Precision in iterative modulation enhanced single-molecule localization microscopy

Kalisvaart, Dylan; Cnossen, Jelmer; Hung, Shih-Te; Stallinga, Sjoerd; Verhaegen, Michel; Smith, Carlas

doi:10.1016/j.bpj.2022.05.027

Cited by 14 publications

(14 citation statements)

References 24 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%

“…In the realm of SMLM, ISM directly enhances the accuracy of single-molecule localizations, thus significantly elevating the ultimate SMLM resolution. From a perspective rooted in physical optics, this mirrors the twofold increase in localization accuracy achieved through patterned illumination techniques like SIMPLE [18, 19], SIMFLUX [20], as well as others [21, 22]. However, ISM offers a substantial reduction in experimental complexity.…”

Section: Introductionmentioning

confidence: 99%

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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%

Section: Introductionmentioning

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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“…Sauer and colleagues recently summarized the three key factors determining image resolution in SMLM: i) localization precision (statistical scattering of the measured position coordinates), ii) localization accuracy (systematic deviation between the measured and true position) and iii) the labeling density ( Helmerich et al, 2022 ). The first of these factors has been the main focus of efforts for improving image resolution, via hardware, e.g., by combining improved structured illumination conditions with single-molecule detection ( Kalisvaart et al, 2022 ; Zhan et al, 2022 ), DNA-PAINT with STORM ( Cnossen et al, 2020 ), using cryo-SMLM and ad hoc software corrections ( Schneider et al, 2020 ; Hinterer et al, 2022 ), or via computational methods that enhance localization precision through engineering of the PSF or other approaches as applied to a single ( Shaw et al, 2022 ) ( Li M. et al, 2022 ) or multiple fluorophores (colocalization precision) ( Koyama-Honda et al, 2005 ; Donnert et al, 2007 ; Lemmer et al, 2009 ; Curthoys et al, 2013 ; Weisenburger et al, 2014 ; Georgieva et al, 2016 ; Willems and MacGillavry, 2022 ).…”

Section: Future Prospectsmentioning

confidence: 99%

“…Another limiting factor on the road to improving the resolution of superresolution microscopies is the size of the fluorophore proper. Last-generation nanoscopies like 2-D MINFLUX ( Balzarotti et al, 2017 ; Eilers et al, 2018 ; Masullo et al, 2021 ), cryogenic nanoscopy ( Furubayashi et al, 2019 ; Furubayashi et al, 2020 ), iterative modulation-enhanced SMLM ( Kalisvaart et al, 2022 ), improved structured-illumination microscopies ( Chen et al, 2018 ; Markwirth et al, 2019 ; Zhanghao et al, 2019 ; Mangeat et al, 2021 ; Qiao et al, 2021 ; Smith et al, 2021 ; Chen et al, 2022 ; Hunter et al, 2022 ; Zhan et al, 2022 ), new high-resolution DNA-PAINT modalities ( Schnitzbauer et al, 2017 ), 3-D MINFLUX ( Gwosch et al, 2020 ; Grabner et al, 2022 ; Gwosch et al, 2022 ) or MINSTED ( Weber et al, 2021 ) are now facing the need to introduce smaller probes to resolve structures at the molecular scale. Successful examples are provided by the recent work in which pyrrolysyl-tRNA synthetase and orthogonal tRNA were matched to introduce clickable amino acids into bacterial and mammalian cell proteins, accomplishing 3-D imaging of β-actin in filopodia with a precision of ∼2 nm ( Mihaila et al, 2022 ).…”

Section: Future Prospectsmentioning

confidence: 99%

Fluorescence microscopy imaging of a neurotransmitter receptor and its cell membrane lipid milieu

Barrantes

2022

Front. Mol. Biosci.

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Hampered by the diffraction phenomenon, as expressed in 1873 by Abbe, applications of optical microscopy to image biological structures were for a long time limited to resolutions above the ∼200 nm barrier and restricted to the observation of stained specimens. The introduction of fluorescence was a game changer, and since its inception it became the gold standard technique in biological microscopy. The plasma membrane is a tenuous envelope of 4 nm–10 nm in thickness surrounding the cell. Because of its highly versatile spectroscopic properties and availability of suitable instrumentation, fluorescence techniques epitomize the current approach to study this delicate structure and its molecular constituents. The wide spectral range covered by fluorescence, intimately linked to the availability of appropriate intrinsic and extrinsic probes, provides the ability to dissect membrane constituents at the molecular scale in the spatial domain. In addition, the time resolution capabilities of fluorescence methods provide complementary high precision for studying the behavior of membrane molecules in the time domain. This review illustrates the value of various fluorescence techniques to extract information on the topography and motion of plasma membrane receptors. To this end I resort to a paradigmatic membrane-bound neurotransmitter receptor, the nicotinic acetylcholine receptor (nAChR). The structural and dynamic picture emerging from studies of this prototypic pentameric ligand-gated ion channel can be extrapolated not only to other members of this superfamily of ion channels but to other membrane-bound proteins. I also briefly discuss the various emerging techniques in the field of biomembrane labeling with new organic chemistry strategies oriented to applications in fluorescence nanoscopy, the form of fluorescence microscopy that is expanding the depth and scope of interrogation of membrane-associated phenomena.

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“…The images contain molecule shapes blurred with the microscope emission pattern, or the point spread function (PSF), which can be experimentally designed and modelled to encode additional positional information in its shape 5,7,8 . The molecule positions are determined through maximum likelihood estimation (MLE) 9 or maximum a posteriori (MAP) estimation 10 of the position parameters within the point spread function (PSF) model. Therefore, creating a flexible PSF model that accurately represents the true experimental PSF is crucial for the ultimate localization accuracy and precision 11 .…”

Section: Introductionmentioning

confidence: 99%

Using splines for point spread function calibration at non-uniform depths in localization microscopy

Korovin,

Kalisvaart,

Hung

et al. 2024

Preprint

Self Cite

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Single-molecule localization microscopy methods extensively leverage the microscope point spread function (PSF) for fitting the molecules. Calibrating an accurate PSF model is especially difficult in the presence of depth-dependent aberrations which alter the PSF shape depending on the imaging depth. The aberrations at depths of a few micrometers become substantial enough to considerably impoverish the conventional calibration methods' performance. In our work, we propose a novel spline model which enables the depth-dependent PSF model calibration by interpolating between the beads at arbitrary depths. We show that diffspline reduces the PSF intensity overestimation by 67.8 percentage points and underestimation by 21.8 percentage points. Moreover, it eliminates the depth-dependent bias and improves the localization precision two-fold compared to previous approaches.

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Precision in iterative modulation enhanced single-molecule localization microscopy

Cited by 14 publications

References 24 publications

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

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

Fluorescence microscopy imaging of a neurotransmitter receptor and its cell membrane lipid milieu

Using splines for point spread function calibration at non-uniform depths in localization microscopy

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