Imaging

Bioimaging

At the John Innes Centre we have state-of-the-art bioimaging facilities, in both light and electron microscopy.

Facility staff provide full training and technical support to suit your needs. From total beginners to those who are more experienced. Training is normally provided 1-to-1 at the instruments. Small groups with similar requirements can be accommodated on request. Remember: microscopy requires a lot of your time and patience.

We specialise in the imaging of plants and microbes but can also cater for a wide-range of imaging requirements for animal cells and material science. We offer advice on choice of techniques, experimental design, image interpretation and image analysis. The bioimaging suite can be used in two ways:

  • Use-it-yourself – once you are fully trained you can use the imaging facilities with ongoing technical support
  • Full service – our highly-skilled staff can provide for your imaging needs, with the emphasis on collaborative projects

We provide:

  • Routine sample preparation service, such as embedding and sectioning
  • Many specialised techniques and bespoke services
  • Various types of fixed and live-cell imaging
  • Dedicated data analysis workstations – you can analyse your data in-house
  • Advice on techniques and appropriate equipment to aid your research

If a specialist technique or our extensive input is required we encourage collaboration, often resulting in a joint publication.

The bioimaging facility primarily supports the microscopy needs of the John Innes Centre but is available to the wider Norwich Research Park and external customers, both academic and industrial.

Bioimaging equipment

Light microscopy

  • Conventional bright-field, phase, DIC (Nomarski), and fluorescence imaging
  • Laser-scanning and spinning-disc confocals, with FRAP, FRET, FLIM and FCS capabilities
  • To go beyond the diffraction limit, we also have a Zeiss LSM880 Airyscan fast and an Elyra PS1 for SIM and STORM

Electron microscopy

  • TEM (transmission electron microscope) and two SEM’s (scanning electron microscopes) and specialist sample preparation equipment
  • Sample preparation by a variety of techniques for TEM, such as high-pressure freezing and freeze-substitution, resin embedding and sectioning, immuno-gold labelling or negative staining
  • 200kV TEM for electron tomography, with high-tilt and dual-tilt holders
  • SEM – we offer critical point drying or cryo-SEM techniques. Both our SEMs have field-emission guns, giving the highest resolution capabilities.
  • High vacuum and low vacuum techniques, SE and BSE detection, STEM and EDS.

Light microscopy

Zeiss LSM 880 confocal with Airyscan

A point-scanning system on an upright Imager Z.2 microscope with a motorised stage and a variety of objectives (air/oil/water/dipping), also suitable for DIC. Available laser lines are: 405, 458, 488, 514, 561, 594 and 633 nm. The system has two standard PMT detectors (tuneable), a more sensitive GaAsP spectral detector array and an Airyscan detector with fast mode.

Leica TCS SP8X confocal

This point-scanning system is based around a fully motorised Leica DM6 upright microscope equipped with a variety of objectives (air/oil/water/dipping) also suitable for DIC. Fluorescence detection is tuneable (400-800nm) with up to five internal (PMT plus HyDs x4) and two external detectors (SPADS). Available laser lines are 405, 440 (pulsed), 457, 488, 514, 561 nm and a pulsed white light laser tuneable from 470 – 670 nm. The scan head has two scanners: conventional (standard) and resonance (fast dynamic). The microscope can be used for fluorescence lifetime imaging (FLIM) and fluorescence correlation spectroscopy (FCS).

Zeiss LSM 780 confocal

A point-scanning system on an upright Imager Z.2 microscope with a motorised stage and a variety of objectives (air/oil/water/dipping), also suitable for DIC. Available laser lines are 405, 458, 488, 514, 561, 633 nm. The system has two standard PMT detectors (tuneable) and a more sensitive GaAsP spectral detector array. It can be used for FCS and raster image correlation spectroscopy (RICS).

Leica SP5 confocal

A point-scanning system on an upright DM6000 microscope with fully motorised stage and a variety of objectives (air/oil/water/dipping), also suitable for DIC. It has four tuneable fluorescence detectors (400-800nm), two of which are standard PMTs and two are hybrid detectors. Available laser lines are 405, 458, 476, 488, 496, 514, 561, 633 nm. The system has two scanning modes; conventional for high resolution imaging and resonance for fast dynamic imaging.

Andor Revolution XD spinning disc confocal

A spinning disc microscope on an inverted Nikon Ti microscope with an incubation chamber for temperature-controlled environments. An EM-CCD camera with 90% quantum efficiency allows high speed imaging and detection of proteins expressed at low levels. Capable of bright field, multi-colour fluorescence, z-series, time lapse and multi-stage position imaging. Six excitation lasers (405, 445, 488, 515, 561 and 640nm). The system has a FRAPPA scanner, used for photoactivation, photoconversion and photobleaching of defined sub-regions.

Zeiss Elyra PS1

This system uses super-resolution imaging methods that break the Abbe resolution limit: 3D structured illumination (SIM) and 2D single molecule localisation techniques (dSTORM and PALM). It is also capable of Total Internal Reflection Fluorescence (TIRF) and can be used as a highly sensitive wide-field system. Built on an inverted Observer Z1 microscope with motorised stage, incubator with temperature control and two EMCCD cameras, one of them back-thinned. Available lasers are 405, 488, 561 and 642 nm.

Zeiss Axio Observer Z1

A high-quality, inverted, wide-field microscope with motorised stage control (for z sectioning, time lapse, tiling), auto focus and a microscope incubator (range from RT to 37oC) making it ideal for live cell imaging.  Equipped with both colour and monochrome cameras. Imaging modalities are bright field, DIC (x100 only), phase contrast (x100 only) and fluorescence over a wide range of wavelengths. The fluorescence illumination is a Zeiss Colibri 7 LED light source giving independent control over 7 LED bands. Additionally, it has a RAPPA UGA42 laser targeting system for FRAP and photo-activation.

Zeiss Axio Imager Z2

This system is equipped with both colour (histology) and monochrome (fluorescence) cameras. The microscope stand is upright with motorised stage control (z sectioning, time lapse, tiling and multiple positions), software auto focus. Acquisition modalities are bright field, DIC, phase contrast (x100 only) and fluorescence (complete visible range). The fluorescence illumination is a Zeiss Colibri 7 LED light source giving independent control over 7 LED bands.

Leica DM6000

Upright, wide-field microscope with fully motorised stage and two digital cameras: a Leica DFC360 FX (monochrome) and a DFC420 (colour). Capable of bright field, polarised, DIC and fluorescence modes. Metal halide lamp for a wide range of visible and fluorescent dyes.

Leica M205A Stereo-Fluorescence microscope

This microscope is computer controlled and can capture images using a Leica DFC310FX colour camera. Imaging modalities are reflective bright field (LED light ring), transmission illumination (bright field, dark field and Rottermann contrast) & fluorescence (LED illumination, for GFP and DSR). It can also perform z sectioning and time lapse.

Electron Microscopy

FEI Talos F200C TEM – A 200 KV transmission electron microscope with an FEG filament (field emission source) and a Gatan OneView camera; a 4k by 4k CMOS bottom-mounted camera. It is capable of Cryo-TEM. High and dual-tilt holders are available for electron tomography.

FEI Nova NanoSEM 450 – A high-resolution, field emission scanning electron microscope with a Gatan cryo-system, EDS,  BSE and STEM detectors. Low voltage (1kV) resolution is 1.4 nm in high vacuum mode. It offers beam deceleration, both high and low vacuum modes, and is the perfect all-round tool for a wide range of samples.

Zeiss Supra 55 VP FEG SEM – A second, high-resolution, field emission scanning electron microscope with a Gatan cryo system.  In addition to the standard secondary electron detector, there is an in-lens SE detector for high resolution work.Optimised for simple imaging at low kV  for biological samples.

We also have a Deben CoolStage (Peltier stage) and variable pressure mode available, allowing imaging at low vacuum or high vacuum.

Carbon thread coater / glow discharge unit – A Leica ACE 200 coater, with quartz crystal thickness monitoring and automated functions, for putting a homogenous, conductive layer of carbon on SEM samples for X-ray microanalysis. More commonly used in glow discharge mode to make TEM grids more hydrophilic.

SEM sample preparation

  • Sputter coater – A high resolution sputter-coater for use with our field emission SEMs. Targets routinely available are Au or Pt
  • Critical Point dryer – A Leica CPD300 used for drying chemically fixed samples for the SEM.

Sectioning facilities

  • Leica ultramicrotomes for ultra-thin and semi-thin resin sectioning for both light and electron microscopy applications. The UC7 is available for general use and, with a camera and monitor, is ideal for training purposes. We have a knife-maker (Leica KMR2) for preparing glass knives, hot-plates for drying slides, and light microscopes for checking sections, along with a slide-staining area.
  • Leica Autocut RM 2055 microtome (for wax or Technovit resin) and a Thermo Fisher cryostat for cutting frozen sections, all for light microscopy applications.

High pressure freezer and freeze substitution unit

A Leica HPM100 high-pressure freezer, with 3mm and 6mm carriers, used for freezing very small samples at pressures of ~2000 bar, to prevent ice-crystal growth. Sample size is extremely limited, but it can provide the best method for membrane-preservation. Due to the challenging nature of this technology, it requires technical assistance.

The Leica AFS2 for freeze substitution machine is generally used after high-pressure freezing but can also be used for the PLT embedding method (progressively lowering temperature). Both these methods have been shown to help retain antigenicity and are therefore used when immuno-gold labelling studies are required for TEM.

Tissue processor

The Leica EM TP embedding machine is used for automatically performing tissue dehydration and resin-infiltration prior to polymerisation of resin. We routinely use it for samples for electron microscopy, embedding in LR White resin. This machine is generally only used by Bioimaging staff, but can be accessed if necessary.

Metal evaporation unit

An EdwardsAuto 306, high-vacuum metal coating unit for carbon-coating grids, plus an electric grinding machine for preparation of the necessary carbon rods. This machine is generally only used by Bioimaging staff, but can be accessed if necessary.