rendering and identification of objects, spots and other features are done using the Surpass view, which will probably be the one you use the most. Imaris does not have a built-in deconvolution package (Bitplane use AutoQuant for that) but it does have the advantage that measurements can be made on the 3D rendered volume itself, which is something that isn't possible in Volocity. It is also possible to use MatLab and Fiji macros and plugins using ImarisXT. The version of Imaris we have also has a Filament Tracer module, which is designed for tracing neurons but that could also be used for branching blood vessels. We are running Imaris 7.7 and have the following modules: MeasurementPro, ImarisTracking, ImarisColoc, ImarisXT, FilamentTracer and ImarisVantage.

Bitplane homepage

Bitplane Learning Zone (includes video tutorials)

Huygens

Licence: Huygens Essential wide-field, confocal and spinning disc on WOPR computer. Huygens Professional confocal and STED on STED and Orac computers

Operating Systems: Windows (but can also run on Mac OS and Linux)

Key features: Deconvolution, but also has tools for 3D reconstruction and visualisation.

Scientific Volume Imaging's Huygens is a modular deconvolution software package. It has a core program that can be used to open, visualise and export data but additional modules must be installed to deconvolve wide-field, confocal, spinning disc and STED data. Multi-photon data can also be deconvolved but we don't have this module yet. We have Huygens on three machines but the licences vary, so you may not be able to deconvolve your data on all the machines (e.g. wide-field, multi-photon and spinning disc data can only be deconvolved on WOPR, confocal data can be deconvolved on all three machines). Additional modules for 3D rendering and image analysis are available but are not installed on our systems so Huygens is mostly used for deconvolution. Huygens is my preferred deconvolution package because it handles low signal-to-noise ratio images well, it is easy to edit parameters in the deconvolution routine and SVI provides a lot of information on their website. You have to register with SVI and get a login to access much of the content on the website.

Scientific Volume Imaging homepage

SVI's YouTube channel - video tutorials

Nikon NIS-Elements

Full NIS-Elements offline packages are available on Son of Beast and Deep Thought. The software is available for PC only but a free NIS-Elements Viewer for PC or Mac is available for download from Nikon's website

Analysis Computers

Beast

Processor: 2x Intel XEON CPU X5650 2.67 GHz, 6 cores, 12 logical processors

RAM: 96 GB

Software: Imaris 7.7, Volocity 6.3, Amira 5.4.1, LAS AF Lite 2.4.1

Contact Jemima Burden or Ian White for training on Amira and Andrew Vaughan for Imaris, LAS AF and Volocity. This machine is administered by the Electron Microscopy Facility and booking is through the MRCLMCB_EM Faces Scheduling System group.

Son of Beast

Processor: 2x Intel XEON CPU X5650 2.67 GHz, 6 cores, 12 logical processors

RAM: 96 GB

Software: Volocity 6.3, Amira 5.4.2

Contact Jemima Burden or Ian White for training on Amira and Andrew Vaughan for training on Volocity. This machine is administered by the Electron Microscopy Facility and booking is through the MRCLMCB_EM Faces Scheduling System group.

Zen (UCL Super-Resolution Facility)

Processor: 2x Intel XEON CPU E5-2643, 3.3 GHz, 4 cores 8 logical processors

RAM: 64 GB

Software: Zen 2012 SP1 (Black Edition) release version 8.1

Contact Andrew Vaughan for training. This machine is administered by the Super-Resolution Facility (SuRF) and booking is through the MRCLMCB_LSM Faces Scheduling System group.

Orac (UCL Super-Resolution Facility)

Processor: Intel Core i7-4770K CPU, 3.5 GHz, 4 cores 8 logical processors

RAM: 16 GB

Software: LAS AF 3.3.0, Huygens Professional 14.10 (confocal and STED licence)

Contact Andrew Vaughan for training. This machine is administered by the Super-Resolution Facility (SuRF) and booking is through the MRCLMCB_LSM Faces Scheduling System group.

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