A simple method to estimate the average localization precision of a single-molecule localization microscopy experiment

Endesfelder, Ulrike; Malkusch, Sebastian; Fricke, Franziska; Heilemann, Mike

doi:10.1007/s00418-014-1192-3

Cited by 222 publications

(190 citation statements)

References 37 publications

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“…The measured distance between actin filaments shows a resolution of at least 334 nm in the case of the low NA objective lens used for chip-based dSTORM. This result is confirmed by localization precision calculations 32 , estimating the average localization precision to be 87.6 nm as shown here. Fig.…”

Section: Supplementary Figure 2 | Mechanical Drift In the Setup For Csupporting

confidence: 73%

“…This shows a resolution of better than 50 nm (Fig. 2b), confirmed by full-width-at-half-maximum (FWHM) values, localization precision 32 , and Fourier ring correlation 33,34 (FRC) calculations ( Supplementary Fig. 1).…”

Section: Chip-based Single Molecule Localization Microscopymentioning

confidence: 72%

“…• Another approach to estimating the experimentally achieved average localization precision is proposed by Endesfelder et al 32 (referred to as [d]). This method relies on computations on the distance to nearest neighbours of localizations in adjacent frames of the raw data as these possibly stem from the same molecule.…”

Section: Supplementary Note 2: Obtainable Resolution For Objective Lementioning

confidence: 99%

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Chip-based wide field-of-view nanoscopy

et al. 2017

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Optical microscopy based on waveguide chips significantly reduces the complexity of the entire optical setup, enabling miniaturization by completely removing the excitation light path from the microscope. Instead, waveguides which tightly confine the guided light by total internal reflection due to a high refractive index contrast (HIC) to the surrounding media such as water and cells are used to deliver the illumination light to the sample. The evanescent field on top of the waveguide can be utilized for total internal reflection fluorescence (TIRF) excitation over an almost arbitrarily wide FOV that is intrinsically independent of the detection objective lens and in principle only limited by the waveguide design. Evanescent field excitation using waveguides was first introduced by Grandin et al. 17 , where a slab waveguide was used to generate an evanescent field over the large stretch of the waveguide chip. Slab waveguides (Fig. 1a) Here, we demonstrate waveguide chip-based super-resolution fluorescence imaging by two complementary approaches using ESI and dSTORM. The high intensity in the evanescent field generated by the HIC waveguide material is used for optical switching of fluorophores as required by dSTORM. In addition, the intrinsically multi-mode interference pattern within the waveguide is used to generate fluctuating intensity patterns for ESI. To demonstrate the applicability of waveguide chip-based super-resolution microscopy we visualize the connection of the actin cytoskeleton and plasma membrane fenestrations in liver sinusoidal endothelial cells (LSECs). RESULTS Chip-based single molecule localization microscopyThe performance of chip-based dSTORM is shown by imaging immunostained microtubules in rat LSECs 26 plated directly on the waveguide (Fig. 2a). Measuring the lateral profile along one straight microtubule filament reveals a hollow structure 27 which has been used earlier in localization microscopy as a benchmark sample [28][29][30] , discussed in detail in 31 . This shows a resolution of better than 50 nm ( Fig. 2b), confirmed by full-width-at-half-maximum (FWHM) values, localization precision 32 , and Fourier ring correlation 33,34 (FRC) calculations ( Supplementary Fig. 1). The resolution capability was further investigated by using DNA origami nanorulers that provide markers at (50 ± 5) nm distance as references. These can be clearly resolved in chip-based dSTORM (Fig. 2c,d, Supplementary Fig. 2) which shows a comparable performance to the widely used architecture of an inverted TIRF dSTORM setup (Fig. 2c,d, Supplementary Fig. 3).As an advantage over conventional setups, waveguide chip-based nanoscopy greatly benefits from the fact that the fluorescence excitation is independent of the detection objective lens. As fluorescence is excited by the evanescent field of the waveguide, the technique provides optical sectioning and excellent signal to background ratios at penetration depths below 200 nm ( Supplementary Fig. 4, Supplementary Fig. 5, Video 1), similar to objective-based TIRF...

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Section: Supplementary Figure 2 | Mechanical Drift In the Setup For Csupporting

confidence: 73%

Section: Chip-based Single Molecule Localization Microscopymentioning

confidence: 72%

Section: Supplementary Note 2: Obtainable Resolution For Objective Lementioning

confidence: 99%

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Chip-based wide field-of-view nanoscopy

et al. 2017

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“…Localization precision was determined to 26 nm using a nearest neighbor approach, according to Ref. 42. The software package used to visualize and derive quantitative data on protein localization and dynamics was custom written for Metamorph (Visitron Systems GmbH, Puchheim, Germany).…”

Section: Single-particle Tracking With Photoactivatedlocalization Micmentioning

confidence: 99%

Single-particle tracking uncovers dynamics of glutamate-induced retrograde transport of NF-κB p65 in living neurons

et al. 2016

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Abstract. Retrograde transport of NF-κB from the synapse to the nucleus in neurons is mediated by the dynein/ dynactin motor complex and can be triggered by synaptic activation. The caliber of axons is highly variable ranging down to 100 nm, aggravating the investigation of transport processes in neurites of living neurons using conventional light microscopy. We quantified for the first time the transport of the NF-κB subunit p65 using high-density single-particle tracking in combination with photoactivatable fluorescent proteins in living mouse hippocampal neurons. We detected an increase of the mean diffusion coefficient (D mean ) in neurites from 0.12 AE 0.05 to 0.61 AE 0.03 μm 2 ∕s after stimulation with glutamate. We further observed that the relative amount of retrogradely transported p65 molecules is increased after stimulation. Glutamate treatment resulted in an increase of the mean retrograde velocity from 10.9 AE 1.9 to 15 AE 4.9 μm∕s, whereas a velocity increase from 9 AE 1.3 to 14 AE 3 μm∕s was observed for anterogradely transported p65. This study demonstrates for the first time that glutamate stimulation leads to an increased mobility of single NF-κB p65 molecules in neurites of living hippocampal neurons.

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“…Ulrike Endesfelder, Mike Heilemann and colleagues (Endesfelder et al 2014) developed a simple method to estimate the average localization precision of a single-molecule localization microscopy experiment based on nearest neighbor analysis. they demonstrate that the method can be used for 2D and 3D single-molecule localization microscopy experiments in a very general context and includes parameters like time dependence, alignment registration and structural labeling factors.…”

mentioning

confidence: 99%