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

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

Molecular Biology of the Cell ◽

10.1091/mbc.e19-09-0499 ◽

2020 ◽

Vol 31 (7) ◽

pp. 619-639 ◽

Cited By ~ 2

Author(s):

Henrietta W. Bennett ◽

Anna-Karin Gustavsson ◽

Camille A. Bayas ◽

Petar N. Petrov ◽

Nancie Mooney ◽

...

Keyword(s):

Molecular Structure ◽

Cell Lines ◽

Single Molecule ◽

Primary Cilia ◽

Three Dimensional ◽

Fibrillar Structure ◽

Systematic Analysis ◽

Superresolution Microscopy ◽

Superresolution Imaging

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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Enhanced-Fluorescence of a Dye on DNA- assembled Gold Nano-Dimers Discriminated by Lifetime Correlation Spectroscopy

10.26434/chemrxiv.5728446.v1 ◽

2017 ◽

Author(s):

Pedro M. R. Paulo ◽

David Botequim ◽

Agnieszka Jóskowiak ◽

Sofia Martins ◽

Duarte M. F. Prazeres ◽

...

Keyword(s):

Single Molecule ◽

Fluorescence Emission ◽

Directed Assembly ◽

Plasmon Mode ◽

Correlation Spectroscopy ◽

Enhanced Fluorescence ◽

Detection Volume ◽

Relaxation Component ◽

Good Agreement ◽

Confocal Detection

<div> <div> <div> <p>We have employed DNA-directed assembly to prepare dimers of gold nanoparticles and used their longitudinally coupled plasmon mode to enhance the fluorescence emission of an organic red-emitting dye, Atto-655. The plasmon- enhanced fluorescence of this dye using dimers of 80 nm particles was measured at single molecule detection level. The top enhancement factors were above 1000-fold in 71% of the dimers within a total of 32 dimers measured, and, in some cases, they reached almost 4000-fold, in good agreement with model simulations. Additionally, fluorescence lifetime correlation analysis enabled the separation of enhanced from non-enhanced emission simultaneously collected in our confocal detection volume. This approach allowed us to recover a short relaxation component exclusive to enhanced emission that is attributed to the interaction of the dye with DNA in the interparticle gaps. </p> </div> </div> </div>

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S09A5 Single-molecule experiments of F1-ATPase correlating with the crystal structures and molecular simulation(Mechanism of F_1-ATPase Molecular Motor-A Cross Talk among Single Molecule, Structural Biology, and Molecular Simulation Studies-)

Seibutsu Butsuri ◽

10.2142/biophys.47.s13_4 ◽

2007 ◽

Vol 47 (supplement) ◽

pp. S13

Author(s):

Hiroyuki Noji

Keyword(s):

Crystal Structures ◽

Molecular Simulation ◽

Structural Biology ◽

Single Molecule ◽

Cross Talk ◽

Molecular Motor ◽

Simulation Studies ◽

F1 Atpase

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OP0178 IMAGING NEOANGIOGENESIS IN RHEUMATOID ARTHRITIS (INIRA): WHOLE-BODY SYNOVIAL UPTAKE OF A 99MTC-LABELLED RGD PEPTIDE IS HIGHLY CORRELATED WITH POWER DOPPLER ULTRASOUND

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.5482 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 110.2-111

Author(s):

L. Attipoe ◽

S. Subesinghe ◽

C. Blanco-Gil ◽

M. Opena ◽

M. Rosser ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Correlation Coefficient ◽

Doppler Ultrasound ◽

Imaging Modality ◽

Power Doppler ◽

Whole Body ◽

Total Power ◽

Acquisition Time ◽

Power Doppler Ultrasound ◽

Highly Correlated

Background:Power Doppler ultrasound (PDUS) is superior to clinical examination in detecting synovitis in patients with rheumatoid arthritis (RA). Although dynamic and cheap it is impractical to scan large numbers of joints in routine clinical settings. MRI, whilst sensitive for synovitis, is expensive and routine use is limited to targeted joints. Bone scintigraphy produces whole body images but due to limited specificity is not routinely used.99mTc-maraciclatide (Serac Healthcare) is a radiolabelled tracer which binds with high affinity to integrin αvβ3, a cell-adhesion molecule up-regulated on neoangiogenic blood vessels. It therefore has the potential to image synovial inflammation at the whole-body level. We previously showed in a pilot study that uptake was seen in the inflamed joints of five RA patients and that this correlated with PDUS. This study explores correlation with PDUS in a larger groups of patients with varied disease activity.Objectives:To determine the correlation between ultrasound and99mTc-maraciclatide imaging in patients with rheumatoid arthritis.Methods:50 patients with RA fulfilling ACR 2010 classification criteria were recruited. Patients underwent an ultrasound scan of 40 joints with grey scale (GS) and PD quantification. Each joint was scored on a scale of 0-3 for GS and PD with a total score calculated for each patient. Within 3 hours of the ultrasound patients were injected with 740 MBq of99mTc-maraciclatide. Using a gamma camera, whole body planar views and dedicated hand and foot views were taken 2 hours after injection (Figure 1). Acquisition time was 20 minutes for whole body and 20 minutes for hand and foot views.99mTc-maraciclatide images were scored as positive or negative uptake for each joint (binary score). A quantitative score was also calculated for each joint where there was uptake with this corrected for background uptake. Total binary and quantitative scores per patient were calculated.Ultrasound and99mTc-maraciclatide scores were tested for correlation with Pearson’s correlation coefficient (r). Interrater agreement for 2 scorers was calculated using kappa (ĸ) and concordance correlation coefficient (Pc).Results:Strong correlation was seen when total PDUS was compared to binary scores (r=0.92, r2=0.85) (Figure 2) and quantitative scores (r=0.85, r2=0.72). ĸ was 0.82 and 0.79 for binary and ultrasound scores respectively.Pcwas 0.82 for quantitative scores. p was <0.0005 for all results.99mTc-maraciclatide uptake was also seen in inflamed tendons/tendon sheaths. The imaging procedure was well-tolerated. There were no tracer-related adverse events.Figure 1.99mTc-maraciclatide imaging with dedicated hand and foot viewsConclusion:99mTc-maraciclatide uptake was highly correlated with PDUS highlighting its potential as an alternative imaging modality.99mTc-based planar imaging has the unique capacity to image the whole body and hence the total synovial inflammatory load in a quick acquisition. The imaging equipment to perform these scans is already widely available in radiology departments. Interpretation of scans is also much simpler compared to US/MRI. It could therefore have a role in key decision-making points in pathways for diagnosis, treatment failure, and remission prior to dose tapering.Figure 2.Correlation between total power doppler and99mTc-maraciclatide binary scoresDisclosure of Interests:None declared

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Extraction of accurate cytoskeletal actin velocity distributions from noisy measurements

10.1101/2020.08.13.247304 ◽

2020 ◽

Author(s):

Cayla M. Miller ◽

Elgin Korkmazhan ◽

Alexander R. Dunn

Keyword(s):

Single Molecule ◽

Actin Filaments ◽

Dynamical Behavior ◽

Jump Process ◽

Pairwise Distance ◽

Actin Dynamics ◽

Cell Cortex ◽

Superresolution Microscopy ◽

Distance Distributions ◽

Primary Fibroblasts

Dynamic remodeling of the actin cytoskeleton allows cells to migrate, change shape, and exert mechanical forces on their surroundings. How the complex dynamical behavior of the cytoskeleton arises from the interactions of its molecular components remains incompletely understood. Tracking the movement of individual actin filaments in living cells can in principle provide a powerful means of addressing this question. However, single-molecule fluorescence imaging measurements that could provide this information are limited by low signal-to-noise ratios, with the result that the localization errors for individual fluorophore fiducials attached to filamentous (F)-actin are comparable to the distances traveled by actin filaments between measurements. In this study we tracked the movement F-actin labeled with single-molecule densities of the fluorogenic label SiR-actin in primary fibroblasts and endothelial cells. We then used a Bayesian statistical approach to estimate true, underlying actin filament velocity distributions from the tracks of individual actin-associated fluorophores along with quantified localization uncertainties. This analysis approach is broadly applicable to inferring statistical pairwise distance distributions arising from noisy point localization measurements such as occur in superresolution microscopy. We found that F-actin velocity distributions were better described by a statistical jump process, in which filaments exist in mechanical equilibria punctuated by abrupt, jump-like movements, than by models incorporating combinations of diffusive motion and drift. A model with exponentially distributed time- and length-scales for filament jumps recapitulated F-actin velocity distributions measured for the cell cortex, integrin-based adhesions, and actin stress fibers, indicating that a common physical model can potentially describe F-actin dynamics in a variety of cellular contexts.

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Single occupancy spectroelectrochemistry of freely diffusing flavin mononucleotide in zero-dimensional nanophotonic structures

Faraday Discussions ◽

10.1039/c5fd00072f ◽

2015 ◽

Vol 184 ◽

pp. 101-115 ◽

Cited By ~ 31

Author(s):

Lawrence P. Zaino ◽

Dane A. Grismer ◽

Donghoon Han ◽

Garrison M. Crouch ◽

Paul W. Bohn

Keyword(s):

Electron Transfer ◽

Single Molecule ◽

Light Emission ◽

Fluorescence Emission ◽

Flavin Mononucleotide ◽

Wide Field ◽

Potential Sweep ◽

Potential Control ◽

Fluorescence Measurements ◽

Electron Transfer Properties

Zero-mode waveguides (ZMW) have the potential to be powerful confinement tools for studying electron transfer dynamics at single molecule occupancy conditions. Flavin mononucleotide contains an isoalloxazine chromophore, which is fluorescent in the oxidized state (FMN) while the reduced state (FMNH2) exhibits dramatically lower light emission, i.e. a dark-state. This allows fluorescence emission to report the redox state of single FMN molecules, an observation that has been used previously to study single electron transfer events in surface-immobilized flavins and flavoenzymes, e.g. sarcosine oxidase, by direct wide-field imaging of ZMW arrays. Single molecule electron transfer dynamics have now been extended to the study of freely diffusing molecules using fluorescence measurements of Au ZMWs under single occupancy conditions. The Au in the ZMW serves both as an optical cladding layer and as the working electrode for potential control, thereby accessing single molecule electron transfer dynamics at μM concentrations. Consistent with expectations, the probability of observing single reduced molecules increases as the potential is scanned negative, Eappl < Eeq, and the probability of observing emitting oxidized molecules increases at Eappl > Eeq. Different single molecules exhibit different electron transfer properties as reflected in the position of Eeq and the distribution of Eeq among a population of FMN molecules. Two types of actively-controlled electroluminescence experiments were used: chronofluorometry experiments, in which the potential is alternately stepped between oxidizing and reducing potentials, and cyclic potential sweep fluorescence experiments, analogous to cyclic voltammetry, these latter experiments exhibiting a dramatic scan rate dependence with the slowest scan rates showing distinct intermediate states that are stable over a range of potentials. These states are assigned to flavosemiquinone species that are stabilized in the special environment of the ZMW nanopore.

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