Quantifying the minimum localization uncertainty of image scanning localization microscopy

Kalisvaart, Dylan; Hung, Shih-Te; Smith, Carlas S.

doi:10.1016/j.bpr.2024.100143

Cited by 3 publications

(1 citation statement)

References 31 publications

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“…This integration leads to a remarkable enhancement in resolution, up to a factor of two, or equivalently, a reduction in the required photon budget for a given resolution by up to four times. A recent theoretical study [46] even suggests that, with advanced data analysis techniques, the resolution enhancement achievable with iSMLM could surpass the factor of two.…”

Section: Discussionmentioning

confidence: 99%

Doubling the resolution of fluorescence-lifetime single-molecule localization microscopy with image scanning microscopy

Radmacher,

Nevskyi,

Gallea

et al. 2023

Preprint

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Single-molecule localization microscopy (SMLM) is a widely used super-resolution microscopy technique, renowned for its simplicity and impressive achievable resolution. It is typically based on a wide-field fluorescence microscope and relies on emitter photoswitching to capture individual snapshots of a sample with sparse distributions of fluorescent labels. These labels can then be precisely identified and localized. Recently, we demonstrated that SMLM can also be realized with a fast confocal laser-scanning microscope (CLSM), opening the door to fluorescence lifetime SMLM. This technique has found applications in lifetime-based image multiplexing and metal-induced energy transfer SMLM. In this work, we present an extension of CLSM-based SMLM by incorporating a single-photon detector array into the CLSM. This enables the combination of CLSM-based SMLM with Image Scanning Microscopy (ISM), a powerful technique for doubling the lateral resolution of a laser-scanning confocal microscope. By doing so, we achieve a two-fold improvement in lateral localization accuracy for SMLM in an easy and straightforward manner. Furthermore, we take advantage of the lifetime information provided by our CLSM for colocalizing different targets without any chromatic aberration. This feature becomes particularly crucial when dealing with the few-nanometer resolution achievable with our system. To demonstrate the capabilities of fluorescence lifetime ISM-SMLM (FL-iSMLM), we perform dSTORM and DNA-PAINT experiments on fluorescently labeled cells, showcasing the resolution-doubling and fluorescence lifetime multiplexing of our system. Finally, we have developed an open-source software that allows any interested user to easily implement (FL)-iSMLM into an existing CLSM equipped with a fast array detector. This user-friendly approach enables widespread adoption and application of (FL)-iSMLM in various research settings.

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Section: Discussionmentioning

confidence: 99%