Abstract
Far-field optical nanoscopy has been widely used to image small objects with sub-diffraction-limit spatial resolution. Particularly, reversible saturable optical fluorescence transition (RESOLFT) nanoscopy with photoswitchable fluorescent proteins is a powerful method for super-resolution imaging of living cells with low light intensity. Here we demonstrate for the first time the implementation of RESOLFT nanoscopy for a biological system using organic fluorophores, which are smaller in size and easier to be chemically modified. With a covalently-linked dye pair of Cy3 and Alexa647 to label subcellular structures in fixed cells and by optimizing the imaging buffer and optical parameters, our RESOLFT nanoscopy achieved a spatial resolution of ~74 nm in the focal plane. This method provides a powerful alternative for low light intensity RESOLFT nanoscopy, which enables biological imaging with small organic probes at nanoscale resolution.
Highlights
RESOLFT nanoscopy with commercially available organic fluorophores remains challenging for a biological system the RESOLFT technique itself has been demonstrated with a few organic dyes[22,23]
The main reason that organic dyes have rarely been used in RESOLFT nanoscopy is the poor resistance to the photobleaching over multiple switching cycles
In order to see if organic fluorophores can be used in RESOLFT nanoscopy, we prepared the Cy3-Alexa[647] heterodimer as described in Methods[28]
Summary
In order to see if organic fluorophores can be used in RESOLFT nanoscopy, we prepared the Cy3-Alexa[647] heterodimer as described in Methods[28]. It shows the absorption maximum at 549 and 652 nm (extinction coefficient: 53,200 and 63,300 M−1cm−1, respectively) and the emission maximum at 564 and 668 nm. We prepared the heterodimer labeled microtubules in primary Human-skin-fibroblast cells and examined its photoswitching capability over many repeated cycles under optimized imaging conditions that will be described below (Fig. 1b,c). In order to successfully implement organic fluorophores in RESOLFT nanoscopy, experimental conditions need to be optimized to improve
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