Novel fibrillar structure in the inversin compartment of primary cilia revealed by 3D single-molecule superresolution microscopy

Using three-dimensional single-molecule superresolution imaging and systematic analysis of knockout cell lines, we have determined the molecular structure and composition of the inversin compartment in primary cilia. INVS establishes fibrillar structures that recruit ANKS6-NEK8 complexes to sequester NPHP3 in this unique periaxonemal compartment.

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Synthetic and genetic dimers as quantification ruler for single-molecule counting with PALM

Molecular Biology of the Cell ◽

10.1091/mbc.e18-10-0661 ◽

2019 ◽

Vol 30 (12) ◽

pp. 1369-1376 ◽

Cited By ~ 9

Author(s):

Tim N. Baldering ◽

Marina S. Dietz ◽

Karl Gatterdam ◽

Christos Karathanasis ◽

Ralph Wieneke ◽

...

Keyword(s):

Membrane Proteins ◽

Single Molecule ◽

Fusion Proteins ◽

Fluorescent Proteins ◽

Superresolution Microscopy ◽

Superresolution Imaging ◽

Molecular Information ◽

Kinetics Of

How membrane proteins oligomerize determines their function. Superresolution microscopy can report on protein clustering and extract quantitative molecular information. Here, we evaluate the blinking kinetics of four photoactivatable fluorescent proteins for quantitative single-molecule microscopy. We identified mEos3.2 and mMaple3 to be suitable for molecular quantification through blinking histogram analysis. We designed synthetic and genetic dimers of mEos3.2 as well as fusion proteins of monomeric and dimeric membrane proteins as reference structures, and we demonstrate their versatile use for quantitative superresolution imaging in vitro and in situ. We further found that the blinking behavior of mEos3.2 and mMaple3 is modified by a reducing agent, offering the possibility to adjust blinking parameters according to experimental needs.

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Obtaining 3D Super-resolution Information from 2D Super-resolution Images through a 2D-to-3D Transformation Algorithm

10.1101/188060 ◽

2017 ◽

Cited By ~ 1

Author(s):

Andrew Ruba ◽

Wangxi Luo ◽

Joseph Kelich ◽

Weidong Yang

Keyword(s):

Single Molecule ◽

Rotational Symmetry ◽

Three Dimensional ◽

Super Resolution ◽

Live Cells ◽

Limited Information ◽

Spatiotemporal Resolution ◽

Superresolution Microscopy ◽

Localization Analysis ◽

Probability Information

AbstractCurrently, it is highly desirable but still challenging to obtain three-dimensional (3D) superresolution information of structures in fixed specimens as well as dynamic processes in live cells with a high spatiotemporal resolution. Here we introduce an approach, without using 3D superresolution microscopy or real-time 3D particle tracking, to achieve 3D sub-diffraction-limited information with a spatial resolution of ≤ 1 nm. This is a post-localization analysis that transforms 2D super-resolution images or 2D single-molecule localization distributions into their corresponding 3D spatial probability information. The method has been successfully applied to obtain structural and functional information for 25-300 nm sub-cellular organelles that have rotational symmetry. In this article, we will provide a comprehensive analysis of this method by using experimental data and computational simulations.

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Particle localization using local gradients and its application to nanometer stabilization of a microscope

10.1101/2021.11.11.468294 ◽

2021 ◽

Author(s):

Anatolii V. Kashchuk ◽

Oleksandr Perederiy ◽

Chiara Caldini ◽

Lucia Gardini ◽

Francesco Saverio Pavone ◽

...

Keyword(s):

Single Molecule ◽

Three Dimensional ◽

Universal Method ◽

Superresolution Microscopy ◽

Accurate Localization ◽

Micro Particles ◽

Localization Algorithms ◽

Gradient Calculation ◽

Particle Localization ◽

Local Gradient

Accurate localization of single particles plays an increasingly important role in a range of biological techniques, including single molecule tracking and localization-based superresolution microscopy. Such techniques require fast and accurate particle localization algorithms as well as nanometer-scale stability of the microscope. Here, we present a universal method for three-dimensional localization of single labeled and unlabeled particles based on local gradient calculation of microscopy images. The method outperforms current techniques in high noise conditions, and it is capable of nanometer accuracy localization of nano- and micro-particles with sub-ms calculation time. By localizing a fixed particle as fiducial mark and running a feedback loop, we demonstrate its applicability for active drift correction in sensitive nanomechanical measurements such as optical trapping and superresolution imaging. A multiplatform open software package comprising a set of tools for local gradient calculation in brightfield and fluorescence microscopy is shared to the scientific community.

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Cby1 promotes Ahi1 recruitment to a ring-shaped domain at the centriole–cilium interface and facilitates proper cilium formation and function

Molecular Biology of the Cell ◽

10.1091/mbc.e14-02-0735 ◽

2014 ◽

Vol 25 (19) ◽

pp. 2919-2933 ◽

Cited By ~ 40

Author(s):

Yin Loon Lee ◽

Joshua Santé ◽

Colin J. Comerci ◽

Benjamin Cyge ◽

Luis F. Menezes ◽

...

Keyword(s):

Primary Cilia ◽

Three Dimensional ◽

Joubert Syndrome ◽

Cystic Kidneys ◽

Superresolution Microscopy ◽

Close Proximity ◽

And Function ◽

Canonical Wnt ◽

Syndrome Protein ◽

Shaped Domain

Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Cby1, the mammalian orthologue of the Drosophila Chibby protein, localizes to mature centrioles, is important for ciliogenesis in multiciliated airway epithelia in mice, and antagonizes canonical Wnt signaling via direct regulation of β-catenin. We report that deletion of the mouse Cby1 gene results in cystic kidneys, a phenotype common to ciliopathies, and that Cby1 facilitates the formation of primary cilia and ciliary recruitment of the Joubert syndrome protein Arl13b. Localization of Cby1 to the distal end of mature centrioles depends on the centriole protein Ofd1. Superresolution microscopy using both three-dimensional SIM and STED reveals that Cby1 localizes to an ∼250-nm ring at the distal end of the mature centriole, in close proximity to Ofd1 and Ahi1, a component of the transition zone between centriole and cilium. The amount of centriole-localized Ahi1, but not Ofd1, is reduced in Cby1−/− cells. This suggests that Cby1 is required for efficient recruitment of Ahi1, providing a possible molecular mechanism for the ciliogenesis defect in Cby1−/− cells.

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High-numerical-aperture cryogenic light microscopy for increased precision of superresolution reconstructions

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618206114 ◽

2017 ◽

Vol 114 (15) ◽

pp. 3832-3836 ◽

Cited By ~ 15

Author(s):

Marc Nahmani ◽

Conor Lanahan ◽

David DeRosier ◽

Gina G. Turrigiano

Keyword(s):

Single Molecule ◽

Room Temperature ◽

Fluorescent Protein ◽

Numerical Aperture ◽

Fluorescent Imaging ◽

Objective Lens ◽

High Numerical Aperture ◽

Superresolution Microscopy ◽

Superresolution Imaging ◽

Photoactivated Localization Microscopy

Superresolution microscopy has fundamentally altered our ability to resolve subcellular proteins, but improving on these techniques to study dense structures composed of single-molecule-sized elements has been a challenge. One possible approach to enhance superresolution precision is to use cryogenic fluorescent imaging, reported to reduce fluorescent protein bleaching rates, thereby increasing the precision of superresolution imaging. Here, we describe an approach to cryogenic photoactivated localization microscopy (cPALM) that permits the use of a room-temperature high-numerical-aperture objective lens to image frozen samples in their native state. We find that cPALM increases photon yields and show that this approach can be used to enhance the effective resolution of two photoactivatable/switchable fluorophore-labeled structures in the same frozen sample. This higher resolution, two-color extension of the cPALM technique will expand the accessibility of this approach to a range of laboratories interested in more precise reconstructions of complex subcellular targets.

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Superresolution imaging reveals structurally distinct periodic patterns of chromatin along pachytene chromosomes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1516928112 ◽

2015 ◽

Vol 112 (47) ◽

pp. 14635-14640 ◽

Cited By ~ 42

Author(s):

Kirti Prakash ◽

David Fournier ◽

Stefan Redl ◽

Gerrit Best ◽

Máté Borsos ◽

...

Keyword(s):

Single Molecule ◽

Histone H3 ◽

Periodic Patterns ◽

Superresolution Microscopy ◽

Superresolution Imaging ◽

Meiotic Chromosomes ◽

Loop Pattern ◽

Active Transcription ◽

Dense Cluster ◽

Small Clusters

During meiosis, homologous chromosomes associate to form the synaptonemal complex (SC), a structure essential for fertility. Information about the epigenetic features of chromatin within this structure at the level of superresolution microscopy is largely lacking. We combined single-molecule localization microscopy (SMLM) with quantitative analytical methods to describe the epigenetic landscape of meiotic chromosomes at the pachytene stage in mouse oocytes. DNA is found to be nonrandomly distributed along the length of the SC in condensed clusters. Periodic clusters of repressive chromatin [trimethylation of histone H3 at lysine (Lys) 27 (H3K27me3)] are found at 500-nm intervals along the SC, whereas one of the ends of the SC displays a large and dense cluster of centromeric histone mark [trimethylation of histone H3 at Lys 9 (H3K9me3)]. Chromatin associated with active transcription [trimethylation of histone H3 at Lys 4 (H3K4me3)] is arranged in a radial hair-like loop pattern emerging laterally from the SC. These loops seem to be punctuated with small clusters of H3K4me3 with an average spread larger than their periodicity. Our findings indicate that the nanoscale structure of the pachytene chromosomes is constrained by periodic patterns of chromatin marks, whose function in recombination and higher order genome organization is yet to be elucidated.

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Three-Dimensional Superresolution Imaging of the FtsZ Ring during Cell Division of the Cyanobacterium Prochlorococcus

mBio ◽

10.1128/mbio.00657-17 ◽

2017 ◽

Vol 8 (6) ◽

Cited By ~ 8

Author(s):

Riyue Liu ◽

Yaxin Liu ◽

Shichang Liu ◽

Ying Wang ◽

Kim Li ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Cell Division ◽

Three Dimensional ◽

Plant Cells ◽

Flowering Plant ◽

Superresolution Microscopy ◽

Superresolution Imaging ◽

Ftsz Ring ◽

Cell Division Protein ◽

Photosynthetic Cells

ABSTRACT Superresolution imaging has revealed subcellular structures and protein interactions in many organisms. However, superresolution microscopy with lateral resolution better than 100 nm has not been achieved in photosynthetic cells due to the interference of a high-autofluorescence background. Here, we developed a photobleaching method to effectively reduce the autofluorescence of cyanobacterial and plant cells. We achieved lateral resolution of ~10 nm with stochastic optical reconstruction microscopy (STORM) in the sphere-shaped cyanobacterium Prochlorococcus and the flowering plant Arabidopsis thaliana. During the cell cycle of Prochlorococcus, we characterized the three-dimensional (3D) organization of the cell division protein FtsZ, which forms a ring structure at the division site and is important for cytokinesis of bacteria and chloroplasts. Although the FtsZ ring assembly process in rod-shaped bacteria has been studied extensively, it has rarely been studied in sphere-shaped bacteria. Similarly to rod-shaped bacteria, our results with Prochlorococcus also showed the assembly of FtsZ clusters into incomplete rings and then complete rings during cell division. Differently from rod-shaped bacteria, the FtsZ ring diameter was not found to decrease during Prochlorococcus cell division. We also discovered a novel double-Z-ring structure, which may be the Z rings of two daughter cells in a predivisional mother cell. Our results showed a quantitative picture of the in vivo Z ring organization of sphere-shaped bacteria. IMPORTANCE Superresolution microscopy has not been widely used to study photosynthetic cells due to their high-autofluorescence background. Here, we developed a photobleaching method to reduce the autofluorescence of cyanobacteria and plant cells. After photobleaching, we performed superresolution imaging in the cyanobacterium Prochlorococcus and the flowering plant Arabidopsis thaliana with ~10-nm resolution, which is the highest resolution in a photosynthetic cell. With this method, we characterized the 3D organization of the cell division protein FtsZ in Prochlorococcus. We found that the morphological variation of the FtsZ ring during cell division of the sphere-shaped cyanobacterium Prochlorococcus is similar but not identical to that of rod-shaped bacteria. Our method might also be applicable to other photosynthetic organisms. IMPORTANCE Superresolution microscopy has not been widely used to study photosynthetic cells due to their high-autofluorescence background. Here, we developed a photobleaching method to reduce the autofluorescence of cyanobacteria and plant cells. After photobleaching, we performed superresolution imaging in the cyanobacterium Prochlorococcus and the flowering plant Arabidopsis thaliana with ~10-nm resolution, which is the highest resolution in a photosynthetic cell. With this method, we characterized the 3D organization of the cell division protein FtsZ in Prochlorococcus. We found that the morphological variation of the FtsZ ring during cell division of the sphere-shaped cyanobacterium Prochlorococcus is similar but not identical to that of rod-shaped bacteria. Our method might also be applicable to other photosynthetic organisms.

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Excitation-multiplexed multicolor superresolution imaging with fm-STORM and fm-DNA-PAINT

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1804725115 ◽

2018 ◽

Vol 115 (51) ◽

pp. 12991-12996 ◽

Cited By ~ 11

Author(s):

Pablo A. Gómez-García ◽

Erik T. Garbacik ◽

Jason J. Otterstrom ◽

Maria F. Garcia-Parajo ◽

Melike Lakadamyali

Keyword(s):

Single Molecule ◽

Cross Talk ◽

Learning Algorithm ◽

Imaging Modality ◽

Fluorescence Emission ◽

Acquisition Time ◽

Color Channel ◽

Superresolution Microscopy ◽

Superresolution Imaging ◽

Frequency Multiplexing

Recent advancements in single-molecule-based superresolution microscopy have made it possible to visualize biological structures with unprecedented spatial resolution. Determining the spatial coorganization of these structures within cells under physiological and pathological conditions is an important biological goal. This goal has been stymied by the current limitations of carrying out superresolution microscopy in multiple colors. Here, we develop an approach for simultaneous multicolor superresolution imaging which relies solely on fluorophore excitation, rather than fluorescence emission properties. By modulating the intensity of the excitation lasers at different frequencies, we show that the color channel can be determined based on the fluorophore’s response to the modulated excitation. We use this frequency multiplexing to reduce the image acquisition time of multicolor superresolution DNA-PAINT while maintaining all its advantages: minimal color cross-talk, minimal photobleaching, maximal signal throughput, ability to maintain the fluorophore density per imaged color, and ability to use the full camera field of view. We refer to this imaging modality as “frequency multiplexed DNA-PAINT,” or fm-DNA-PAINT for short. We also show that frequency multiplexing is fully compatible with STORM superresolution imaging, which we term fm-STORM. Unlike fm-DNA-PAINT, fm-STORM is prone to color cross-talk. To overcome this caveat, we further develop a machine-learning algorithm to correct for color cross-talk with more than 95% accuracy, without the need for prior information about the imaged structure.

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