A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM)

Abstract

The use of cryosectioning facilitates the morphological analysis and immunocytochemistry of cells in tissues in atomic force microscopy (AFM). The cantilever can access all parts of a tissue sample in cryosections after the embedding medium (sucrose) has been replaced with phosphate-buffered saline (PBS), and this approach has enabled the production of a type of high-resolution image. The images resembled those obtained from freeze-etching replica electron microscopy (EM) rather than from thin-section EM. The AFM images showed disks stacked and enveloped by the cell membrane in rod photoreceptor outer segments (ROS) at EM resolution. In addition, ciliary necklaces on the surface of connecting cilium, three-dimensional architecture of synaptic ribbons, and the surface of the post-synaptic membrane facing the active site were revealed, which were not apparent using thin-section EM. AFM could depict the molecular binding of anti-opsin antibodies conjugated to a secondary fluorescent antibody bound to the disk membrane. The specific localization of the anti-opsin binding sites was verified through correlation with immunofluorescence signals in AFM combined with confocal fluorescence microscope. To prove reproducibility in other tissues besides retina, cryosectioning-AFM was also applied to elucidate molecular organization of sarcomere in a rabbit psoas muscle.

Micro-mechanical fingerprints of the rat bladder change in actinic cystitis and tumor presence

“…Previous studies have shown little impact on the mechanical properties on freezing tissue 46 . However, working with frozen tissues has several advantages, and different AFM studies used fresh-frozen clinical specimens 27,[47][48][49][50] . Furthermore, frozen tissue allows to prepare semithin sections, which allows histological analysis and correlation with histological examination.…”

Subcellular Imaging of Liquid Silicone Coated-Intestinal Epithelial Cells

“…During electron microscopy studies of fixed cells the samples are protected from ambient contamination but at the cost of losing soluble cell contents 11 . Consequently, an alternative method is required, which leverages recent advances in instrumentation, preserves cellular morphology and simultaneously detects subcellular topological features without the requirement for multistep sample preparation procedures as in cryosectioning for AFM based cellular imaging 13 . Such a facile and robust methodology will have a direct impact on clinical level screening of pathological diseases 14 characterised by cell structure malfunction, e .…”

Easy ultrastructural insight into the internal morphology of biological specimens by Atomic Force Microscopy

“…C. elegans 32 ) but suffered from specific limitations concerning embedding resins remaining in the analyzed sections, deeming the application of a topographical technique like AFM especially difficult. Cryo-sectioning techniques have also been applied in some studies to elucidate internal features of tissues by AFM 33 , 34 . These techniques altogether suffered to a certain extend from being experimentally highly complex concerning the sample preparation as well as the needed expensive machinery e.g.…”