HIV taken by STORM : super-resolution fluorescence microscopy of a viral infection

<p>Abstract</p> <p>Background</p> <p>The visualization of viral proteins has been hindered by the resolution limit of conventional fluorescent microscopes, as the dimension of any single fluorescent signal is often greater than most virion particles. Super-resolution microscopy has the potential to unveil the distribution of proteins at the resolution approaching electron microscopy without relying on morphological features of existing characteristics of the biological specimen that are needed in EM.</p> <p>Results</p> <p>Using direct stochastic optical reconstruction microscopy (dSTORM) to achieve a lateral resolution of 15–20 nm, we quantified the 2-D molecular distribution of the major structural proteins of the infectious human immunodeficiency virus type 1 (HIV-1) before and after infection of lymphoid cells. We determined that the HIV-1 matrix and capsid proteins undergo restructuring soon after HIV-1 infection.</p> <p>Conclusions</p> <p>This study provides the proof-of-concept for the use of dSTORM to visualize the changes in the molecular distribution of viral proteins during an infection.</p

Three-dimensional topology-based analysis segments volumetric and spatiotemporal fluorescence microscopy

Image analysis techniques provide objective and reproducible statistics for interpreting microscopy data. At higher dimensions, three-dimensional (3D) volumetric and spatiotemporal data highlight additional properties and behaviors beyond the static 2D focal plane. However, increased dimensionality carries increased complexity, and existing techniques for general segmentation of 3D data are either primitive, or highly specialized to specific biological structures. Borrowing from the principles of 2D topological data analysis (TDA), we formulate a 3D segmentation algorithm that implements persistent homology to identify variations in image intensity. From this, we derive two separate variants applicable to spatial and spatiotemporal data, respectively. We demonstrate that this analysis yields both sensitive and specific results on simulated data and can distinguish prominent biological structures in fluorescence microscopy images, regardless of their shape. Furthermore, we highlight the efficacy of temporal TDA in tracking cell lineage and the frequency of cell and organelle replication

The relationship between cognitive ability and BOLD activation across sleep–wake states

The sleep spindle, a waxing and waning oscillation in the sigma frequency range, has been shown to correlate with fluid intelligence; i.e. the ability to use logic, learn novel rules/patterns, and solve problems. Using simultaneous EEG and fMRI, we previously identified the neural correlates of this relationship, including activation of the thalamus, bilateral putamen, medial frontal gyrus, middle cingulate cortex, and precuneus. However, research to date has focussed primarily on non-rapid eye movement (NREM) sleep, and spindles per se, thus overlooking the possibility that brain activity that occurs in other sleep–wake states might also be related to cognitive abilities. In our current study, we sought to investigate whether brain activity across sleep/wake states is also related to human intelligence in N = 29 participants. During NREM sleep, positive correlations were observed between fluid intelligence and blood oxygen level dependent (BOLD) activations in the bilateral putamen and the paracentral lobule/precuneus, as well as between short-term memory (STM) abilities and activity in the medial frontal cortex and inferior frontal gyrus. During wake, activity in bilateral postcentral gyri and occipital lobe was positively correlated with short-term memory abilities. In participants who experienced REM sleep in the scanner, fluid intelligence was positively associated with midbrain activation, and verbal intelligence was associated with right postcentral gyrus activation. These findings provide evidence that the relationship between sleep and intellectual abilities exists beyond sleep spindles

Cluster analysis for localisation-based data sets: dos and don’ts when quantifying protein aggregates

Many proteins display a non-random distribution on the cell surface. From dimers to nanoscale clusters to large, micron-scale aggregations, these distributions regulate protein-protein interactions and signalling. Although these distributions show organisation on length-scales below the resolution limit of conventional optical microscopy, single molecule localisation microscopy (SMLM) can map molecule locations with nanometre precision. The data from SMLM is not a conventional pixelated image and instead takes the form of a point-pattern—a list of the x, y coordinates of the localised molecules. To extract the biological insights that researchers require cluster analysis is often performed on these data sets, quantifying such parameters as the size of clusters, the percentage of monomers and so on. Here, we provide some guidance on how SMLM clustering should best be performed

Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations

Despite significant progress, high-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination. Here, we describe a new analytical approach, super-resolution radial fluctuations (SRRF), provided as a fast graphics processing unit-enabled ImageJ plugin. In the most challenging data sets for super-resolution, such as those obtained in low-illumination live-cell imaging with GFP, we show that SRRF is generally capable of achieving resolutions better than 150 nm. Meanwhile, for data sets similar to those obtained in PALM or STORM imaging, SRRF achieves resolutions approaching those of standard single-molecule localization analysis. The broad applicability of SRRF and its performance at low signal-to-noise ratios allows super-resolution using modern widefield, confocal or TIRF microscopes with illumination orders of magnitude lower than methods such as PALM, STORM or STED. We demonstrate this by super-resolution live-cell imaging over timescales ranging from minutes to hours

Does the early bird really get the worm? How chronotype relates to human intelligence

Objectives: Chronotype impacts our state at a given time of day, however, chronotype is also heritable, trait-like, and varies systematically as a function of age and sex. However, only a handful of studies support a relationship between chronotype and trait-like cognitive abilities (i.e., intelligence), and the evidence is sparse and inconsistent between studies. Typically, studies have: (1) focused on limited subjective measures of chronotype, (2) focused on young adults only, and (3) have not considered sex differences. Here, using a combination of cognitive aptitude and ability testing, subjective chronotype, and objective actigraphy, we aimed to explore the relationship between trait-like cognitive abilities and chronotype. Design: Participants (N = 61; 44 females; age = 35.30 ± 18.04 years) completed the Horne-Ostberg Morningness-Eveningness Questionnaire (MEQ) to determine subjective chronotype and wore an activity monitor for 10 days to objectively assess bedtime, rise-time, total sleep time, inter-daily stability, intra-daily variability, and relative amplitude. Cognitive ability (e.g., Verbal, Reasoning and Short-Term Memory) testing took place at the completion of the study. Results: Higher MEQ scores (i.e., more morning) were associated with higher inter-daily stability scores. Superior verbal abilities were associated with later bedtimes, younger age, but paradoxically, higher (i.e., more morning) MEQ scores. Superior STM abilities were associated with younger age only. The relationships between chronotype and trait-like cognitive abilities were similar for both men and women and did not differ between younger and older adults. Conclusions: The present study demonstrates that chronotype, measured by the MEQ, is highly related to inter-daily stability (i.e., the strength of circadian synchronization). Furthermore, although evening types have superior verbal abilities overall, higher (i.e., more morning) MEQ scores were related to superior verbal abilities after controlling for “evening type” behaviours

Phagocytosis in the Brain: Homeostasis and Disease

Microglia are resident macrophages of the central nervous system and significantly contribute to overall brain function by participating in phagocytosis during development, homeostasis, and diseased states. Phagocytosis is a highly complex process that is specialized for the uptake and removal of opsonized and non-opsonized targets, such as pathogens, apoptotic cells, and cellular debris. While the role of phagocytosis in mediating classical innate and adaptive immune responses has been known for decades, it is now appreciated that phagocytosis is also critical throughout early neural development, homeostasis, and initiating repair mechanisms. As such, modulating phagocytic processes has provided unexplored avenues with the intent of developing novel therapeutics that promote repair and regeneration in the CNS. Here, we review the functional consequences that phagocytosis plays in both the healthy and diseased CNS, and summarize how phagocytosis contributes to overall pathophysiological mechanisms involved in brain injury and repair

Functional differences in cerebral activation between slow wave-coupled and uncoupled sleep spindles

Spindles are often temporally coupled to slow waves (SW). These SW-spindle complexes have been implicated in memory consolidation that involves transfer of information from the hippocampus to the neocortex. However, spindles and SW, which are characteristic of NREM sleep, can occur as part of this complex, or in isolation. It is not clear whether dissociable parts of the brain are recruited when coupled to SW vs. when spindles or SW occur in isolation. Here, we tested differences in cerebral activation time-locked to uncoupled spindles, uncoupled SW and coupled SW-spindle complexes using simultaneous EEG-fMRI. Consistent with the “active system model,” we hypothesized that brain activations time-locked to coupled SW-spindles would preferentially occur in brain areas known to be critical for sleep-dependent memory consolidation. Our results show that coupled spindles and uncoupled spindles recruit distinct parts of the brain. Specifically, we found that hippocampal activation during sleep is not uniquely related to spindles. Rather, this process is primarily driven by SWs and SW-spindle coupling. In addition, we show that SW-spindle coupling is critical in the activation of the putamen. Importantly, SW-spindle coupling specifically recruited frontal areas in comparison to uncoupled spindles, which may be critical for the hippocampal-neocortical dialogue that preferentially occurs during sleep

Platelet actin nodules are podosome-like structures dependent on Wiskott-Aldrich syndrome protein and ARP2/3 complex

The actin nodule is a novel F-actin structure present in platelets during early spreading. However, only limited detail is known regarding nodule organization and function. Here we use electron microscopy, SIM and dSTORM super-resolution, and live-cell TIRF microscopy to characterize the structural organization and signalling pathways associated with nodule formation. Nodules are composed of up to four actin-rich structures linked together by actin bundles. They are enriched in the adhesion-related proteins talin and vinculin, have a central core of tyrosine phosphorylated proteins and are depleted of integrins at the plasma membrane. Nodule formation is dependent on Wiskott-Aldrich syndrome protein (WASp) and the ARP2/3 complex. WASp(-/-) mouse blood displays impaired platelet aggregate formation at arteriolar shear rates. We propose actin nodules are platelet podosome-related structures required for platelet-platelet interaction and their absence contributes to the bleeding diathesis of Wiskott-Aldrich syndrome