Dark-field microscopy | EPFL Graph Search

Dark-field microscopy (also called dark-ground microscopy) describes microscopy methods, in both light and electron microscopy, which exclude the unscattered beam from the image. As a result, the field around the specimen (i.e., where there is no specimen to scatter the beam) is generally dark. In optical microscopes a darkfield condenser lens must be used, which directs a cone of light away from the objective lens. To maximize the scattered light-gathering power of the objective lens, oil immersion is used and the numerical aperture (NA) of the objective lens must be less than 1.0. Objective lenses with a higher NA can be used but only if they have an adjustable diaphragm, which reduces the NA. Often these objective lenses have a NA that is variable from 0.7 to 1.25. Light microscopy applications In optical microscopy, dark-field describes an illumination technique used to enhance the contrast in unstained samples. It works by illuminating the sample with light that wi

Water as a contrast agent for imaging interfacial structure and ion transport in giant vesicles

David Roesel

Hydrated lipid bilayer membranes and their asymmetry play a fundamental role in living cells by maintaining and regulating concentration gradients between cells, their environment, and their compartments. They achieve this not only through various channels and transporters, but also by being inherently semi-permeable to various molecules, including water and ions. However, molecular information about passive ion transport, as well as the complex structure of membrane hydration remain elusive, mainly due to a lack of experimental methods that can access this information, and relate it to micro- and macroscopic membrane properties. In this work, we study the molecular structure of lipid bilayer membranes and ion transport across them with high throughput wide-field second harmonic (SH) microscopy by utilizing water as the contrast agent. We show that by detecting small amounts of asymmetry in the structure of interfacial water, it is possible to measure the distribution of charged species across the membrane with high sensitivity. We apply this surface-selective technique to measure ion-lipid-water interactions, track ion transport, and quantify electro-chemical surface properties at the interfaces of lipid bilayer membranes in the form of giant unilamellar vesicles (GUVs). We start by improving the throughput of wide field SH microscopy in order to probe low-asymmetry interfaces with high contrast in a label-free manner. To do this, we design a custom optical parametric amplifier (OPA) with a tunable output in the 670-1000 nm range, up to a 1 MHz repetition rate, and an ultra-short 23 fs pulse duration. We then experimentally demonstrate the achieved throughput improvement. Next, we establish a way to probe interfacial hydration of GUVs. We quantify the surface properties of vesicles composed of different ratios of zwitterionic and anionic lipids, and show that only a few percent of anionic headgroups are ionized. We also observe spatial and temporal fluctuations in surface properties, and demonstrate that these fluctuations are universally found in lipid bilayer systems.Following that, we demonstrate a direct link between membrane potential fluctuations and divalent ion transport. Molecular dynamics simulations reveal that these fluctuations reduce the free energy cost of transient pore formation and increase the ion flux across an open pore. These transient pores can act as conduits for ion transport, which we SH image for a series of divalent cations (Cu2+, Ca2+, Ba2+, Mg2+) passing through GUV membranes. Combining the experimental and computational results, we show that permeation through pores formed via an ion-induced electrostatic field is a viable mechanism for unassisted ion transport.Then, we focus on unassisted Ca2+ translocation in more detail. We vary the hydrophobic core of bilayer membranes and observe different types of behavior in high throughput wide-field SH images. Ca2+ translocation is observed through mono-unsaturated membranes, significantly reduced upon adding cholesterol, and completely inhibited for branched and poly-unsaturated membranes. We propose, using molecular dynamics simulations, that ion transport occurs through ion induced transient pores, which require non-equilibrium membrane restructuring. This results in different transport rates at different locations and suggests that the hydrophobic structure of lipids plays a much more sophisticated regulating role than previously thought.

EPFL2022

Mapping of Chemical Heterogeneity at Surfaces with High-Throughput Wide-Field Second Harmonic Microscopy

Igor Nahálka

Many industrial processes are based on electrochemical reactions using engineered electrocatalysts. However, the current lack of theoretical knowledge obscures efforts to maximize catalysts' efficiency, and the optimization process is mostly empirical. To fully understand the structure/chemical function relationship on complex surfaces, new experimental tools are needed that can evaluate electrochemical processes on nm to tens of ÎŒm length scales. These tools have to be surface-specific and ideally label-free in order not to influence the reactions. In this thesis, we apply high-throughput wide-field second harmonic (SH) microscopy together with in-situ electrochemical techniques to i) study the extent of surface heterogeneity, ii) surface changes during electrochemical cycling, and iii) to identify and characterize surface active sites. We first investigated surface heterogeneity at the glass/electrolyte interface with the novel SH microscope developed in our laboratory, as this interface has been widely discussed in the literature with inconsistent results. The spatial resolution of our SH microscope, ~400 nm, together with the temporal resolution of 250 ms, allowed us to spatially resolve the surface pKa,s for the silica deprotonation reaction with values ranging from 2.3 to 10.7. The average value, 6.7, coincides well with the reported values from experimental studies lacking spatial resolution. Furthermore, we demonstrated the ability of SH microscopy to image the orientation of bulk water molecules under an externally-applied electric field in a confined 1 ÎŒm-sized glass pore. We further applied SH imaging coupled in-situ with cyclic voltammetry to characterize the surface of gold electrodes during electrochemical cycling. Analyzing the voltage dependence of the SH signal and utilizing a novel correlation coefficient procedure for wide-field SH imaging, we have identified two types of surface areas on the polycrystalline gold. The first type remains stable during electrochemical cycling, while the second type undergoes surface reconstruction. We assign the second type of gold surface areas to domains of higher roughness, where anion adsorption occurs at lower potentials than expected. Lastly, we focused on studying the oxygen evolution reaction (OER) on polycrystalline gold working electrodes by coupling SH imaging with electrochemical methods. We observe a large spatial heterogeneity of the OER, and we quantify that only < 1 % of the electrode surface is responsible for oxygen production. We further identify two types of active sites. The first type is observed at potentials > 2 V vs. the reverse hydrogen electrode (RHE), which is the onset of the OER. This type is stable under potential cycling, presumably extending multiple layers deep into the gold electrode. The second, anomalous type, is observed at potentials < 2 V vs. RHE. This type is removed by potential cycling, suggesting it involves a structural motif only 1-2 gold layers deep. The findings in this thesis highlight the importance of surface heterogeneity on a sub-ÎŒm level, showing that the surface structure influences the chemical properties and reactivity. This heterogeneity needs to be considered and utilized in the industrial context to establish more efficient electrochemical processes with lower energy requirements.

EPFL2020

Transmission Microsphere-Assisted Dark-Field Microscopy

Hongyu Li, Stéphane Perrin, Giorgio Quaranta

Microsphere-assisted microscopy allows the limit of the diffraction of light to overcome while being non-invasive, full-field, label-free, and easy-to-implement. However, the observation of translucent samples remains difficult using a classical bright-field illumination. In this work, a method is presented for the inspection of quasi-transparent sub-diffraction-limited structures by using dark-field illumination in the transmission mode. Glass-imprint features, having a size of 250 nm, as well as fixed mouse brain cells have been visualized using the dark-field microsphere-assisted technique. The possibility to observe feature sizes up to 100 nm has been demonstrated in air using a 25-mu m-diameter glass microsphere combined with an optical microscope, opening new possibilities for biological imaging.

WILEY-V C H VERLAG GMBH2019