Abstract
Single-Molecule Localization Microscopy (SMLM) has become one of the most important methods of super-resolution fluorescence microscopy. It is based on the precise localization of single molecules in wide-field microscopy images. It is well known that the localization accuracy can show a significant bias if the imaged molecules have a fixed orientation and are located either close to an interface or not exactly within the focal plane of the microscope. In this Letter, we propose a simple solution to this problem, which is based on polarization-resolved imaging. This method can be easily implemented into any existing SMLM setup, and we demonstrate its performance by imaging single dye molecules embedded into a polymer film, which fixes their orientation in space.
Highlights
Single-Molecule Localization Microscopy (SMLM) has become one of the most powerful techniques of super-resolution fluorescence microscopy
By labeling a sample with fluorophores, which can be switched between dark and bright fluorescent states, and taking many sequential images of the sample, where each time a different small subset of identifiable and well-separated molecules are switched on, one can localize them in each recorded frame with high accuracy and frame by frame, obtain a super-resolved image of the sample
The different methods of SMLM mostly differ by their approach of how to switch fluorophores between dark and bright states
Summary
Single-Molecule Localization Microscopy (SMLM) has become one of the most powerful techniques of super-resolution fluorescence microscopy. Lew and co-worker presented an elegant solution to this problem: By using a custom-build polarizer (broadband metasurface mask) that transmits only the azimuthally polarized component of the fluorescence light, they could demonstrate that the resulting images of single molecules do no longer show orientation-dependent lateral shifts with respect to their actual positions.. It is compatible with any type of emitter, labeling procedure, sample geometry, or rotational mobility of emitters It will be suitable for correcting the polarization-induced localization bias even under imperfect imaging conditions (e.g., refractive index mismatch) as often found in biological samples and is in all other characteristics similar to other polarization-resolved SMLM techniques (see Ref. 19).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
