From tissue to cellular ultrastructure: closing the gap between micro- and nanostructural imaging.

Structural investigation of tissue biopsies requires the coupling of optimal structural sample preservation with detailed information collected at the light and electron microscopic level. Unfortunately, although cryo-immobilization by high-pressure freezing provides the best structural preservation, it is used routinely only for electron microscopic (EM) investigations, whereas for light microscopy chemical fixation protocols have been established. These chemically invasive fixation protocols have the drawback of introducing unpredictable fixation artefacts. Therefore, comparative histopathological (i.e. light microscopic) and ultrastructural (i.e. EM) results are usually obtained from parallel samples that have not been prepared identically and never by examining exactly the same features in exactly the same, optimally preserved sample. Finally, finding an area of interest for EM investigation within a complex tissue is like searching for a needle in a haystack. To overcome these handicaps, we modified the well-established freeze-substitution technique (FS) to allow us to investigate resin-embedded cryo-immobilized tissue by confocal laser scanning microscopy (CLSM) prior to EM examination. Thus (1) selected cells throughout the whole tissue block can be depicted by CLSM and subsequently (2) selectively prepared by targeted sectioning for follow-up investigation of the identical structure by transmission electron microscopy. This is facilitated by the addition of specific fluorescent dyes during the first FS exchange step. Selective binding properties of various dyes to different cellular structures allow a direct histological description of the tissue at the light microscope level. After embedding and preparation of a blockface, the specimen can first be examined by CLSM. For areas of interest, the depth in the resin block is determined followed by removal of the tissue lying above. Then, the cell layer can be cut into a series of ultrathin sections and examined by EM for determination of the subcellular and nanostructural organization.

Combined use of confocal laser scanning microscopy and transmission electron microscopy for visualisation of identical cells processed by cryotechniques.

Successive visualisation of identical plant cells by light and electron microscopy is reported. For this purpose segments of pea and barley leaves were prepared by high-pressure freezing, freeze-substitution, and low-temperature embedding. The use of Safranin O during low-temperature dehydration allowed, on one hand, staining of all cellular components as investigated by confocal laser scanning microscopy and, on the other hand, excellent ultrastructural and antigenic preservation. A newly constructed specimen holder enabled precise relocation of the target cells for electron microscopic investigations. Transmission electron microscopy and immunohistochemistry revealed that during the whole procedure the ultrastructure of the cells as well as the antigenicity of cell constituents were preserved.