Super‐resolution imaging with the Nanoimager can provide images of cellular features with 20 nm and better resolution. This level of detail allows an unprecedented understanding of the interaction and ultrastructure of molecules in cells. Complimentary analysis such as colocalization and clustering, which are included in the Nanoimager custom software, allow in‐depth and quantitative analysis of interacting species and consequently molecular function. The ease of use of the Nanoimager combined with its super‐resolution capability allows the user to gain greater insight into important biological questions.
Super‐resolution techniques break the 200 nm limit on image resolution imposed by the diffraction of light in the optical path. The particular method employed by the Nanoimager to achieve super‐resolution information is single‐molecule localization. Localization‐based methods include direct stochastic optical reconstruction microscopy (dSTORM) and photo‐activated localization microscopy (PALM). These techniques involve localizing only subsets of fluorophores in consecutive frames, to a high precision (under 20 nm laterally and 50 nm axially depending on the experiment), and reconstructing an image from the positions of the localizations.
This principle is illustrated in the figure above, where localization‐based super‐resolution imaging is applied to actin in an MDBK cell, labeled with AlexaFluor647‐phalloidin. In a conventional diffraction‐limited TIRF image (A), the fluorophores on the labeled structure are too close to spatially resolve. To solve this problem, the fluorophores are imaged with high intensity under specific buffer conditions, causing them to blink on and off as they switch between a dark and a fluorescent active state. In each frame, only a subset of fluorophores is in the active state, and these are localized with nanometer accuracy (B). This process is repeated for each frame in the acquisition (C), when different fluorophores are in the active state. The super‐resolved image (D) is a pointillistic rendering of all the localized spots from throughout the acquisition.
Localization‐based super‐resolution requires labeling with fluorophores that have the capacity to switch between an active and a dark state; there are a wide range of commercially available options and these have been reviewed heavily in the literature. But once the procedure is established, this method offers the ultimate precision and specificity when it comes to quantifying the organization of molecules in cells.