Abstract

Fluorescence-lifetime single molecule localization microscopy (FL-SMLM) adds the lifetime dimension to the spatial super-resolution provided by SMLM. Independent of intensity and spectrum, this lifetime information can be used, for example, to quantify the energy transfer efficiency in Förster Resonance Energy Transfer (FRET) imaging, to probe the local environment with dyes that change their lifetime in an environment-sensitive manner, or to achieve image multiplexing by using dyes with different lifetimes. We present a thorough theoretical analysis of fluorescence-lifetime determination in the context of FL-SMLM and compare different lifetime-fitting approaches. In particular, we investigate the impact of background and noise, and give clear guidelines for procedures that are optimized for FL-SMLM. We do also present and discuss our public-domain software package “Fluorescence-Lifetime TrackNTrace,” which converts recorded fluorescence microscopy movies into super-resolved FL-SMLM images.

Highlights

  • The advent of super-resolution microscopy (Hell, 2007; Huang et al, 2009) has revolutionized optical microscopy over the last ca. 30 years, pushing the limits of spatial resolution by three orders of magnitude down to the molecular length scale

  • Using Monte-Carlo simulations, we have demonstrated that maximum likelihood estimator (MLE)-based fitting outperforms common LSQ-based fitting and achieves close to shot-noise limited accuracy

  • In SMLM, the localization uncertainty derived from the Cramér-Rao lower bound (CRLB) has become an indispensable parameter for data filtering

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Summary

Introduction

The advent of super-resolution microscopy (Hell, 2007; Huang et al, 2009) has revolutionized optical microscopy over the last ca. 30 years, pushing the limits of spatial resolution by three orders of magnitude down to the molecular length scale. 30 years, pushing the limits of spatial resolution by three orders of magnitude down to the molecular length scale The first of these super-resolution methods was STimulated Emission Depletion (STED) microscopy (Hell and Wichmann, 1994; Klar et al, 2000), developed by Stefan Hell and co-workers since the nineties of the last century, and later extended to Ground State Depletion IMaging (GSDIM) (Fölling et al, 2008; Hell, 2009) and REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) imaging (Keller et al, 2007; Schwentker et al, 2007). By recording many images of well-separated molecules (by using fluorescent labels that can be switched between non-fluorescent and fluorescent states), one can generate a super-resolved image, the resolution of which is only limited by the number of photons detectable from a single molecule

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