Adding to the super-resolution arsenal
Structured illumination microscopy (SIM) uses light intensities that are orders of magnitude lower than other super-resolution methods. SIM is also far faster ...over cellular-sized fields of view. Li
et al.
used two approaches to improve the resolution of SIM to allow live cell imaging of dynamic cellular processes, including endocytosis and cytoskeleton remodeling. The contrast in performance between SIM and other techniques is due to a few key differences. Defining the practical resolution at the limited signal-to-noise ratios necessary for live cell imaging will require better imaging metrics.
Science
, this issue
10.1126/science.aab3500
Super-resolution imaging of fast dynamic processes in living cells is facilitated by improvements to structured illumination microscopy.
INTRODUCTION
Various methods of super-resolution (SR) fluorescence microscopy have the potential to follow the dynamic nanoscale interactions of specific macromolecular assemblies in living cells. However, this potential is often left unfulfilled, either owing to the method’s inability to follow these processes at the speeds dictated by nature or because they require intense light that can substantially perturb the very physiology one hopes to study. An exception is structured illumination microscopy (SIM), which can image live cells far faster and with orders of magnitude less light than required for other SR approaches. However, SIM’s resolution is usually limited to only a twofold gain beyond conventional optical microscopes, or ~100 nm with visible light.
RATIONALE
We endeavored to find ways to extend SIM to the sub-100-nm regime while retaining, to the greatest extent possible, the advantages that make it the preferred SR method for live-cell imaging. Our first solution used an ultrahigh numerical aperture (NA) lens and total internal reflection fluorescence (TIRF) to achieve 84-nm resolution at subsecond acquisition speeds over hundreds of time points in multiple colors near the basal plasma membrane. Our second exploited the spatially patterned activation of a recently developed, reversibly photoswitchable fluorescent protein to reach 45- to 62-nm resolution, also at subsecond acquisition, over ∼10 to 40 time points.
RESULTS
We used high-NA TIRF-SIM to image the dynamic associations of cortical filamentous actin with myosin IIA, paxillin, or clathrin, as well as paxillin with vinculin and clathrin with transferrin receptors. Thanks to the combination of high spatial and temporal resolution, we were able to measure the sizes of individual clathrin-coated pits through their initiation, growth, and internalization. We were also able to relate pit size to lifetime, identify and characterize localized hot spots of pit generation, and describe the interaction of actin with clathrin and its role in accelerating endocytosis. With nonlinear SIM by use of patterned activation (PA NL-SIM), we monitored the remodeling of the actin cytoskeleton and the dynamics of caveolae at the cell surface. By combining TIRF-SIM and PA NL-SIM for two-color imaging, we followed the dynamic association of actin with α-actinin in expanding filopodia and membrane ruffles and characterized shape changes in and the transport of early endosomes. Last, by combining PA NL-SIM with lattice light sheet microscopy, we observed, in three dimensions and across the entire volume of whole cells, the dynamics of the actin cytoskeleton, the fusion and fission of mitochondria, and the trafficking of vesicles to and from the Golgi apparatus, each at axial resolution fivefold better than that of conventional widefield microscopy.
In addition, through direct experimental comparisons, we demonstrated that the resolution for our methods is comparable with or better than other SR approaches yet allowed us to image at far higher speeds, and for far longer durations. To understand why this is so, we developed a detailed theoretical model showing that our methods transmit the information encoded in spatial frequencies beyond the diffraction limit with much greater strength than do other alternatives and hence require far fewer photons emitted from the specimen, using far less intense light.
CONCLUSION
High-NA TIRF-SIM and PA NL-SIM fill an unmet need for minimally invasive tools to image live cells in the gap between the 100-nm resolution traditionally associated with SIM and the sub-60-nm regime of protein-specific structural imaging served by single-molecule localization microscopy.
Two approaches for improved live-cell imaging at sub-100-nm resolution.
(
Left
) Association of cortical actin (purple) with clathrin-coated pits (green), the latter seen as rings (
inset
) at 84-nm resolution via a combination of total internal reflection fluorescence and structured illumination microscopy at ultrahigh numerical aperture (high-NA TIRF-SIM). (
Right
) Progression of resolution improvement across the actin cytoskeleton of a COS-7 cell, from conventional, diffraction-limited TIRF (220-nm resolution), to TIRF-SIM (97-nm resolution), and nonlinear SIM based on the patterned activation of a reversibly photoswitchable fluorescent protein (PA NL-SIM, 62 nm resolution). (Left and right represent single frames from time-lapse movies over 91 and 30 frames, respectively. Scale bars, 2 μm (left); 3 μm (right).
Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.
Long-term antibiotic use for managing chronic respiratory disease is increasing; however, the role of the airway resistome and its relationship to host microbiomes remains unknown.
To evaluate airway ...resistomes and relate them to host and environmental microbiomes using ultradeep metagenomic shotgun sequencing.
Airway specimens from 85 individuals with and without chronic respiratory disease (severe asthma, chronic obstructive pulmonary disease, and bronchiectasis) were subjected to metagenomic sequencing to an average depth exceeding 20 million reads. Respiratory and device-associated microbiomes were evaluated on the basis of taxonomical classification and functional annotation including the Comprehensive Antibiotic Resistance Database to determine airway resistomes. Co-occurrence networks of gene-microbe association were constructed to determine potential microbial sources of the airway resistome. Paired patient-inhaler metagenomes were compared (
= 31) to assess for the presence of airway-environment overlap in microbiomes and/or resistomes.
Airway metagenomes exhibit taxonomic and metabolic diversity and distinct antimicrobial resistance patterns. A "core" airway resistome dominated by macrolide but with high prevalence of β-lactam, fluoroquinolone, and tetracycline resistance genes exists and is independent of disease status or antibiotic exposure.
and
are key potential microbial reservoirs of macrolide resistance including the
,
, and
genes. Significant patient-inhaler overlap in airway microbiomes and their resistomes is identified where the latter may be a proxy for airway microbiome assessment in chronic respiratory disease.
Metagenomic analysis of the airway reveals a core macrolide resistome harbored by the host microbiome.
Dietrich Bonhoeffer was a uniquely reluctant and distinctly German Lutheran revolutionary. In this volume, the author, an Anglican priest and historian, argues that Bonhoeffer’s powerful critique of ...Germany’s moral derailment needs to be understood as the expression of a devout Lutheran Protestant. Bonhoeffer gradually recognized the ways in which the intellectual and religious traditions of his own class - the Bildungsbürgertum - were enabling Nazi evil. In response, he offered a religiously inspired call to political opposition and Christian witness—which cost him his life. The author investigates Bonhoeffer’s stance in terms of his confrontation with the legacy of Hegelianism and Neo-Rankeanism, and by highlighting Bonhoeffer’s intellectual and spiritual journey, shows how his endeavor to politicially reeducate the German people must be examined in theological terms.
Undoubtedly, cyclooctane has been investigated theoretically and experimentally, however, considering the vast application of cycloalkanes and limited research for the higher members, the reactivity ...and structural investigation of cyclooctane, fluorocyclooctane, bromocyclooctane, chlorocyclooctane, azocane, oxocane and thiocane have been explored using density functional theory (DFT) method. In this work, we reported elaborately on some molecular and electronic properties. Interestingly, the condensed dual descriptors (Δf); Br24 of bromocyclooctane compound and N22 of azocane compound has the highest positive values of 0.3561eV and 0.1618eV respectively and hence, potential sites for nucleophilic reaction compared to chlorocyclooctane, fluorocyclooctane, oxocane, thiocane and the parent cyclooctane. In the UV-VIS spectroscopic analysis, σ-π* nor π to π* transitions where observed for all the studied molecules and these molecules are expected to be colorless due to the absence of an absorption band in the visible region (380–760 nm). In all the substituted cyclooctane molecules investigated, C–F bond of fluorocyclooctane is more polar than C–O, C–S, C–N, C–Cl, C–Br bonds of other compounds. The interactions between the lone pair (LP (1) N22) and antibonding; σ*(1)C4–C5 of azocane gave the highest stabilization energy (10.44 kcal/mol) compared to other compounds investigated. We believe our detail reported work on this eight-membered ring compound and its derivatives will widen the scope of these molecules to other researchers.
•Br24 of bromocyclooctane compound with highest positive value is a site for nucleophilic reaction•The studied molecules are expected to be colorless due to the absence of an absorption band in the visible region (380–760 nm).•A closed look at cyclooctane and the substituted cyclooctane shows that the electrons are less binded than benzene•C–F bond of fluorocyclooctane is more polar than C–O, C–S, C–N, C–Cl, C–Br bonds of other compounds.•Azocane gave the highest stabilization energy (10.44 kcal/mol) compared to other compounds investigated.
This study sought to accurately describe the success rate, risks, and patient-reported benefits of contemporary chronic total occlusion (CTO) percutaneous coronary intervention (PCI).
In light of the ...evolving techniques to successfully revascularize CTO lesions, there remains a compelling need to more accurately quantify the success rates, risks, and benefits of these complex procedures.
Using a uniquely comprehensive, core-lab adjudicated, single-arm, multicenter registry of 1,000 consecutive patients undergoing CTO PCI by the hybrid approach, we evaluated the technical success rates, complication rates, and raw and adjusted health status benefits at 1 month among successfully as compared to unsuccessfully treated patients.
Technical success was high (86%). In-hospital and 1-month mortality was 0.9% and 1.3%, respectively, and perforations requiring treatment occurred in 48 patients (4.8%). Among those who survived and completed the 1-month interview (n = 947), mean ± SEM Seattle Angina Questionnaire quality of life scores improved from 49.4 ± 0.9 to 75.0 ± 0.7 (p < 0.01), mean Rose Dyspnea Scale scores improved (decreased) from 2.0 ± 0.1 to 1.1 ± 0.1 (p < 0.01), and physician health questionnaire (for depression) scores improved (decreased) from 6.2 ± 0.2 to 3.5 ± 0.1 (p < 0.01) at 1 month. After adjusting for baseline differences the mean group difference in Seattle Angina Questionnaire quality of life between successful and unsuccessful CTO PCI was 10.8 (95% confidence interval: 6.3 to 15.3; p < 0.001).
Clarifying the success rates, risks, and benefits of CTO PCI will help to more accurately contextualize the informed consent process for these procedures so that patients with appropriate indications for CTO PCI can more effectively share in the decision to pursue this or other therapeutic options.
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This study sought to describe the angiographic characteristics, strategy associated with perforation, and the management of perforation during chronic total occlusion percutaneous coronary ...intervention (CTO PCI).
The incidence of perforation is higher during CTO PCI compared with non-CTO PCI and is reportedly highest among retrograde procedures.
Among 1,000 consecutive patients who underwent CTO PCI in a 12-center registry, 89 (8.9%) had core lab–adjudicated angiographic perforations. Clinical perforation was defined as any perforation requiring treatment. Major adverse cardiac events (MAEs) were defined as in-hospital death, cardiac tamponade, and pericardial effusion.
Among the 89 perforations, 43 (48.3%) were clinically significant, and 46 (51.7%) were simply observed. MAE occurred in 25 (28.0%), and in-hospital death occurred in 9 (10.1%). Compared with nonclinical perforations, clinical perforations were larger in size, more often at a collateral location, had a high-risk shape, and less likely to cause staining or fast filling. Compared with perforations not associated with MAE, perforations associated with MAE were larger in size, more proximal or at collateral location, and had a high-risk shape. When the core lab attributed the perforation to the approach used when the perforation occurred, 61% of retrograde perforations by other classifications were actually antegrade.
Larger size, proximal or collateral location, and high-risk shapes of a coronary perforation were associated with MAE. Six of 10 perforations occurred with antegrade approaches among patients who had both strategies attempted. These finding will help emerging CTO operators understand high-risk features of the perforation that require treatment and inform future comparisons of retrograde and antegrade complications.
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Serial Angiographic Follow-Up of Sirolimus-Eluting Stents for Unprotected Left Main Coronary Artery Revascularization
Matthew J. Price, Ecaterina Cristea, Neil Sawhney, John A. Kao, Jeffrey W. Moses, ...Martin B. Leon, Ricardo A. Costa, Alexandra J. Lansky, Paul S. Teirstein
The goal of this study was to evaluate the serial angiographic outcomes of patients who underwent sirolimus-eluting stent (SES) implantation for unprotected left main coronary artery (LMCA) stenosis. Surveillance angiography was performed at three and nine months follow-up. Of 50 patients, most of whom had distal LMCA disease, 21 (42%) had restenosis, the majority of which was focal and involved the left circumflex ostium. Target lesion revascularization occurred in 19 patients (38%), although only 7 patients (14%) had signs or symptoms of ischemia. In conclusion, restenosis after unprotected LMCA SES intervention is typically focal, involves the branch ostia, and frequently occurs without symptoms.
This study was performed to evaluate the clinical and serial angiographic outcomes of patients undergoing sirolimus-eluting stent (SES) implantation for unprotected left main coronary artery (LMCA) stenosis.
The efficacy of SES has led to their expanded use for off-label indications, including LMCA disease.
Unprotected LMCA intervention with SES was attempted in 50 patients. Surveillance angiography was performed at three and nine months’ follow-up.
The target lesion involved the distal LMCA in 47 patients (94%). In-lesion restenosis occurred in 21 patients (42%), was focal in 85% of cases, and in 82% involved the branch ostia, sparing the LMCA itself. Target lesion revascularization (TLR) occurred in 19 patients (38%) over a mean follow-up of 276 ± 57 days; TLR was ischemia-driven in 7 patients (14%). Late loss was significantly greater within the left circumflex (LCX) ostium compared to the parent vessel (PV) of the LMCA bifurcation (0.83 ± 0.89 mm vs. 0.49 ± 0.72 mm, p = 0.04). Late loss continued to increase between three- and nine-month follow-up. Final minimal luminal diameter and maximal balloon pressure were independent predictors of restenosis of the PV.
Restenosis is a frequent finding when serial angiographic follow-up is performed after SES implantation for unprotected distal LMCA lesions. Restenosis is usually focal, most often involves the LCX ostium, and often occurs without symptoms.
The aromaticity and CDFT properties of naphthalene and its aza-derivatives were theoretically investigated using density functional theory (DFT) electronic structure method. The reactivity and ...chemistry of Azanaphthalene (1-AN), 1, 2-diazanaphthalene (1, 2-DAN), 1, 3-diazanaphthalene (1, 3-DAN), 1, 4-diazanaphthalene (1,4-DAN), 1, 5-diazanaphthalene (1, 5-DAN), 1, 6-diazanaphthalene (1, 6-DAN), 1, 7-diazanaphthalene (1,7-DAN) and 1, 8-diazanaphthalene (1, 8-DAN) were thoroughly explored and predicted focusing more on the fuzzy atomic space analysis, quantum chemical descriptors (CDFT), natural bond orbital (NBO), and structural electronic properties. The CDFT is focused on predicting the condensed Fukui function and dual descriptors along with condensed local electrophilicity and nucleophilicity investigation. From the aromaticity computational study, 1,7-DAN gave PDI, FLU, FLU-π, PLR, HOMA, BIRD and LOLIPOP values of approximately one (1) was found to be the most aromatic in the group, and strongest π-stacking ability. The aromaticity follows the trend: 1, 7-DAN > 1, 8-DAN > 1, 5-DAN > 1, 6-DAN > 1, 4-DAN > 1, 2-DAN > 1-AN > naphthalene. The second order perturbation energy NBO analysis revealed that the 3 highest stabilization energies in the molecules are C6–Na to C3–C4(π∗−π∗ 236.90 kcal/mol) of 1, 6-DAN, C3–C4 to C1–C2 (π∗−π∗236.37 kcal/mol) of 1-AN and C7–N10 to C2–C4 (π∗−π∗235 kcal/mol) of 1, 3-DAN.
Diazanaphthalenes, CDFT, Aromaticity, NBO, Structural
Demyelination causes slowed or failed neuronal conduction and is a driver of disability in multiple sclerosis and other neurological diseases. Currently, the gold standard for imaging demyelination ...is MRI, but despite its high spatial resolution and sensitivity to demyelinated lesions, it remains challenging to obtain specific and quantitative measures of molecular changes involved in demyelination. To understand the contribution of demyelination in different diseases and to assess the efficacy of myelin-repair therapies, it is critical to develop new in vivo imaging tools sensitive to changes induced by demyelination. Upon demyelination, axonal K+ channels, normally located underneath the myelin sheath, become exposed and increase in expression, causing impaired conduction. Here, we investigate the properties of the K+ channel PET tracer
18
F3F4AP in primates and its sensitivity to a focal brain injury that occurred three years prior to imaging.
18
F3F4AP exhibited favorable properties for brain imaging including high brain penetration, high metabolic stability, high plasma availability, high reproducibility, high specificity, and fast kinetics.
18
F3F4AP showed preferential binding in areas of low myelin content as well as in the previously injured area. Sensitivity of
18
F3F4AP for the focal brain injury was higher than
18
FFDG,
11
CPiB, and
11
CPBR28, and compared favorably to currently used MRI methods.
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