Stimulated Emission Depletion Fluorescence Microscopy Research Articles

Stimulated Raman-induced beam focusing

Stimulated Raman scattering, employing a pump and a Stokes beam, exhibits itself through both the Raman loss observed in the pump beam and the Raman gain in the Stokes beam. This phenomenon finds application in spectroscopy for chemical analyses and microscopy for label-free bioimaging studies. Recent efforts have been made to implement super-resolution Raman microscopy using a doughnut-shaped pump, Stokes, or depletion beam. In this study, it is shown that the amplitude and phase of the pump or Stokes beam undergo significant modulation through the stimulated Raman process when they are configured as one of the higher-order Laguerre-Gauss modes, achieved using appropriate spiral phase plates or spatial light modulators. The resulting intensity distributions of the pump and Stokes beams are determined by a superposition of multiple Laguerre-Gauss modes that are coupled through nonlinear Raman gain and loss processes. Calculation results are used to elucidate the limitations associated with super-resolution coherent Raman imaging with a toroidal pump or Stokes beam. This stands in contrast with the stimulated emission depletion fluorescence microscopy technique, which has no fundamental limit in the spatial resolution enhancement.

Rapid Internalization of Nanoparticles by Human Cells at the Single Particle Level.

Nanoparticle uptake by cells has been studied for applications both in nanomedicine and in nanosafety. While the majority of studies have focused on the biological mechanisms underlying particle internalization, less attention has been given to questions of a more quantitative nature, such as how many nanoparticles enter cells and how rapidly they do so. To address this, we exposed human embryonic kidney cells to 40-200 nm carboxylated polystyrene nanoparticles and the particles were observed by live-cell confocal and super-resolution stimulated emission depletion fluorescence microscopy. How long a particle remained at the cell membrane after adsorbing onto it was monitored, distinguishing whether the particle ultimately desorbed again or was internalized by the cell. We found that the majority of particles desorb, but interestingly, most of the particles that are internalized do so within seconds, independently of particle size. As this is faster than typical endocytic mechanisms, we interpret this observation as the particles entering via an endocytic event that is already taking place (as opposed to directly triggering their own uptake) or possibly via an as yet uncharacterized endocytic route. Aside from the rapidly internalizing particles, a minority of particles remain at the membrane for tens of seconds to minutes before desorbing or being internalized. We also followed particles after cell internalization, observing particles that appeared to exit the cell, sometimes as rapidly as within tens of seconds. Overall, our results provide quantitative information about nanoparticle cell internalization times and early trafficking.

Electrogenerated Chemiluminescence in Submicrometer Wells for Very High-Density Biosensing.

Inspired by the mechanism in stimulated emission depletion fluorescence microscopy, we established for the first time electrogenerated chemiluminescence (ECL) generated from indium tin oxide (ITO) submicrometer electrodes in submicrometer wells with aluminum walls. Aluminum is observed to have the ability to absorb ECL from luminol and, thus, removes the ECL from the diffused species away from the electrode surfaces. Accordingly, the size of the individual ECL spots is restricted to 4 μm in diameter, enabling the density of the ECL based array to be over 3 × 104/mm2. The conceptual detection of hydrogen peroxide and glucose exhibits the feasibility in the application of luminol ECL for very high-density biosensing. The realization of this ECL in submicrometer wells and the resultant biosensing offer a new strategy for high-density ECL analysis and might initiate the thought for super-resolution ECL microscopy.

Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent

Photobleaching remains a limiting factor in superresolution fluorescence microscopy. This is particularly true for stimulated emission depletion (STED) and reversible saturable/switchable optical fluorescence transitions (RESOLFT) microscopy, where adjacent fluorescent molecules are distinguished by sequentially turning them off (or on) using a pattern of light formed as a doughnut or a standing wave. In sample regions where the pattern intensity reaches or exceeds a certain threshold, the molecules are essentially off (or on), whereas in areas where the intensity is lower, that is, around the intensity minima, the molecules remain in the initial state. Unfortunately, the creation of on/off state differences on subdiffraction scales requires the maxima of the intensity pattern to exceed the threshold intensity by a large factor that scales with the resolution. Hence, when recording an image by scanning the pattern across the sample, each molecule in the sample is repeatedly exposed to the maxima, which exacerbates bleaching. Here, we introduce MINFIELD, a strategy for fundamentally reducing bleaching in STED/RESOLFT nanoscopy through restricting the scanning to subdiffraction-sized regions. By safeguarding the molecules from the intensity of the maxima and exposing them only to the lower intensities (around the minima) needed for the off-switching (on-switching), MINFIELD largely avoids detrimental transitions to higher molecular states. A bleaching reduction by up to 100-fold is demonstrated. Recording nanobody-labeled nuclear pore complexes in Xenopus laevis cells showed that MINFIELD-STED microscopy resolved details separated by <25 nm where conventional scanning failed to acquire sufficient signal.

Molecular Interactions on the Plasma Membrane Studied by STED Fluorescence Microscopy

The direct and non-invasive observation of a whole range of cellular functionalities is impeded by the resolution limit of >200nm of a conventional far-field optical microscope. Prominent examples are molecular interactions on the plasma membrane such as the integration into lipid nanodomains (‘rafts’), which are considered to play a functional part in a whole range of membrane-associated processes. We report the detection of single diffusing lipid and protein molecules in nanosized areas in the plasma membrane of living cells using the superior spatial resolution of stimulated emission depletion (STED) far-field nanoscopy or of fast single-molecule tracking. By combining a (tunable) resolution of down to 30 nm with tools such as fluorescence correlation spectroscopy (FCS) or by spatio-temporally following the movement of single lipids, we obtain new details of molecular membrane dynamics. For example, we reveal transient (∼ 10 ms) trapping of certain sphingolipids on the nanoscale in cholesterol-mediated molecular complexes. However, distinct differences show up between different lipids and molecules. Molecules such as certain transmembrane proteins show both trapping and a kind of hopping diffusion. The novel observations may highlight new details on lipid-protein interactions and their role in membrane bioactivity.

High- and Low-Mobility Stages in the Synaptic Vesicle Cycle

Synaptic vesicles need to be mobile to reach their release sites during synaptic activity. We investigated vesicle mobility throughout the synaptic vesicle cycle using both conventional and subdiffraction-resolution stimulated emission depletion fluorescence microscopy. Vesicle tracking revealed that recently endocytosed synaptic vesicles are highly mobile for a substantial time period after endocytosis. They later undergo a maturation process and integrate into vesicle clusters where they exhibit little mobility. Despite the differences in mobility, both recently endocytosed and mature vesicles are exchanged between synapses. Electrical stimulation does not seem to affect the mobility of the two types of vesicles. After exocytosis, the vesicle material is mobile in the plasma membrane, although the movement appears to be somewhat limited. Increasing the proportion of fused vesicles (by stimulating exocytosis while simultaneously blocking endocytosis) leads to substantially higher mobility. We conclude that both high- and low-mobility states are characteristic of synaptic vesicle movement.

Statistical Analysis of High-Resolution Light Microscope Images Reveals Effects of Cytoskeleton-Disrupting Drugs on the Membrane Organization of the Nicotinic Acetylcholine Receptor

Extensive evidence supports the notion that the cytoskeleton participates in the immobilization and membrane clustering of the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. Stimulated emission depletion fluorescence microscopy has revealed the supramolecular organization of AChR nanoclusters at the surface of CHO-K1/A5 cells with subdiffraction resolution (Kellner et al., Neuroscience 144:135-143 2007). We studied the effect of two cytoskeletal-disrupting drugs (cytochalasin D and jasplakinolide) on the nanoscale distribution of muscle-type AChR expressed in these cells by means of mathematical and statistical analysis of images obtained with the same high-resolution microscopy. AChR nanoclusters were found to be randomly distributed in both controls and cells treated with either drug for distances larger than 500 nm. Treatments altered the distribution of AChR nanoclusters according to their brightness/size. Cytochalasin D and jasplakinolide produced a statistically significant increase in the proportion of medium-size nanoclusters and a diminution of small nanoclusters, indicating higher disrupting activity on the latter. This was further corroborated by the diminution of the brightness/diameter ratio of nanoclusters (a measure of the intracluster density of AChR molecules) and by Ripley's analysis applied to simulated patterns with intracluster aggregation of AChR molecules. The combined analytical tools bring out subtle changes in the two-dimensional organization of the AChR nanoaggregates on disruption of the cytoskeletal network and throw light on the possible link between the cytoskeleton and the distribution of the AChR at the cell surface.

Effects of coherence and vector properties of the light on the resolution limit in stimulated emission depletion fluorescence microscopy

Stimulated emission depletion (STED) fluorescence microscopy is a diffraction-unlimited microscopy. We report a method of analyzing the intensity distribution in the focal region. The method takes both the coherence and the vector properties of the light into account. By using the Gaussian Schell model to describe the cross-spectral density function of the incident beam, we show that the coherence that exists between the electric field at any two points is one of the factors that limit further increase of the spatial resolution in STED fluorescence microscopy.

Cholesterol effects on nicotinic acetylcholine receptor

This short overview addresses the effects of cholesterol (Chol) on the stability, supramolecular organization, and function of the murine muscle-type nicotinic acetylcholine receptor (AChR). The biophysical properties of Chol-AChR interaction are discussed first, as they constitute, in our view, the very source of the modulation exerted by Chol on receptor structure and function. In the absence of innervation, AChRs occur in the form of aggregates that remain stable at the cell-surface membrane of mammalian model cells over a period of hours. Acute Chol depletion drastically reduces (approximately 50%) the number of receptor aggregates by accelerating the rate of endocytosis in a receptor-expressing clonal cell line. Chol depletion also results in ion channel gain-of-function of the remaining cell-surface AChRs, whereas Chol enrichment has the opposite effect. Wide-field and confocal microscopy show AChR clusters as diffraction-limited puncta of approximately 200 nm diameter. Stimulated emission depletion fluorescence microscopy resolves these puncta into nanoclusters with an average diameter of approximately 55 nm. Exploiting the enhanced resolution, the effect of acute Chol depletion is shown to alter the short- and long-range organization of AChR nanoclusters. In the short range, the 50% AChRs remaining at the cell surface form larger nanoclusters. On larger scales (0.5-3.5 microm) Ripley's K-test on stimulated emission depletion images reveal changes in nanocluster distribution, attributable to the Chol-related abolition of cytoskeletal physical barriers normally preventing the lateral diffusion of AChR nanoclusters. We discuss these observations in the light of the so-called 'raft' hypothesis and surmise that Chol content at the plasmalemma homeostatically modulates cell-surface organization and stability of receptor nanodomains and fine tunes receptor channel function to temporarily compensate for acute AChR loss from the cell surface.