In the past decades, high-throughput technologies were developed with the aim to unravel the secret of life. In the genomics project many genomes were sequenced, including the human genome. We ended up with a lot of data but no real clue if and at what point of time a certain illness will manifest, finally not the gene but the presence or absence of the responsible protein will be decisive. Therefore, proteomics came into reach and focus of research. Again we ended up with a wealth of data but still the final answer cannot be found. Finally, not only the presence of the protein is important but also its modification and, even more importantly, its location, whether it is ‘just' stored in a cell or whether it is active at the place the where action is needed. The future will therefore be structomics.

Our Approach

We decided to tackle this new challenge first on the cellular level. Seldom is the whole tissue affected by the disease, especially in an early stage only a small area possibly down to one cell is affected. To find the place of interest, we are developing methods on the base of correlative microscopy. With early markers, aberrant cells will be labelled and traced by light microscopy. For in depth structure analysis, the light microscope can not provide the resolution capability and therefore we want to zoom in by electron microscopy to analyze the changed morphology, e.g., leaky junctions, and the location of important proteins, e.g., those involved in ­junction formation, in comparison to the healthy tissue.

Sample Preparation

We will make use of the whole palette of electron microscopic methods available. First of all, we will aim at cryo-fixation and freeze-substitution [1]. After freeze-substitution the samples are embedded in epoxy resin for morphological analysis especially in combination with electron tomography [2] or in methacrylate for immunolocalization [1]. This method, though well-established, is still in deve­lopment to improve contrast and higher efficiency of immunolabelling. To overcome the problems in label sensitivity recently, a new method was developed to rehydrate freeze-substitution fixed spe­cimens and process them by the Tokuyasu cryo-sectioning method.

This approach proved to retain most of the structural benefits of cryo-fixation and freeze-substitution and combines it with the high efficiency of labelling the Tokuyasu method is known for [3]. In many cases, however, proper cryo-fixation cannot be done because the tissue is too large or even infectious, then the other standard preparation methods based on chemical fixation will be valid as well: epoxy embedding [4]; progressive lowering of the temperature [5] or Tokuyasu cryo-sectioning [6].

Correlative Light and Electron Microscopy

For correlative light microscopy [7] a set of one or two semi-thin sections (ca. 500 nm) and several ultra-thin sections
(50-100 nm) will be cut. The semi-thin section will be mounted on a glass coverslip stained with a histological stain, e.g., Toluidine blue, to allow for differentiation of the tissue. A few ultra-thin sections are also mounted on coverslips and the remaining on Formvar/carbon coated grids. The protein of interest is labelled and detected with a fluorescence dye (coverslips) or gold probe (EM grids). Inspection in the fluorescence microscope will guide us to the place where the protein of interest is present and thereafter facilitate its location in the electron ­microscope. There the fine localization will be done.

Focussed Ion Beam Technology

A dual beam scanning electron microscope (FIB-SEM) is equipped with two columns, one with an electron source like a conventional scanning electron microscope (SEM) and one with an ion source. Both beams are focussed onto the same spot. The ion beam can be used to cut and dig trenches in a specimen in the nanometre to tenths of micrometre range and the electron beam is used to image the area.
This year, we embarked on a new challenge: we want to implement the dual beam technology in our quest of finding the place of interest, this not only for biological but also for material science applications especially in the field of geology. Here we will focus on the biological applications. The final aim is to localize the place of interest by specific markers, which can be visualized in the scanning electron microscope. The SEM has the great advantage of the large magnification range. Pieces of tissue as large as several millimetres, even centimetres, can be scanned. When the place of interest is found, it can be magnified and imaged at high-resolution close to that of a TEM. After the interesting area has been identified, there are two possibilities to continue [8-10]: