Facilities at JIC

Here you'll find a list of the main equipment we have in the Microscopy department at The John Innes Centre.  These are only for use by internal staff and students but are listed here to give you an idea of the typical facilities a biological research laboratory requires.

• General Light Microscopy
• CCD camera system
• Confocal Microscopy
• Transmission Electron Microscopy
• Sectioning facilities
• Scanning Electron Microscopy
• Miscellaneous

General Light Microscopy

Nikon Eclipse 800 with Pixera Pro 600ES and Nikon Coolpix 995 digital cameras.
Capabilities are bright field (Köhler) illumination, dark field, Nomarski (DIC) (high resolution only) and fluorescent filters UV-2A (330-380nm > 420LP); BV-2A (400-440 > 470LP); G-2A (510-560nm > 590LP); FITC (465-495nm > 515-555nm).

Nikon Microphot-SA (1) with 35mm film and with Nikon Coolpix 990 digital cameras.
Capabilities are bright field (Köhler) illumination, and fluorescent filters DAPI (365nm > 418-483nm); Rhod (546nm > 590LP); FITC (450-490nm > 520LP).

Nikon Microphot-SA (2) with 35mm film and with Nikon Coolpix 990 digital cameras.
Capabilities are bright field (Köhler) illumination , dark field, phase contrast (No. 1 & 3 phase rings), Nomarski (DIC) and fluorescent filters DAPI (330-380nm > 450LP); Rhod (546nm > 590LP); FITC (450-490nm > 520LP).

Leica Stereo-Fluorescence microscope
Fluorescent filters UV (340-380nm>420LP), GFP2 (460-500nm > 510LP) and GFP3 (450-490nm > 500-550nm).

Cairn CCD system

Nikon Eclipse 600  with Hamamatsu Orca HQ cooled CCD digital camera.
The system has a motorised XY stage, Z focus drive, automated fluorescence filters and electronic shutters on both bright-field and UV light sources. The system is computer controlled via MetaMorph software and is capable of doing bright field (b/w), multi-colour fluorescence, z-series, time lapse and multi-stage position imaging. The fluorescence filters available on the system can been seen in the table below.

Confocal Microscopy

Leica SP .
The confocal scan head is mounted on a Leica DMR upright microscope and has three tuneable fluorescence detectors (400-800nm); plus three excitation lasers: Argon ion (457, 477, 488, and 514nm), Green HeNe (543nm) and Red HeNe (633nm), which can be used with a wide range of visible dyes (acquired 1998).

Leica SP2
The confocal scan head is mounted on a Leica DM IRB inverted microscope and has three tuneable fluorescence detectors (400-800nm); plus four excitation lasers: Violet laser diode(405nm), Argon ion (457, 477, 488, and 514nm), Green HeNe (543nm) and Red HeNe (633nm) which can be used with a wide range of UV and visible dyes (acquired 2000).

VisiTech spinning disc
This system is a lower resolution confocal microscope and is better suited for for fast image acquisition (sample dependant) and imaging living cells for extended periods of time. System is computer controlled via MetaMorph software and is capable of doing bright field (b/w), multi-colour fluorescence, z-series, time lapse and multi-stage position imaging. The confocal scan head is mounted on a Nikon E800 upright microscope and has a Hamamatsu Orca ER cooled CCD detector. The system has three excitation lasers: Violet laser diode(405nm), Argon Ion (457, 488, 514nm), Argon Krypton laser 568 and 647nm) and emission filters [432-482nm, 465-505nm, 500-550nm, 520-550nm, 580-654nm, 662-738nm], which can be used to image a wide range of UV and visible dyes (acquired 2003).

Zeiss 510 META confocal
The newest confocal microscope at JIC (installed around Easter 2007), is the Zeiss LSM 510 META which is a high-end laser scanning microscope from Carl Zeiss. A completely new detection technology overcomes the limits of existing detection methods. The new system allows many more fluorescent markers to be used simultaneously and their fluorescent emissions to be assigned despite spectral overlaps. The LSM 510 META thus specifically addresses the growing need of users in biomedical research to discover the complex relations and interactions between cellular structures and components and to trace them in living cells or tissues. This milestone development is based on a confocal detection technique combining innovative detector technology with intelligent analytical methods. By splitting the fluorescent light and projection onto a multi-channel detector, the LSM 510 META delivers the spectral distribution of the fluorescence signals as a parameter of every pixel in the object under investigation. In a second step, this information can be used for the digital separation of the fluorescence emissions in a multifluorescence experiment. All functions needed for this are provided by the user-friendly LSM 510 META software.

Transmission Electron Microscopy

The transmission electron microscope uses a beam of electrons, controlled by electromagnetic lenses, to image very thin samples – usually sections of around 100nm – which the beam has to pass through. To provide contrast, the specimen is usually stained with heavy metals such as lead and uranium. Such heavy metals will absorb or scatter the electrons, thus removing them from the primary beam as it passes through the sample, and before it can strike a phosphor screen. When the resultant electron beam hits the screen it gives off visible light, thereby forming an image that we can see.  

We have an FEI Tecnai G2 20 Twin TEM. This is a 200 KV instrument installed in spring 2008. It is suitable for all routine TEM specimens on 3.05 mm grids, such as negatively stained samples and immunogold labelled sections. It is fitted with two Deben digital cameras; a 1.3 megapixel AMT HRL side-mounted system and a 4k AMT XR60M CCD bottom-mounted camera, both for collection of TIFF digital image files. It has a LaB6 filament as the electron source and also has a dual-axis, high-tilt, holder suitable for electron tomography of thick resin sections to 70° tilt. There is an additional support computer in the Bioimaging facilities with FEI's Inspect3D Xpress software for post-acquisition processing and reconstruction of the tomograms acquired on the microscope. The resolution of our TEM is considerably better than the SEMs, with a line resolution of 0.144nm.

Sectioning facilities

See further information on Embedding and Sectioning.

We have two ultramicrotomes for ultra-thin (approx 100nm) sectioning for the TEM's and semi-thin (approx 0.5 micron) resin sectioning for light microscopy. The older, Reichert-Jung machine is an Ultracut E (acquired around 1985), is for general use and has an attachment for cryo sectioning. The second machine is a Leica UC6 with a touchscreen computer to operate the controls (purchased in 2004). In the same room, there is a Leica knife-maker (KMR2) apparatus (acquired 2000) and a hot-wax plate for preparing glass knives, aswell as two light microscopes (a Leica MZ16 and a Vickers) for checking samples and slides.

Next door, there are two Leica Autocut microtomes (an RM 2125RT and an RM 2055) for making thick sections (from wax, Historesin or Technovit embedded material) for light microscopy work (up to 25 microns). There is also a large hotplate and a light microscope for drying and checking slides.

Embedding Facilities

There are two standard embedding ovens for routine heat polymerisation of resin-infiltrated material used for ultrastructural studies. We also have a "cold box" apparatus designed for low temperature embedding, which has been shown to improve antigenicity and is therefore always used when immuno-labelling studies are required for the TEM.

Grid preparation

An EdwardsAuto 306, high-vacuum metal coating unit (acquired 2002) for low-angle rotary shadowing and carbon-coating grids, plus an electric grinding machine for preparation of the necessary carbon rods.

See further information on Embedding and Sectioning.

Cryo-techniques

One of the Reichert microtomes in the sectioning room has an attachment for cryo-ultramicrotomy using the Tokuyasu technique (first cryoprotecting aldehyde fixed samples with sucrose).

Both the SEM's are fitted with cryo-stages so that samples can be cryo-fixed and imaged in the SEM whilst in the frozen-hydrated state.

Scanning Electron Microscopy

The scanning electron microscopes at JIC are used for looking at surfaces of samples, providing images of three-dimensional objects with a greater depth of focus than a light microscope and a maximum resolution of around 2nm.

In the SEM, the sample is coated with a thin layer of metal (known as sputter coating) and scanned by an electron beam, which is brought to a focus on the sample by means of the electromagnetic lenses. The resultant secondary electrons (SE’s) that are emitted from the sample surface are measured by the SE detector and are used to control to the intensity of successive points in an image built up on a monitor. We also have a backscattered electron detector which can be used for collection of atomic number composition of a sample.

Philips XL30 FEG  
This SEM (acquired 1996) has a field emission electron gun (FEG) for high resolution, with an Oxford Instruments CT1500 HF cryo-stage for cryo-fixed samples and freeze-fracture work. This instrument is equipped with a 120 roll film camera for analogue image capture, but can also capture digital images of up to a maximum of 3k by 4k pixels. The system is computer controlled and fully networked to permit easy transfer of images.

Zeiss Supra 55 VP FEG
A second high-resolution SEM was installed in 2004. It is a Zeiss Supra 55 VP FEG SEM with a Gatan Alto 2500 cryo system for cryo-fixed samples and freeze-fracture work. It also has a Deben CoolStage which is basically a Peltier stage and can be used to hold samples at specific temperatures in the range +50°C to -25°C. The microscope also has variable pressure (VP) mode which allows imaging at low vacuum (in the range 2-133Pa). This, in combination with the CoolStage, helps reduce charging artefacts and permits some samples to be imaged without sputter-coating and at ambient, or close to 0°C, temperatures which would otherwise have had to be cryo-fixed if imaged at high vacuum, such as insects and even some leaves.

When working in VP mode, the microscope uses a special detector (VPSE) for imaging. In addition to the normal secondary electron (SE) detector, there is also an in-lens detector and therefore this microscope offers higher resolution than our other SEM. It has no camera so all images must be captured digitally. The system is fully computer-controlled and has a CD-writer for users to write their images to a CD before leaving the microscope room.

Other SEM equipment

A critical point dryer apparatus used for drying chemically fixed samples.

An Agar, high resolution sputter-coater apparatus with planetary-motion stage, specifically for coating samples for high-resolution, field emission scanning electron microscopy (acquired 2000).  

Miscellaneous

Linkam PE120 system:  temperature controlled stage (-25 to 120oC) for upright optical microscopes with a Prior motorised stage.

Two 200 litre self-pressurising liquid nitrogen tanks to provide a supply of nitrogen required by much of the above EM apparatus.