The CryoCapsule: simplifying correlative light to electron microscopy.

Correlating complementary multiple scale images of the same object is a straightforward means to decipher biological processes. Light microscopy and electron microscopy are the most commonly used imaging techniques, yet despite their complementarity, the experimental procedures available to correlate them are technically complex. We designed and manufactured a new device adapted to many biological specimens, the CryoCapsule, that simplifies the multiple sample preparation steps, which at present separate live cell fluorescence imaging from contextual high-resolution electron microscopy, thus opening new strategies for full correlative light to electron microscopy. We tested the biological application of this highly optimized tool on three different specimens: the in vitro Xenopus laevis mitotic spindle, melanoma cells over-expressing YFP-langerin sequestered in organized membranous subcellular organelles and a pigmented melanocytic cell in which the endosomal system was labeled with internalized fluorescent transferrin.

A new approach to improve the quality of ultrathin cryo-sections; its use for immunogold EM and correlative electron cryo-tomography.

Cryo-ultramicrotomy can be used to obtain ultrathin cryo-sections from cryo-fixed or aldehyde-fixed cryo-protected vitreous biologic samples. For immuno-gold EM, cryo-sections are retrieved from the cryo-chamber on a droplet of a pick-up solution (paste-like and almost frozen) to which the sections attach. The sections are then placed on an EM specimen grid at room temperature. This procedure compromises the ultrastructure, resulting in folds, holes, and loss of the original material. In this paper we show the critical influence of humidity, stretching, and relief of compression during thawing of the sections. We show a new lift-up hinge device for semi-automated retrieval of cryo-sections that results in significantly improved section quality. This approach was also applied successfully to vitreous sections from high pressure frozen samples. An important advance is that these vitreous cryo-sections can now successfully be post-fixed and immunolabelled after thawing; this allows cryo-EM comparison with adjacent ribbons of sections still in the frozen hydrated state. These findings call for technical innovations aiming at automated cryo-ultramicrotomy in a fully controlled environment for improved localization of proteins within their 'close to native' cellular context and correlative electron cryo-tomography of consecutive ribbons of sections of one frozen hydrated sample.

Gelatin-embedded cell-polymer constructs for histological cryosectioning.

Many tissue-engineering strategies involve the delivery of cells via porous polymer scaffolds. Obtaining histological sections of the emerging tissue is often necessary to analyze numerous characteristics of the microscopic environment. However, difficulties arise upon applying standard histological techniques to cell-seeded polymer scaffolds. This report describes a simple and reliable method for cryosectioning cell-polymer constructs embedded in gelatin. Solvent-soluble (PLGA) and insoluble (PGA) scaffolds were cultured in vitro with preosteoblasts, followed by histological processing with paraffin, OCT, or gelatin. Although paraffin-embedded PGA scaffolds withstood standard sectioning and rinsing steps, paraffin-embedded PLGA scaffolds were partially dissolved during the clearing step. OCT-embedded scaffolds produced sections that did not adhere well to slides, and most of the sample was lost during rinsing steps. In contrast, gelatin-embedded scaffolds exhibited adequate structural integrity during cryosectioning, adhered well to the slides, retained the actual polymer morphology, and exhibited compatibility with common stains.

Recent progress in whole-body radioautography.

We review whole-body radioautography for water-soluble substances and its applications studied to date in our laboratory. To transfer the whole-body section onto a glass slide, Japanese paper was inserted between frozen block and adhesive tape before sectioning. Then the section was dried together with the paper and adhesive tape in a cryotome and applied onto the glass slide coated with a mixture of egg-albumin and glycerin. The whole-body section was transferred to slide and contacted with the film in vacuum. By using these techniques, we review the glucose and taurine metabolism, the glucose-6-phosphatase activity and the insulin receptor.