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

Abstract

SummaryStructural 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 highpressure 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.

Multi-dimensional correlative imaging of subcellular events: combining the strengths of light and electron microscopy

“…These have been successfully applied to a range of different samples, moving from tissues and cells towards the subcellular level (for overview papers, see Biel et al 2003;Braet and Geerts 2009;Giepmans 2008;Plitzko et al 2009;Sosinsky et al 2007;Swanson and Peters 2005), as is evident from a literature search on the topic of 'correlative microscopy' (Fig. 2).…”

“…Rhodamine-dextran-amine (Fluoro-Ruby) has been exploited in labeling axons and has been used successfully to photo-oxidize DAB (Schmued and Snavely 1993). In another approach, fluorescent histostains, such as acridine orange, Nile blue sulfate, and safranine T, have been applied during freeze substitution, alongside conventional uranyl-acetate staining, for confocal microscopy and 3-D reconstruction, followed by correlation with EM imaging (Biel et al 2003). This route provided excellent fluorescent labeling and high-quality preservation for highresolution EM observation.…”

Reevaluation of the documentation of the technique used for the work “Combined use of confocal laser scanning microscopy and transmission electron microscopy for visualization of identical cells processed by cryotechniques”

“…Nevertheless, it is basically possible to correlate light and electron microscopy. Prerequisite is the use of internal references (Biel et al 2003).…”

Plastic embedding immunolabeled large-volume samples for three-dimensional high-resolution imaging

“…In this case, whether the whole-mount immunolabelling method is compatible with plastic embedding should be examined first, as plastic embedding of the sample is a pre-requisite to enable precise and ultra-thin slicing. In fact, plastic embedding has been applied to immunolabelled thin samples like cells and tissues [19][20][21][22], and could improve the axial resolution of immunolabelled sections [23]. However, procedures and resins for immunostaining and embedding the thin sample are unsuitable for large-volume sample.…”