In our research, we develop a new theory for modulation enhanced localization in image scanning microscopy (ISM), enabling up to 3.5-fold precision improvements over single-molecule localization microscopy. In the cover image of the March 13 issue of Biophysical Reports, we illustrate the design of such an image scanning localization microscope, which we name SpinFlux.
The cover image demonstrates how a SpinFlux image scanning localization microscope forms an image of a fluorescent sample. Excitation light is windowed by pinholes in the spinning disk of an image scanning microscope. Because of this windowing, the sample is illuminated with patterned excitation, resulting in bright and dark spots in the sample. As a result of this, the emission intensity of fluorescent labels in the sample will be modulated, proportional to the brightness of the excitation pattern at the emitter location.
We sequentially record the raw emission signal on the camera for varying rotations of the spinning disk. In contrast to existing ISM approaches, SpinFlux obtains its precision improvement directly from the raw data, by retrieving molecule positions from each individual camera frame. This direct processing is made possible by the windowing effect of the spinning disk. By combining knowledge about the emitter position with knowledge about the excitation patterns, the position of each emitter can be estimated with improved precision.
The research shown in this article explores the theoretical minimum uncertainty achievable through localization on ISM reconstruction data and through SpinFlux. This directly addresses a fundamental question in the field of single-molecule localization microscopy, namely the determination of the precision with which single molecules can be localized.
Our study has notable implications. Our findings indicate that even minor adjustments to existing ISM setups, such as phase masks, can substantially improve localization precision. In addition, our modeling framework allows for the evaluation of various spinning disk setups, offering insights for optimized spinning disk designs.
You can find more information on our work at https://www.tudelft.nl/me/over/afdelingen/delft-center-for-systems-and-control/research/numerics-for-control-identification/quantitative-nanoscopy and https://github.com/qnano.
— Dylan Kalisvaart, Shih-Te Hung, and Carlas S. Smith