Ultra-high-resolution images of nestin and vimentin in pancreatic carcinoma cells using 2 novel microscopy systems.

Nestin, a type VI intermediate filament protein, heterodimerizes with vimentin, desmin, or internexin and forms intermediate filaments 10 nm in diameter. Because the limit of resolution of conventional microscopes is approximately 250 nm, the ability to demonstrate the locations of these proteins has previously been restricted. In the present study, we determined the locations of nestin and vimentin in PANC-1 human pancreatic carcinoma cells by means of 2 novel ultra-high-resolution imaging systems: structured illumination microscopy (SIM, 100-nm resolution) and stochastic optical reconstruction microscopy (STORM, 20-nm resolution). The SIM system improves resolution by processing moiré images arising from samples excited by structured illumination. STORM makes high-resolution images with the center of mass of individual molecules determined with high precision. Although confocal laser scanning microscopy (CLSM) has demonstrated the filamentous expression of nestin and vimentin (Fig. 1), it produces uninterpretable images at high magnification (Fig. 2a). A corresponding-magnification image captured with SIM clearly resolved filamentous structures, and nestin and vimentin appeared to be merged (Fig. 2b). STORM images revealed the filamentous network created by aggregates of small dots indicating nestin and vimentin molecules (Fig. 2c, d). The nestin and vimentin dots