Ryanodine receptor cluster fragmentation and redistribution in persistent atrial fibrillation enhance calcium release

In atrial fibrillation (AF), abnormalities in Ca(2+) release contribute to arrhythmia generation and contractile dysfunction. We explore whether RyR cluster ultrastructure is altered and is associated with functional abnormalities in AF.status: publishe

Fluorescence microscopy for fast and local scale dynamics in suspensions

Nanomaterials are all around us. They are widely used as they show some extraordinary properties as catalysts (showing unexpected reactivity or needing less starting material), as additives to change properties of bulk materials (exhibiting luminescent properties like in quantum dots, or altering wetting behavior for self cleaning effect), biomedical applications for target drug delivery, potential investigation tool or new treatment procedure. Most of these applications seem futuristic, yet nanomaterials are present in many household objects such as sunblock (sun burn protection), toothpaste (for improved cleansing and protection), in fridges (antimicrobial) and in computers (for miniaturization). To study their behavior, interaction and influence one needs to be able to visualize these materials, and their dynamics, at the real size. For years biologists have used optical microscopy to study microscopic life, such as bacteria and viruses. Hence optical microscopy has proven itself as a convenient tool for studying microscale materials. Especially fluorescence microscopy, such as confocal microscopy, has been shown to be extremely useful in investigatingnbsp;of (sub)micronnbsp;with limiting disturbance of the sample. Nowadays, improvements in the field of optical microscopy, such as STED, allownbsp;to obtainnbsp;of nanometers in resolution. These techniques are classified as super-resolution fluorescence microscopy techniques or optical nanoscopy techniques. As to control the location of the material of interest, droplets can be used. Droplets are a commonly encountered, threenbsp;system (of liquid, gas and solid) and are or a side effect or a goal in many industrial and academic research. Droplets are an easy way to assemble nano and biomaterials, in a controlled way.nbsp;This thesis aims to show the usefulness of fluorescence microscopy for studyingnbsp;particles, of any origin, in a controllable model system: i.e. evaporatingnbsp; The studies in this PhD deal with droplet induced substrate assembly of nanoparticles, microorganisms or DNA (a biomolecule well-known for carrying genetic information). Additional, supplementary information about the results is put atnbsp;end of this dissertation, in the respective appendices. The well-known coffee ring effect was exploited. This is a flow inside a dropletnbsp;is present when it is pinned to a substrate and creating, upon drying, ring like deposits of the present suspended matter. Dropsnbsp;fluorescent bacteria andnbsp;show this coffee ring flow. Upon addition of surfactants this flow profilenbsp;and subsequently also the final deposition. Remarkably, we see that bacteria that produce surfactants themselves show comparable flows and altered deposits.nbsp; A follow-up studynbsp;a higher surfactant concentration showed a macroscopic repetitivenbsp;of the colloids is reached, along the droplet edge, and this in the shape of a flower. In a third study, droplets containing fluorescentnbsp;were used to deposit linear fragments ofnbsp;on a substrate. Tuning the substrate influences the efficiency ofnbsp;retention and stretching. Further optimization, by droplet pulling, allowed efficient DNA linearization and binding, even at extremely low concentrations of DNA. nbsp;can be linearly stretched and can reach lengths of micrometers. Yet DNA is 1D nanoscale system, as the base pairs only are a few nanometers wide. In the last study, tSTED (an extension of the high resolution STED technique) is used to investigate DNA assemblies on dry substrates. Varying buffer parameters allowed alternating DNAnbsp;visual via tSTED in high resolution. Additional experiments showed that the DNA probe, YOYO-1nbsp;interactnbsp;itself and deteriorate the image quality of STED.nbsp;imaging allows us to overcomenbsp;deterioration and the life time information provides an additional resolution improvement. This laserscanning tSTED platform will allow studying DNA stretching by droplets, in high spatial and temporal resolution, where other techniques would fail.status: publishe

PANDA: capturing fast dynamics of interfacial surfactant loading

I. Oscillating pendant drops: indirect imaging of surface tension Surfactant molecules are important in household, industrial procedures (such as cleaning in semiconductor production) and, nowadays, as a study object for biological1 and micro-organism related processes 2. Many use pendant drop experiments to measure dynamic surface tension. However, the method is severly limited by inertia and tends to be slow. Hence, it is hard to probe for the fast molecular dynamics of surfactant ad- and desorption to the interface. II. Fast surfactant dynamics: Interfacial surfactant loading dominates flow and its time scale An oscillating pendant droplet was used, yet instead of a pendant droplet, a small volume of air was trapped into a aqueous surfactant solution (see figure 1, left). This scheme with improved temporal resolution (i.e. 20 Hz) is named PANDA or Pressure Assisted Nucleation of a Droplet of Air. To prove its strength, a systematic PANDA study of varying concentrations and surfactant types was done. Furthermore, a complementary fluorescence microscopy study of the internal flow in geometrical identical drops, of those in the PANDA study, was done (see figure 2, left). Astonishingly (even above the CMC) time and concentration depending properties influence the internal flow and the deposition pattern in the sessile droplets (such as an altered coffee ring effect of the (bio)colloidal fronts). Further experiments in bacterial colonies of surfactant producing micro-organisms (Pseudomonas aeruginosa) show comparable flows. The flow properties were heavily influenced by the soluble surfactants; and their ad- and desorption dominate the observed time scale in the oscillating flow behaviour (see figure 2, right). This could be clarified by the PANDA technique as the needed time of loading and unloading of the interface by the surfactant, steered by the capillary force (induced by evaporation) in combination with a surfactant induced Marangoni force.status: publishe

Recent advances in super-resolution fluorescence microscopy

Super-resolution fluorescence microscopy has attracted much attention in recent years as a technology which enables noninvasive observation of fine structures with more than lambda/10 resolution in far-field microscopy. The resolution is enhanced based on detection of a subpopulation of fluorescent molecules within a diffraction-limited area by switching off fluorescence in various ways. Many conventional and new dyes are applicable for realizing super-resolution fluorescence microscopy. Super-resolution fluorescence microscopy also makes new nanofabrication and recording techniques possible. In this review, the brief history of the field is overviewed, and the key features of individual microscopy techniques are explained in terms of photochemistry with most recent papers. Future outlook of super-resolution fluorescence microscopy is also discussed.status: publishe

PANDA: capturing fast dynamics of interfacial surfactant loading

An oscillating pendant droplet was used, yet instead of a pendant droplet, a small volume of air was trapped into a aqueous surfactant solution. This scheme with improved temporal resolution (i.e. 20 Hz) is named PANDA or Pressure Assisted Nucleation of a Droplet of Air. To prove its strength, a systematic PANDA study of varying concentrations and surfactant types was done. Furthermore, a complementary fluorescence microscopy study of the internal flow in geometrical identical drops, of those in the PANDA study, was done. Astonishingly (even above the CMC) time and concentration depending properties influence the internal flow and the deposition pattern in the sessile droplets (such as an altered coffee ring effect of the (bio)colloidal fronts). Further experiments in bacterial colonies of surfactant producing micro-organisms (Pseudomonas aeruginosa) show comparable flows. The flow properties were heavily influenced by the soluble surfactants; and their ad- and desorption dominate the observed time scale in the oscillating flow behavior. This could be clarified by the PANDA technique as the needed time of loading and unloading of the interface by the surfactant, steered by the capillary force (induced by evaporation) in combination with a surfactant induced Marangoni force.http://ima7.conf.tuwien.ac.at/AbstractsIMA7.pdfstatus: accepte

Structured deposition by Marangoni flows in droplets: thermo-capillary fingering

A drying, pinned droplet induces aninhomogeneous evaporation rate sets up a capillary flow, directed towards the edges. This flow drags all suspended matter to these edges and creates an accumulation, commonly referred to as the “coffee ring effect” All matter, even present surfactants move to the edge. The heap up and adsorption at the contact line create a surface tension gradient at the interface and induce a Marangoni stress, resulting in a recirculating flow in the droplet As time increases, these vortices become unstable and zones with a distinctly higher concentration of tracer particles start to move outward, in distinct finger-like depositions.status: publishe

STimulated Emission Depletion Microscopy

Fluorescence microscopy is used extensively in life sciences and has lead to unique discoveries, yet is limited by the diffraction barrier. Each image is blurred by projecting point emitters as broad spots of a few 100 nm in size. All high resolution techniques are based on spatial modulation and transistions between multiple states. STED or STimulated Emission Depletion microscopy was the first high resolution fluorescence (or nanoscopy) technique that circumvented this barrier, allowing high spatio-temporal visualisation of cellular structures.status: publishe

BIOSURFACTANT OF A BACTERIAL SYSTEM REVERSES THE COFFEE RING EFFECT

The deposition of material at the edge of evaporating droplets, known as the ”coffee ring effect”, is caused by a radially outward capillary flow. This phenomenon is common to a wide array of systems including colloidal and bacterial systems. The role of surfactants in counteracting these coffee ring depositions is related to the occurrence of local vortices known as Marangoni eddies. We have shown that these swirling flows are universal, and not only lead to a uniform deposition of colloids but also occur in living bacterial systems. Experiments on Pseudomonas aeruginosa suggest that the auto-production of biosurfactants plays an essential role in creating a homogeneous deposition of the bacteria upon drying. Moreover, at biologically relevant conditions, intricate time dependent flows are observed in addition to the vortex regime, which are also effective in reversing the coffee ring effect at even lower surfactant concentrations.status: publishe

Self-produced biosurfactants inhibit the bacterial coffee ring effect

status: publishe

Thermocapillary Fingering in Surfactant-Laden Water Droplets

The drying of sessile droplets represents an intriguing problem, being a simple experiment to perform but displaying complexities that are archetypical for many free surface and coating flows. Drying can leave behind distinct deposits of initially well dispersed colloidal matter. For example, in the case of the coffee ring effect, particles are left in a well-defined macroscopic pattern with particles accumulating at the edge, controlled by the internal flow in the droplet. Recent studies indicate that the addition of surfactants strongly influences this internal flow field, even reversing it and suppressing the coffee ring effect. In this work, we explore the behavior of droplets at high surfactant loadings and observe unexpected outward fingering instabilities. The experiments start out with droplets with a pinned contact line, and fast confocal microscopy is used to quantify a radially outward surfactant-driven Marangoni flow, in line with earlier observations. However, the Marangoni flows are observed to become unstable, and local vortex cells are now observed in a direction along the contact line. The occurrence of these vortices cannot be explained on the basis of the effects of surfactants alone. Thermal imaging shows that thermocapillary effects are superimposed on the surfactant-driven flows. These local vortex cells acts as little pumps and push the fluid outward in a fingering instability, rather than an expected inward retraction of the drying droplet. This leads to a deposition of colloids in a macroscopical flower-shaped pattern. A scaling analysis is used to rationalize the observed wavelengths and velocities, and practical implications are briefly discussed