We provide light and electron microscopy technologies to facilitate the control, standardisation and testing of existing biological medicines and reference reagents and to further research and development of novel products.
We are equipped with a selection of high specification light and electron microscopes and associated preparation equipment, which fall into two main areas:
Electron microscopes use a high energy beam of electron to image samples rather than light. The resolution of electron microscopes is greater than that of light microscopes due to the small wavelength of electrons allowing smaller features to be seen, such as proteins, DNA, viruses and even atoms. There are two basic types of electron microscope: transmission electron microscope (TEM) and scanning electron microscope (SEM).
JEOL JEM 2100 cryoTEM equipped with a GIF Quantum, two Gatan ultrascan 4000’s and one Erlangshen ES500W camera.
Electrons are transmitted through the sample creating contrast in the image where they interact with the sample; this necessitates thin samples, in the region of >300nm to allow the electron beam to fully penetrate. The contrast can be enhanced by the use of heavy metal stains; however the resolution achievable is limited. Unstained samples can be visualised with the contrast created from the difference between water and the sample. These samples are embedded in vitreous ice, which requires cooling of the microscope and sample holder to -170°C and is commonly called cryoTEM.
TEM is primarily used at NIBSC for the analysis of vaccine components, viruses, bacteria and bacterial products and for the localisation of cellular components.
JEOL 7401-F cryoSEM equipped with an Oxford Inca.
The electron beam is scanned across the surface of the sample interacting with the surface of the sample and producing signals which form an image of the surface of the sample. The most common of these signals is secondary scattered electrons, there are released from the sample in response to the electron beam. Unfortunately most biological samples do not produce enough secondary electron to create an image, so samples are coated with a fine layer of metal (tungsten, chromium or tantalum) which readily produced secondary electrons. Samples can be imaged at room temperature or in cryoSEM mode, where the microscope is cooled to -150°C.
SEM is primarily used at NIBSC for the analysis of cell surface proteins and cellular morphology.
Water and vacuums do not mix, which makes imaging of biological samples in the vacuum of an electron microscope a little difficult. In order to get around this problem water is removed completely, substituted for something else or frozen. Our sample preparation uses one or a combination of these techniques to allow samples to be imaged. Our preparation techniques for electron microscopy include:
Additional software available in the group allows us to collect electron tomograms and to run automatic data acquisition sessions; this data can then be used to calculate three-dimensional reconstructions of proteins, viruses, cells and tissues. We currently have:
Light microscopy uses visible light which is transmitted through the sample to form an image a series of lenses magnify the image. The resolution limit of light microscopes is limited to the wavelength of visible light (approximately 0.2micrometers) and is ideal for the visualisation of cells and tissue sections where viewing fine details is not required. Fluorescent microscopes use Specimens are illuminated with light of a specificwavelength, which is absorbed by the fluorophores, causing them to emit light of longer wavelengths (a different colour to that of the emitted light). Samples are required to be auto-fluorescent, have been stained with fluorescent dyes or have been immuno-labelled with an antibody conjugated to a fluorophore. In the imaging group we have two main fluorescent microscopes:
The epifluorescent microscope is a basic screening microscope, which also has a Linkam cryo stage allowing correlative light and electron microscopy (CLEM) to be carried out on TEM samples. CLEM allows imaging of the same area of a sample but both fluorescent and electron microscope creating a high magnification image correlating to a region of interest in the lower magnification image. Dynamic cellular events can also be captured under the light microscope, immobilised and then viewed in the electron microscope.
Leica TCS SP8X MP OPO Spectral Confocal Microscope
Confocal microscopes provide greater optical resolution and contrast than conventional fluorescent microscopes by eliminating out of focus light; thus allowing only the focal plane to be illuminated and for 3-dimensional images to be calculated. The confocal microscope at NIBSC is predominantly used for fluorescence imaging and it is equipped with:
The imaging group has a wide range of research, focussed on the characterisation and control of biological medicines by microscope and fundamental research into novel vaccine products and other biological medicines by microscopy. The group has formed collaborations with both external researchers and internal researchers to pursue these aims.
Kirsty MacLellan-Gibson, Principal ScientistRobert J. Francis - Biological Imaging ScientistLudovic Renault - shared staff member