Nonblinking and long-lasting single-molecule fluorescence imaging

Photobleaching and blinking of fluorophores pose fundamental limitations on the information content of single-molecule fluorescence measurements. Photoinduced blinking of Cy5 has hampered many previous investigations using this popular fluorophore. Here we show that Trolox in combination with the enzymatic oxygen-scavenging system eliminates Cy5 blinking, dramatically reduces photobleaching and improves the signal linearity at high excitation rates, significantly extending the applicability of single-molecule fluorescence techniques.     

Blinking effect and the use of quantum dots in single molecule spectroscopy

Luminescent semiconductor nanocrystals (quantum dots, QD) have unique photo-physical properties: high photostability, brightness and narrow size-tunable fluorescence spectra. Due to their unique properties, QD-based single molecule studies have become increasingly more popular during the last years. However QDs show a strong blinking effect (random and intermittent light emission), which may limit their use in single molecule fluorescence studies. QD blinking has been widely studied and some hypotheses have been done to explain this effect. Here we summarise what is known about the blinking effect in QDs, how this phenomenon may affect single molecule studies and, on the other hand, how the “on”/“off” states can be exploited in diverse experimental settings. In addition, we present results showing that site-directed binding of QD to cysteine residues of proteins reduces the blinking effect. This option opens a new possibility of using QDs to study protein–protein interactions and dynamics by single molecule fluorescence without modifying the chemical composition of the solution or the QD surface.     

Blinking suppresses the neural response to unchanging retinal stimulation

Blinks profoundly interrupt visual input but are rarely noticed, perhaps because of blink suppression, a visual-sensitivity loss that begins immediately prior to blink onset. Blink suppression is thought to result from an extra-retinal signal that is associated with the blink motor command and may act to attenuate the sensory consequences of the motor action. However, the neural mechanisms underlying this phenomenon remain unclear. They are challenging to study because any brain-activity changes resulting from an extra-retinal signal associated with the blink motor command are potentially masked by profound neural-activity changes caused by the retinal-illumination reduction that results from occlusion of the pupil by the eyelid. Here, we distinguished direct top-down effects of blink-associated motor signals on cortical activity from purely mechanical or optical effects of blinking on visual input by combining pupil-independent retinal stimulation with functional MRI (fMRI) in humans. Even though retinal illumination was kept constant during blinks, we found that blinking nevertheless suppressed activity in visual cortex and in areas of parietal and prefrontal cortex previously associated with awareness of environmental change. Our findings demonstrate active top-down modulation of visual processing during blinking, suggesting a possible mechanism by which blinks go unnoticed.     

Single-Molecule Imaging and Spectroscopy Using Fluorescence and Surface-Enhanced Raman Scattering

We extended single molecule fluorescence imaging and time-resolved fluorometry from the green to the violet-excitation regime to find feasibility of detecting and identifying fluorescent analogs of nucleic-acid bases at the single-molecule level. Using violet excitation, we observed fluorescent spotsfrom single complexes composed of a nucleotide analogue and the Klenow fragmentof DNA polymerase I. Also, we implemented Raman imaging and spectroscopy of adenine molecules adsorbed on Ag colloidal nanoparticles to find feasibility of identifying nucleic-acid bases at the single-molecule level. Surface enhanced Raman scattering (SERS) of adenine molecules showed an intermittent on-and-off behavior called blinking. The observation of blinking provides substantial evidence for detecting single adenine molecules.     

Detection and correction of blinking bias in image correlation transport measurements of quantum dot tagged macromolecules

Semiconductor nanocrystals or quantum dots (QDs) are becoming widely used as fluorescent labels for biological applications. Here we demonstrate that fluorescence fluctuation analysis of their diffusional mobility using temporal image correlation spectroscopy is highly susceptible to systematic errors caused by fluorescence blinking of the nanoparticles. Temporal correlation analysis of fluorescence microscopy image time series of streptavidin-functionalized (CdSe)ZnS QDs freely diffusing in two dimensions shows that the correlation functions are fit well to a commonly used diffusion decay model, but the transport coefficients can have significant systematic errors in the measurements due to blinking. Image correlation measurements of the diffusing QD samples measured at different laser excitation powers and analysis of computer simulated image time series verified that the effect we observe is caused by fluorescence intermittency. We show that reciprocal space image correlation analysis can be used for mobility measurements in the presence of blinking emission because it separates the contributions of fluctuations due to photophysics from those due to transport. We also demonstrate application of the image correlation methods for measurement of the diffusion coefficient of glycosyl phosphatidylinositol-anchored proteins tagged with QDs as imaged on living fibroblasts.     

A songbird inhibits blinking behaviour in flight

Visual attention plays a fundamental role in avian flight but attention is likely limited whenever birds blink. Because blinks are necessary to maintaining proper vision, this study tested the hypothesis that birds strategically inhibit their blinks in flight. The blinks of captive great-tailed grackles (Quiscalus mexicanus) were recorded before, during and after they flew a short distance in an open environment. The grackles spent the least amount of time blinking in flight (take-off, during flight and landing) and the most amount of time blinking at impact. Their blinking behaviour was similar before and after flight. These results suggest that grackles strategically inhibit their blinking behaviour in flight, potentially because blinks impose costs to avian flight.     

Electroencephalography as implicit communication channel for proximal interaction between humans and robot swarms

Search and rescue, autonomous construction, and many other semi-autonomous multirobot applications can benefit from proximal interactions between an operator and a swarm of robots. Most research on proximal interaction is based on explicit communication techniques such as gesture and speech. This study proposes a new implicit proximal communication technique to approach the problem of robot selection. We use electroencephalography (EEG) signals to select the robot at which the operator is looking. This is achieved using steady-state visually evoked potential (SSVEP), a repeatable neural response to a regularly blinking visual stimulus that varies predictively based on the blinking frequency. In our experiments, each robot was equipped with LEDs blinking at a different frequency, and the operator’s SSVEP neural response was extracted from the EEG signal to detect and select the robot without requiring any conscious action by the user. This study systematically investigates several parameters affecting the SSVEP neural response: blinking frequency of the LED, distance between the robot and the operator, and color of the LED. Based on these parameters, we study two signal processing approaches and critically analyze their performance on 10 subjects controlling a set of physical robots. Our results show that despite numerous artifacts, it is possible to achieve a recognition rate higher than 85 % on some subjects, while the average over the ten subjects was 75 %.     

Real-time nanoscopy by using blinking enhanced quantum dots

Superresolution optical microscopy (nanoscopy) is of current interest in many biological fields. Superresolution optical fluctuation imaging, which utilizes higher-order cumulant of fluorescence temporal fluctuations, is an excellent method for nanoscopy, as it requires neither complicated optics nor illuminations. However, it does need an impractical number of images for real-time observation. Here, we achieved real-time nanoscopy by modifying superresolution optical fluctuation imaging and enhancing the fluctuation of quantum dots. Our developed quantum dots have higher blinking than commercially available ones. The fluctuation of the blinking improved the resolution when using a variance calculation for each pixel instead of a cumulant calculation. This enabled us to obtain microscopic images with 90-nm and 80-ms spatial-temporal resolution by using a conventional fluorescence microscope without any optics or devices.     

Bayesian localization microscopy reveals nanoscale podosome dynamics

We describe a localization microscopy analysis method that is able to extract results in live cells using standard fluorescent proteins and xenon arc lamp illumination. Our Bayesian analysis of the blinking and bleaching (3B analysis) method models the entire dataset simultaneously as being generated by a number of fluorophores that may or may not be emitting light at any given time. The resulting technique allows many overlapping fluorophores in each frame and unifies the analysis of the localization from blinking and bleaching events. By modeling the entire dataset, we were able to use each reappearance of a fluorophore to improve the localization accuracy. The high performance of this technique allowed us to reveal the nanoscale dynamics of podosome formation and dissociation throughout an entire cell with a resolution of 50 nm on a 4-s timescale.     

The intercentral relations of the biopotentials in the rabbit brain during the creation of a blinking dominant]

On rabbits in chronic experiment intercentral relations were studied of biopotentials of the sensorimotor cortical zone (blinking presentation in the cortex), VPM of the thalamus and motor nucleus of the oculomotor nerve at formation of blinking dominant created by serial stimulation of one eye by air jet. By the method of spectral-correlation analysis an increase was revealed of spectrum power and increase of COG of the potentials in delta-range in the structures connected with blinking function of the stimulated eye. At transition of the dominant focus to the inhibitory state and activation of the symmetrical centre of the other eye, in centers constellation of this induced focus a reconstruction of the electrical processes took place typical for the dominant.     

Blinking behavior in great-tailed grackles (Quiscalus mexicanus) increases during simulated rainfall

Animals often adjust their behavior in response to changes in environmental conditions, and these behavioral adjustments may result from sensory constraints. In particular, rainfall influences behavior but our understanding of its effects on visual abilities is limited. This study, therefore, tested the hypothesis that rainfall influences blinking behavior, a major component of visual processing, in captive great-tailed grackles (Quiscalus mexicanus). The blinking behavior of the grackles was recorded when they were exposed to simulated rain that was direct (water falling directly atop them) or indirect (water falling at a distance from them). The grackles exhibited increased blinking behavior when they were exposed to the direct rain but not the indirect rain. These results suggest that rainfall may impact visual processing in birds through sensory impairments.     

Increased iris-lens contact following spontaneous blinking: Mathematical modeling

The purpose of this work was to study in silico how iris root rotation due to spontaneous blinking alters the iris contour. An axisymmetric finite-element model of the anterior segment was developed that included changes in the iris contour and the aqueous humor flow. The model geometry was based on average values of ocular dimensions. Blinking was modeled by rotating the iris root posteriorly and returning it back to the anterior. Simulations with maximum rotations of 2°, 4°, 6°, and 8° were performed. The iris-lens contact distance and the pressure difference between the posterior and anterior chambers were calculated. When the peak iris root rotation was 2°, the maximum iris-lens contact increased gradually from 0.28 to 0.34mm within eight blinks. When the iris root was rotated by 6° and 8°, the pressure difference between the posterior and anterior chambers dropped from a positive value (1.23Pa) to negative values (-0.86 and -1.93Pa) indicating the presence of reverse pupillary block. Apparent iris-lens contact increased with steady blinking, and the increase became more pronounced as posterior rotation increased. We conclude that repeated iris root rotation caused by blinking could maintain the iris in a posterior position under normal circumstances, which would then lead to the clinically observed anterior drift of the iris when blinking is prevented.     

Electroneuromyographic studies of pain sensitivity

We analyze the published information on the techniques of studies of pain sensitivity using objective electroneuromyographic approaches and the results of these studies. In particular, we describe works where the H reflex, nociceptive flexor and blinking reflexes, and exteroceptive suppression of voluntary muscle activity were examined. Some aspects of using somatosensory evoked potentials in the studies of pain phenomena are also discussed.     

A photoprotection strategy for microsecond-resolution single-molecule fluorescence spectroscopy

Time resolution of current single-molecule fluorescence techniques is limited to milliseconds because of dye blinking and bleaching. Here we introduce a photoprotection strategy that affords microsecond resolution by combining efficient triplet quenching by oxygen and Trolox with minimized bleaching via the oxygen radical scavenger cysteamine. Using this approach we resolved the single-molecule microsecond conformational fluctuations of two proteins: the two-state folder α-spectrin SH3 domain and the ultrafast downhill folder BBL.     

Voltage regulation of single green fluorescent protein mutants

We report the analysis of the fluorescence intensity fluctuations of single proteins of a GFP mutant, GFPmut2, embedded in a polyelectrolyte nanocapsule adsorbed on thin conductive layers. Our results, based on single molecule fluorescence spectroscopy, indicate that the fluorescence blinking dynamics of GFP is strongly dependent on the bulk conductivity of the metal layer substrate, on the distance from the conductive surfaces and on the amplitude of the voltage applied to the poly-electrolyte layers. These findings suggest that fluorescence blinking itself might be employed as a reporter signal in nano-bio-technology applications.     

Analysis method for measuring submicroscopic distances with blinking quantum dots

A method is described that takes advantage of the intermittency ("blinking") in the fluorescence of quantum dots (QDs) to measure absolute positions of closely spaced QDs. The concept is that even if two QDs are separated by only tens of nanometers, the position of each QD is resolvable if the point spread function of each can be imaged independently of the other. In the case of QDs, this is possible if each QD separately blinks completely on and off during a time-lapse sequence. To demonstrate the principle of this method, time-lapse sequences of single blinking QDs were acquired and the centroids of the point spread functions determined. Images of the blinking QDs were then overlapped in software, pixel by pixel, generating a range of submicroscopic distances between QD pairs. Methods were developed for analyzing the overlapped time sequences of the QD pairs so that the positions of the QDs and the distances between them could be determined without prior knowledge of the single QD positions. We subsequently used this method to measure the end-to-end length of a 122-basepair double-stranded DNA fragment.     

Sscillatory Cortical Activity during Visual Hallucinations

From a theoretical point of view we investigate cortical activity patterns causing dynamic visual hallucinations. For this reason we analyze an oscillatory instability of the dynamics ofthe activator-inhibitor model of Ermentrout and Cowan.Such an oscillatory instability occurs as a result of several disease mechanisms.We explicitly derive the order parameter equation. By means of the averaging theorem, we obtain the averaged order parameter equation.The latter enables us to determine stable and unstable bifurcating cortical activity patterns analytically in lowest order.Depending on model parameters as well as on initial conditions two types of cortical activity patterns occur: travelling waves and blinking rolls, i.e.standing waves oscillating with the same frequency and with a phase shift of /2. In contrast to cortical activitypatterns caused by non-oscillatory instabilitiesanalyzed by Ermentrout and Cowan and by the author the travelling waves and the blinking rolls lead to a variety of dynamic visual hallucinations which are discussed indetail.     

Optical methods for exploring dynamics of single copies of green fluorescent protein.

Single copies of four different phenolate ion mutants of the green fluorescent protein (GFP) exhibit a complex blinking and fluctuating behavior, a phenomenon that is hidden in measurements on large ensembles. Both total internal reflection microscopy and scanning confocal microscopy can be used to study the blinking dynamics, and autocorrelation analysis yields histograms of the correlation times for many individual molecules. While the total internal reflection method can follow several single molecules simultaneously, the confocal method offers higher time resolution at the expense of parallelism. We compare and contrast the two methods in terms of the ability to follow the complex dynamics of this system.     

Cellular Uptake of Gold Nanoparticles and Their Behavior as Labels for Localization Microscopy

Gold nanoparticles (GNPs) enhance the damaging absorbance effects of high-energy photons in radiation therapy by increasing the emission of Auger-photoelectrons in the nm-μm range. It has been shown that the incorporation of GNPs has a significant effect on radiosensitivity of cells and their dose-dependent clonogenic survival. One major characteristic of GNPs is also their diameter-dependent cellular uptake and retention. In this article, we show by means of an established embodiment of localization microscopy, spectral position determination microscopy (SPDM), that imaging with nanometer resolution and systematic counting of GNPs becomes feasible, because optical absorption and plasmon resonance effects result in optical blinking of GNPs at a size-dependent wavelength. To quantify cellular uptake and retention or release, SPDM with GNPs that have diameters of 10 and 25 nm was performed after 2 h and after 18 h. The uptake of the GNPs in HeLa cells was either achieved via incubation or transfection via DNA labeling. On average, the uptake by incubation after 2 h was approximately double for 10 nm GNPs as compared to 25 nm GNPs. In contrast, the uptake of 25 nm GNPs by transfection was approximately four times higher after 2 h. The spectral characteristics of the fluorescence of the GNPs seem to be environment-dependent. In contrast to fluorescent dyes that show blinking characteristics due to reversible photobleaching, the blinking of GNPs seems to be stable for long periods of time, and this facilitates their use as an appropriate dye analog for SPDM imaging.     

Electrophysiologic study of the cerebral control of swallowing

Swallowing dominant, created by serial pouring of water into rabbit's mouth cavity was tested in after-action from water supply. As a result of summation in the dominant focus of excitations elicited by stimuli of various modalities, swallowing took place. The study of swallowing dominant revealed by its stimulus which elicits its own unconditioned reflex (blinking), has shown that temporary feedback is characteristic for the dominant as well as for the conditioned reflex. Summation in the swallowing center during dominant testing by stimulation of the eye cornea takes place without conjugated inhibition of the blinking center. During discrete pouring of water into the animal's mouth, evoked potentials appear in the cortical-subcortical structures of the swallowing center. Identical potentials appear in corresponding structures before the summation swallowing. Appearance of these potentials in the electric activity of the dominant focus testifies to its readiness for summation.