Imaging subcellular structures of rat mammary carcinoma cells by scanning force microscopy

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Abstract
Scanning force microscopy (SFM) was used for imaging subcellular structures of cultured rat mammary carcinoma cells dried in air. Identification of cellular substructures was achieved by immunofluorescence and specific fluores-cence probes. Cells grown attached to a glass support exhibited submicrometer thickness in the dried state. Inside the nuclear domain the nucleoli appeared as prominent conical protrusions. Membrane extensions, microspikes and microvilli were well preserved at the cell periphery after fixation in glutaraldehyde vapor and air-drying and were distinguishable either as isolated elements or intercellular communications. The plasma membrane and soluble proteins were selectively removed with non-ionic detergent in a buffer system. The mitochondria were concentrated primarily in the perinuclear space and exhibited a well defined filamentous shape. Their identity was confirmed by specific fluorescence staining with rhodamine 123. In the membrane-free system achieved by dry-cleaving of the sample surface, the cytoskeletal network was resolved as a complex mesh of actin-containing fiber bundles interwoven with a filigree arrangement of thinner filaments. The smallest fibrous substructures revealed by SFM with the scanning tips used to date were ∼8 to 10 nm in height and 80 nm in width.