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Quantifying the minimum localization uncertainty of image scanning localization microscopy.

Dylan Kalisvaart¹, Shih-Te Hung¹, Carlas S Smith^1,2

¹Delft Center for Systems and Control, Delft University of Technology, Delft, the Netherlands.

Biophysical Reports

|February 21, 2024

Summary

This summary is machine-generated.

Modulation enhanced localization for spinning disk confocal microscopy (SpinFlux) improves single-molecule localization precision. This technique offers significant advancements for super-resolution microscopy, enhancing localization accuracy in biological imaging.

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Area of Science:

Super-resolution microscopy
Optical imaging techniques
Biophysics

Background:

Single-molecule localization microscopy (SMLM) achieves nanoscale resolution but has limitations in precision.
Modulation-enhanced SMLM (meSMLM) improves precision by using patterned illumination to activate emitters sequentially.
Existing meSMLM methods require specialized setups and may not be compatible with all microscopy platforms.

Purpose of the Study:

Introduce SpinFlux, a novel modulation-enhanced localization method for spinning disk confocal microscopy.
Enhance the localization precision of SMLM using a spinning disk confocal setup.
Compare the performance of SpinFlux with other advanced localization techniques.

Main Methods:

SpinFlux utilizes a spinning disk with pinholes in illumination and emission paths for sequential region illumination.
An intensity-modulated emission signal is analyzed for each pattern to determine emitter positions.
A statistical image formation model and Cramér-Rao lower bound were derived to quantify localization uncertainty.

Main Results:

SpinFlux achieves a twofold localization precision improvement over SMLM with three illumination patterns focused around the emitter.
Using four donut-shaped patterns, SpinFlux demonstrated a maximum local precision improvement of 3.5 over SMLM.
Localization on Fourier reweighted image scanning microscopy reconstructions offers ideal global precision improvement (2.1x over SMLM).

Conclusions:

SpinFlux significantly enhances localization precision in spinning disk confocal microscopy.
SpinFlux is a valuable tool for local precision refinements in super-resolution imaging.
Image scanning microscopy reconstructions provide the greatest potential for global precision improvements, while SpinFlux excels in local refinements.