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Light microscopy

State-of-the-art microscopy is a vital tool in understanding the critical mechanisms of cell growth and division. We provide the latest in live cell imaging technology, enabling ICR scientists to study these processes in real time and immaculate detail.

In our world-class microscopy suites, researchers have access to a fleet of temperature and CO2-controlled microscopes capable of performing high-resolution confocal and super-resolution microscopy, single-molecule and multi-position time-lapse experiments.

In 2017, the ICR became the first research organisation in the UK to acquire a lattice light sheet microscope. Using ultra-thin lattices of Bessel beams, the instrument enables very rapid imaging of cellular sub-sections without the effects of photo toxicity.

“By working with industry to acquire the lattice light sheet scope, we can now analyse highly dynamic processes such as cell division with unprecedented resolution in both time and space.”

Professor Jon Pines, Head of Cancer Biology

For more information, contact Kai Betteridge, Light Microscopy Facility Manager (Chelsea) or Louise Howell, Light Microscopy Facility Manager (Sutton).

Light Microscopy Facility Chelsea

The light microscopy facility in Chelsea is managed by Kai Betteridge and supported by Ana Stojiljkovic.

It consists of 13 State of the art microscopes for live and fixed cell analysis. The facility is composed of 3 parts:

  1. The advanced light microscopy facility
  2. The high content screening facility
  3. A state of the art image analysis suite

The Advanced Light Microscopy Facility Equipment

Two widefield microscopes

For widefield imaging of samples and for long time lapse experiments of live cells.

  • Marianas 100 Widefield Fluorescent Microscope:
    The Mariana 100 widefield is a Zeiss Axio Observer Z1 MarianasTM Microscope attached with a Cool LED pE 300 fluorescence excitation source. The system is equipped LED excitation light sources at wavelengths best suited for live cell imaging (405nm, 488nm, and 561nm, and 647).. Bright field imaging is set up on this system and it is capable of Differential Interference Contrast (DIC) imaging. The system is also equipped with a colour camera, which makes it suitable for looking at standard histology specimens, or any experiment that may need to detect colour using transmitted light.
    Marianas 100 Widefield
  • Nikon Eclipse Ti Widefield Fluorescent Microscope: 
    The Nikon widefield is a Nikon Eclipse Ti inverted Microscope attached with a Cool LED pE 300 fluorescence excitation source. The system is equipped LED excitation light sources at wavelengths best suited for live cell imaging (405nm, 445, 488nm, 515, and 561nm, and 647). Bright field imaging is set up on this system and it is capable of phase imaging. The equipment is also set up and built around a Hypoxia set up and is connected to a 3 way gas mixer that can mix 100% N2, Air (with 10% CO2), and 100% CO¬2. The OKOlabs LEO console is a pump that measures CO2 and O2 within the incubation chamber during imaging allowing you to mix gases for Hypoxia conditions.
    Nikon Eclipse Ti Widefield microscope

Three confocal microscopes

For different confocal imaging and for time-lapse experiments of live cells.

  • Basic spinning disk confocal microscope:
    The Basic Spinning Disk is a Zeiss Axio Observer Z1 MarianasTM Microscope attached with a CSU-X1 spinning disk unit built by Intelligent Imaging Innovations (3i). The system is equipped with 6 solid state lasers at wavelengths best suited for live cell imaging and FRET (405nm, 445nm, 488nm, 525nm, and 561nm, and 640nm).
    Basic spinning disk confocal
  • Advanced spinning disk confocal microscope:
    The Advanced Spinning Disk is a Zeiss Axio Observer Z1 MarianasTM Microscope attached with a CSU-W spinning disk unit built by Intelligent Imaging Innovations (3i). The system is equipped with 6 solid state lasers at different wavelengths for a range of imaging modalities (405nm, 445nm, 488nm, 525nm, 561nm, and 647nm). The system is also equipped with a 355nm UV laser which can be controlled simultaneously with the spinning disk (using an mSwitcher). The UV laser and any of the other solid state lasers can be quickly scanned over specified Regions Of Interest (ROI’s) during spinning disk image acquisition using a diffraction limited high speed X, Y scanner called a VectorTM , this allows interactive examination of living specimens using photoactivation, photoablation, and Florescence Recovery after Photobleaching (FRAP). This feature is most commonly used by our users to locally cause DNA damage and observe the effects of this damage in real time.
    Advanced spinning disk confocal microscope
  • Leica SP8 Point Scanning Confocal Microscope: 
    The Leica SP8 is a point scanning confocal microscope ideal for imaging fixed cells and tissue mounted to a slide. It is equipped with 4 gas lasers with different wavelengths for a range of imaging modalities (405nm, 445nm/488nm 561nm, and 647nm). The microscope has a fully adjustable pinhole and uses a combination of Photomultiplier tubes (PMT’s) and Hybrid Detectors (HyD) for detecting image.
    Leica SP8 Point Scanning Confocal Microscope

Three light sheet microscopes

  • Lattice light sheet microscope: 
    The lattice light sheet utilises extremely thin lattices (0.5 μm) made from Bessel beams to illuminate the sample. The thin sheet makes it possible to sub-section through a cell but still collecting all the light. The main benefits are extremely low photo toxicity and extremely fast imaging. The system does support one super resolution method, structured illumination microscopy (SIM).
    Lattice light sheet microscope 
  • Marianas Light Sheet (MLS):
    The MLS is a Dual-View inverted Single Plane Illumination Microscope (DiSPIM) that uses a Gaussian light sheet. Because it is possible to image from two sides this microscope can provide images with iso-tropic resolution. It is very useful for larger structures such as organoids as well as for single cells.
    Marianas Light Sheet (MLS)
  • The Cleared Tissue Lightsheet (CTLS):
    The CTLS is a dual-side illumination lightsheet microscope designed for imaging whole cleared organs or tissues. It incorporates a macro zoom microscope with high NA objectives and a Spatial Light Modulator (SLM) to optimise lightsheet for high resolution imaging.
    The Cleared Tissue Lightsheet (CTLS)

One TIRF microscope

  • Total internal reflection fluorescence (TIRF):
    The TIRF is a Zeiss Axio Observer Z1 MarianasTM Microscope attached with a 3i Vector TIRF for Total Internal Reflection of Fluorescence Microscopy. The microscope is also capable of super resolution imaging (STORM and PALM).
    Total internal reflection fluorescence

The High Content Screening Facility Equipment

Four plate based high-throughput microscopes

  • Celigo S: Widefield system that can scan plates very fast. This instrument also comes with an automated plate loading robot and is great for performing very large screens quickly.
    Celigo S
  • Opera Phenix Plus: High content spinning disc confocal microscope. This instrument comes with an automated plate loading robot as well as a liquid handling system. It is ideal for performing very large high content screens with unrivalled resolution and speed.
    Opera Phenix Plus
  • ImageXpress: High content spinning disc confocal microscope.
    ImageXpress
  • Oblique Plane Microscope: Homebuild microscope for automated lightsheet imaging of multiwell plates. This instrument has been built as part of a collaboration between Christopher Dunsby’s Lab (at Imperial College London) and Chris Bakal’s Lab (ICR).

The Image Analysis Suite Equipment and Software

Dedicated image analysis computers

  • Analysis Computer 1: for open source analysis and use of MetaXpress and Slidebook
  • Analysis Computer 2: for open source analysis and use of slidebook. This computer is specially designed for visualising and processing data acquired on theb lightsheet microscopes
  • Celigo Analysis computer: For offline analysis of Celigo data
  • MetaXpress Analysis computer: For offline analysis of data captured on the Image Xpress
  • Aivia Analysis Computer: For use of the commercial software Aivia – AI powered 3D segmentation and visualisation
  • Imaris Analysis Computer: For use of the commercial software Imaris –3D segmentation and visualisation

Commercial software

  • SlideBook: Image acquisition and analysis software from 3i.
  • Aivia: A single license is available in the facility for this software. Particularly useful for rendering of very large images (e.g. lightsheet). Also good for AI powered segmentation of 3D data
  • MetaXpress: Operates the ImageXpress system, and can also be used for image analysis. 
  • Harmony: Similar to MetaXpress, Harmony is the software used to run the Operetta system, and can be used for analysis.
  • Signals Image Artist (SiMA): Separate analysis software from Perkin Elmer to analysis data from the Operetta, which can also be used for data from other systems. This belongs to Professor Chris Bakal's group but can be used by the users of the facility.
  • Celigo: Operates the Celigo plate-based imager and can be used to analyse the data.
  • Imaris: A single licence for 3D segmentation and visualisation of 3D images. Particularly useful for lightsheet data.
  • Volocity: Flexible image analysis software for visualisation and analysis of data from any system.

Open-source software

  • FIJI (ImageJ): Perhaps the most popular image analysis software, with hundreds of plugins to carry out nearly every kind of image analysis. Fairly easy to use (with no prior experience needed), but requires some trial and error.
  • Cell Profiler and Cell Profiler Analyst: Complementary to FIJI in that Cell Profiler allows automatic analysis of thousands or even millions of images, particularly from high-content, plate-based systems. Cell profiler analyst is designed to work with data processed with Cell Profiler and can be used for automatic, machine-learning based image classification.
  • Python/Napari: a python based open-source platform for visualising 2D and 3D images during automated analysis pipelines using python scripts.
  • Ilastik: Has a number of features, but the unique feature is pixel-based classification. This is a machine learning approach which is useful when features of interest cannot be detected using intensity alone (such as brightfield images).

Other resources

  • Custom scripting (e.g. MATLAB/Python): For any image analysis that is not well catered to by existing software packages, custom programs can be written to perform image processing, image analysis and statistical analysis.
  • High performance computing (HPC): For any analysis that is impractical to run on desktop PCs such as huge screening experiments, or light sheet imaging with large individual file sizes, HPC can be used to greatly speed up parallelisable computational problems.
  • ICR research data storage (RDS): Data storage in the facility is temporary and so scientific computing provides high capacity storage when the needs of a lab outgrow local solutions (e.g. external hard drives).

Sutton facility

The Sutton Light Microscopy Facility is managed by Louise Howell. It consists of 6 advanced light microscopes geared towards state of the art automated slide scanning and live cell confocal and fluorescence imaging.

Facility Equipment

Confocal microscopes

  • Zeiss LSM980 with Airyscan 2: 
    The Zeiss LSM980 Airyscan 2 is built on a fully motorised Axio Observer 7 inverted microscope stand with environmental chamber for control for temperature and C02 and uses Zen blue 3 acquisition and analysis software. It has a fast multiplexed Airyscan 2 detector which allows super-resolution imaging (1.7x increase in spatial resolution compared to confocal) at faster speed. It is suitable for imaging fixed cells/tissues and live cell imaging/time courses, photo-manipulation techniques such as FRAP, imaging in multiple positions and stitching of large areas. The system is equipped with 7 solid state lasers at different wavelengths for a range of imaging modalities (405, 445, 488, 514, 561, 639 and 730nm). The microscope uses a combination of PMT’s, GaAsP detectors and Airyscan for detecting images. The Airyscan 2 detector is ideally designed to perform fast sensitive (4 to 8x higher SNR) imaging applications.  The system also has a Colibri LED widefield illuminator for DAPI to Cy5.
    Zeiss LSM980 with Airyscan 2
  • Zeiss LSM700:
    The Zeiss LSM700 is a point scanning confocal microscope on a motorised inverted Zeiss Axio Observer Z1, with stage top incubation for temperature and CO2 control of samples so both fixed and live samples can be imaged. The system is equipped with 4 solid state lasers at different wavelengths: 405, 488, 555 and 639nm.
    Zeiss LSM700

Slide scanners

  • Zeiss Axio Scan Z1:
    The Zeiss Axio Scan Z1 is a high end brightfield and fluorescent slide scanner with a 100 slide capacity. It has one preview specimen/label camera and two imaging cameras (2MP 3CCD colour camera and a Hamamatsu Orca flash 4MP s-CMOS camera for fluorescence). High end fluorescence illumination with HXP and fast separated filter wheels (with filters for DAPI, GFP, Cy3, TexRed and Cy5). Zeiss Plan-Apochromat objectives lenses (10x, 20x and 40x) and a Zeiss Fluar 5x for overview and coarse focus. Zeiss Zen2 Slidescan imaging and analysis software.
    Zeiss Axio Scan Z1
  • Zeiss Metasystems automated fluorescence and brightfield scanning system:
    Automated system for the unattended detection and analysis of signals in interphase cells and tissue sections based on the scanning platform Metafer 4. With Zeiss Axio Imager Z2, fully motorised microscope with 10 position filter turret, 8 FISH filter sets (DAPI, Aqua, Green, Gold/Orange, TexRed, Cy5), 5x, 20x, 40x, 63x and 100x objective lenses, with auto oiling pump and nozzle. Metacyte software finds and identifies nuclei or cells based on morphologic criteria, automatically stores position data, acquires fluorescence signals within objects from different focal planes and in up to 6 colour channels.
    Zeiss Metasystems automated fluorescence and brightfield scanning system
  • Hamamatsu Nanozoomer-XR C12000:
    The Hamamatsu Nanozoomer is a high throughout automated slide scanner for brightfield and fluorescent slides with a 320 slide capacity. Fast scanning speeds (approx. 35s at 40x mode: area 15mm x 15mm) make this system ideal for quickly acquiring your H&E and IHC images.
    Hamamatsu Nanozoomer-XR C12000
  • Nikon TE-2000-s Widefield Fluorescent Microscope:
    An inverted widefield system with incubation for temperature and C02 - suitable for live cell/time lapse imaging. Can acquire fluorescence signals from DAPI to Cy5.

Image Analysis Equipment and Software

Dedicated image analysis computers

  • Analysis computer 1: Zeiss Zen blue 3 software and Zen Intellesis machine learning software for image segmentation. Definiens tissue studio software. Open source software.
  • Analysis computer 2: Processing PC for images acquired on the Zeiss LSM980 Airyscan 2, with Zen blue 3.4 image processing and Zen analysis software, Definiens tissue studio software for brightfield and fluorescent images. Open source software.
  • Analysis computer 3: Processing PC with WACOM pen screen for interactive and automated TissueFISH segmentation, for images acquired on the Zeiss Metasystems slide scanner. Open source software. 
  • Zeiss Axio Scan Analysis computer: For analysis of images acquired on the Axio Scan and other slide scanners. Zen image analysis modules, Definiens tissue studio software. Open source software.

Commercial software

  • Zeiss Zen software: image acquisition, processing, and analysis software
  • Zeiss Zen Intellesis: machine learning segmentation software
  • Metacyte: Finds and identifies nuclei or cells based on morphologic criteria, automatically stores position data, acquires fluorescence signals within objects from different focal planes and in up to 6 colour channels.
  • In Situ Imaging System software: For manual capture, processing, archiving and analysis of single plane or Z-stacked fluorescence images.
  • Tissue FISH module: For automated analysis of cell signal in tissue sections. Includes optional pre-marking of regions of interest and dedicated tools for interactive and automated separation of cells.
  • V-slide software and VSViewer: Digitises slides in brightfield and/or fluorescence.  
  • Zeiss Zen Intellesis: machine learning based segmentation software.
  • Definiens Tissue Studio software: Advanced image analysis software for brightfield and immunofluorescent images of tissue and cells.
  • Hamamatsu NZ Acquire and NDP view 2: for image acquisition and viewing of images acquired on the Nanozoomer slide scanner.
  • Duolink Image Tool: Image analysis software to quantify Duolink (proximity ligation assay) signals and cell nuclei.

Open source software

  • FIJI (ImageJ): Perhaps the most popular image analysis software, with hundreds of plugins to carry out nearly every kind of image analysis. Fairly easy to use (with no prior experience needed) but requires some trial and error.
  • QuPath: Open source software for bioimage analysis. QuPath is often used for digital pathology applications because it offers a powerful set of tools for working with whole slide images - but it can be applied to lots of other kinds of image as well. Features include: Powerful annotation & visualization tools using a modern JavaFX interface, built-in algorithms for common tasks, including cell and tissue detection, interactive machine learning, both for object and pixel classification, compatibility with other popular open tools, including ImageJ, OpenCV, Java Topology Suite and OMERO.
  • Cell Profiler and Cell Profiler Analyst: Complementary to FIJI in that Cell Profiler allows automatic analysis of thousands or even millions of images, particularly from high-content, plate-based systems. Cell profiler analyst is designed to work with data processed with Cell Profiler and can be used for automatic, machine-learning based image classification.