Biological Electron Microscopy

January 11, 2013

The Electron Microscopy (EM) laboratory in the School of Biological and Biomedical Sciences at Durham University is an integral part of a wider facility spanning a range of advanced imaging tools (laser scanning and spinning disc confocal microscopes, TIRF microscopy and live cell imaging) as well as an ultra high resolution (<0.5 nm) field emission in-lens scanning electron microscope (Hitachi S5200) and a routine transmission EM with high tilt stage for tomography (Hitachi H7600).

Microscopes

These instruments are supported by a comprehensive range of sample preparation equipment including a Leica EM PACT high pressure freezer, Leica metal mirror and plunge freezers (MM80 and CPC), Leica EM AFS freeze substitution instrument, Leica cryo-ultramicrotome, Baltech (now Leica) CPD030 critical point drier, Cressington vacuum units for carbon evaporation and for ultra high resolution metal coating (e.g. chromium). Our aim is to develop the best possible methods for examining and analysing biological ultrastructure both in thin sections (TEM) and on biological surfaces (SEM), and then to offer these techniques to internal and external clients.

Specimen preparation

Most importantly, methods are generally developed as part of intensively EM oriented internal research projects. We work with a wide range of samples including plant tissues, single molecules, polymers, all types of animal tissue and culture cells, which may be processed for surface imaging, thin sectioning and immuno-gold labelling. One particular research interest is to understand how nuclear pore complexes (NPCs) carry out the controlled transport of molecules such as proteins and RNAs to and from the cell nucleus. We look at NPC structure using SEM by isolating the nuclear envelope (which encloses the nucleus and contains the NPCs), critical point drying them and then coating with a 1.5 nm thick film of chromium (Figure 1). Such samples can be labelled with gold-tagged antibodies to determine the position of specific proteins (Figure 2).

Range of specimens

Most importantly, methods are generally developed as part of intensively EM oriented internal research projects. We work with a wide range of samples including plant tissues, single molecules, polymers, all types of animal tissue and culture cells, which may be processed for surface imaging, thin sectioning and immuno-gold labelling.

Fig. 1: Field emission scanning electron micrograph of an isolated Xenopus laevis oocyte nuclear envelope showing the outer surface the outer nuclear membrane and the nuclear pore complexes.<br><br>
Fig. 2: Field emission scanning electron micrograph of an isolated Xenopus laevis oocyte nuclear envelope showing the inner face the inner nuclear membrane and the nuclear pore complex baskets immuno-gold labelled for a NPC protein. The secondary electron image showing the sample structure is in red and the backscatter image which detects the position of the gold particle is in yellow.<br><br>

High pressure freezing and immuno-gold labelling of nuclear transport

To look at how molecules travel through the NPC we have to look at cross sections in the TEM. We use baker’s yeast because in this organism it is easy to genetically manipulate the proteins that make up the NPC. We can then discover how those proteins are involved in NPC structure and function. The problem however is that yeast has a cell wall which is resistant to chemical fi xation. Transport through the NPC is also extremely rapid, so molecules are rarely caught in transit. Both these problems are solved by high pressure freezing followed by low temperature fixation and embedding (Figure 3).

High resolution immuno-scanning electron microscopy of nuclear pore complexes

One particular research interest is to understand how nuclear pore complexes (NPCs) carry out the controlled transport of molecules such as proteins and RNAs to and from the cell nucleus. We look at NPC structure using SEM by isolating the nuclear envelope (which encloses the nucleus and contains the NPCs), critical point drying them and then coating with a 1.5 nm thick film of chromium (Figure 1). Such samples can be labelled with gold-tagged antibodies to determine the position of specific proteins (Figure 2).

Such sample preparation is also amenable to immuno-gold labelling, so that we can use antibodies to locate cargoes and transporters in transit, as well as the proteins that make up this gateway (the NPC) (Figure 4).

Fig. 3: High magnification transmission electron micrograph of high pressure frozen, freeze substituted yeast cell, clearly showing both leaflets of the inner and the outer nuclear membranes, with ribosomes docked on the outer membrane and NPCs at points where the two membranes are joined.<br><br>
Fig. 4: Transmission electron micrograph of high pressure frozen, freeze substituted yeast cell immuno-gold labelled for a NPC protein.

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