Bioimaging - Equipment
Cryo TEM Preparation.
The Gatan Cryo Plunge can be used prepare frozen hydrated specimens for cryo TEM. Samples are loaded onto EM grids, blotted and rapidly frozen using ethane to produce specimens embedded in a thin layer of vitreous ice allowing them to be imaged as close to the native state as possible, without any staining artefacts. The Gatan 626 cryo holder maintains frozen samples at liquid nitrogen temperatures for TEM imaging.
Equipped with three work stations, software including Metamorph, Zeiss Zen 2011, Image J, and Leica LAS-X are available for further image analysis without having to book a microscope.
Energy Dispersive Spectroscopy.
All three of our electron microscopes are equipped with an Oxford EDS. This allows characterisation of the elemental composition of samples by detecting the x rays emitted by the sample during bombardment by the electron beam. Qualitative and quantitative analysis, and elemental mapping can be performed.
JEOL 2011 Transmission Electron Microscope.
The JEOL 2011 transmission electron microscope can be used to image both resin embedded and cryo samples. Samples must be sufficiently thin for the electron beam to penetrate them – around 200nm or less. It is equipped with a Gatan Ultrascan CCD camera, suitable for collecting high resolution images. The 2011 is also fitted with a high tilt holder for tomography.
JEOL JSM-6360 Low Vacuum Scanning Electron Microscope.
The JEOL 6360 scanning electron microscope provides routine imaging of samples at 10nm, several hundreds of times greater resolution than optical microscopes. The LV SEM has an ALTO 2100 cryo system allowing biological specimens to be imaged without dehydration. It also features a low vacuum mode suitable for samples with excessive water content or that cannot be frozen. The backscatter detector provides additional information regarding composition of the sample.
JEOL JSM-6700F Scanning Electron Microscope.
The JEOL 6700F scanning electron microscope uses a cold field emission gun and semi in lens objective to generate high resolution images. It is fitted with an ALTO 2500 cryo system allowing imaging of biological samples. This SEM has a resolution limit of 3nm and magnification range of 350-100,000x making it suitable for visualising detailed features of specimens.
There are two laboratories for sample preparation, allowing users to use Bioimaging for more complex protocols without having to work between two lab spaces. One is used mainly as a clean lab for electron microscopy specimen preparation, the other is well equipped for chemical fixation protocols, wax and resin embedding.
Leica CM1850 Cryostat.
The Leica Cryostat is designed for rapid freezing and sectioning of fresh or fixed tissue samples. The Cryostat will cool from 0 to -35oC and section from 1- 60µm, to give high-quality sections without the need for sample embedding.
Leica EM Auto Freeze Substitution unit.
The Leica AFS is used to perform freeze substation on high pressure frozen samples. This process is a form of dehydration, removing the water from the sample and replacing it with a solvent prior to resin embedding.
The HPM100 high pressure freezer uses liquid nitrogen and high pressure to rapidly freeze samples as an alternative to chemical fixation that avoids fixation induced artefacts. Samples prepared with the HPM100 are suitable for embedded for both light and electron microscopy.
Leica M205 FA Stereomicroscope.
Suitable for observing whole organisms with precise depth of field imaging, the M205 FA is fully motorised to give excellent experiment reproducibility. The M205 has a digital camera capable of colour imaging for both reflected or transmitted brightfield light and monochrome imaging for fluorescence. Fluorescence filters available on the M205 FA stereomicroscope: UV, DAPI, Violet, CFP, GFP, GFP plus, GFP plants, YFP, TxRed, DsRed and mCherry.
PALM Microbeam laser dissection microscope.
The PALM Microbeam allows microdissection and micro-ablation of small cell clusters or single cells using a 337nm laser. Isolated cell areas can be catapulted into sample tube for further processing. Live samples or fixed sectioned material can be used.
Thick sections are defined as tissue sections of 0.5µm thickness or above, and are mainly suitable for light and laser confocal microscopy. Samples can be either fresh or fixed and embedded in wax or resin before sectioning.
Semi-thin (0.5µm -100nm) and ultra-thin sections (100nm or less) are cut from samples that are resin embedded using a glass or diamond knife. Semi-thin sections can be viewed with a light microscope, but ultra-thin are only suitable for transmission electron microscopy. Cryo-sectioning is a similar technique for thin sectioning samples that have been frozen between -20oC and -185oC, without the requirement of embedding in resin.
The Axioiphot is equipped with Q-Imaging Retiga digital camera with RGB colour filter for monochrome and colour imaging. It has an automated stage for multi-image aqusition and MetaMorph image software. Zeiss plan-Apochromat objectives with the best colour correction and highest numerical apertures, give high resolution light and fluorescence imaging. The Axipohot can also be set up for DIC, polarised light and phase contrast imaging.
The Zeiss LSM780 confocal microscope is used for live cell imaging, multi-fluorescence, spectral imaging and unmixing, co-localisation, FRET, FRAP and FLIP. The high sensitivity of LSM 780 is achieved with the 32-channel GaAsP detector which significantly benefits the imaging of plant systems and low signal strength fluorescence.