Diffusivity Measurement by Single-Molecule Recycling in a Capillary Microchannel.

Microfluidic devices have been extensively investigated in recent years in fields including ligand-binding analysis, chromatographic separation, molecular dynamics, and DNA sequencing. To prolong the observation of a single molecule in aqueous buffer, the solution in a sub-micron scale channel is driven by a electric field and reversed after a fixed delay following each passage, so that the molecule passes back and forth through the laser focus and the time before irreversible photobleaching is extended. However, this practice requires complex chemical treatment to the inner surface of the channel to prevent unexpected sticking to the surface and the confined space renders features, such as a higher viscosity and lower dielectric constant, which slow the Brownian motion of the molecule compared to the bulk solution. Additionally, electron beam lithography used for the fabrication of the nanochannel substantially increases the cost, and the sub-micron dimensions make the molecule difficult to locate. In this paper, we propose a method of single-molecule recycling in a capillary microchannel. A commercial fused-silica capillary with an inner diameter of 2 microns is chopped into a 1-inch piece and is fixed onto a cover slip. Two o-rings on the sides used as reservoirs and an o-ring in the middle used as observation window are glued over the capillary. The inner surface of the capillary is chemically processed to reduce the non-specific sticking and to improve capillary effect. The device does not require high-precision fabrication and thus is less costly and easier to prepare than the nanochannel. 40 nm Fluospheres® in 50% methanol are used as working solution. The capillary is translated by a piezo stage to recycle the molecule, which diffuses freely through the capillary, and a confocal microscope is used for fluorescence collection. The passing times of the molecule through the laser focus are calculated by a real-time control system based on an FPGA, and the commands of translation are given to the piezo stage through a feedback algorithm. The larger dimensions of the capillary overcomes the strong sticking, the reduced diffusivity, and the difficulty of localizing the molecule. We have achieved a maximum number of recycles of more than 200 and developed a maximum-likelihood estimation of the diffusivity of the molecule, which attains results of the same magnitude as the previous report. This technique simplifies the overall procedure of the single-molecule recycling and could be useful for the ligand-binding studies in high-throughput screening.

Three-dimensional feedback-driven trapping of a single nanoparticle or molecule in aqueous solution with a confocal fluorescence microscope.

Control of electroosmotic flows in a two-layer microfluidic device with crossed channels is used to counteract Brownian diffusion in aqueous solution for three-dimensional trapping of a single nanoparticle or molecule within the probe volume of a confocal fluorescence microscope. A field programmable gate array sorts and counts photons into four channels synchronous with laser pulses in four beams focused to waists slightly offset from the center of the confocal volume and uses the counts to update voltages between the four fluidic inlets every 13.5 µs. Trapping is demonstrated for 40 nm nanoparticles for up to 240 s, 20 nm nanoparticles for up to 25 s, and single molecules of streptavidin-Alexa 647 for up to 1.2 s.

The Wearable Cardioverter Defibrillator: an Early Single Centre Australian Experience. Some Pitfalls and Caveats for Use.

BACKGROUND: Wearable Cardioverter Defibrillators (WCD) have been effectively used for more than a decade in North America and Europe for prevention of sudden cardiac death (SCD) due to ventricular arrhythmias. This device has only recently been available in Australia. METHOD: At Westmead hospital, WCD has been used since 2013 as a bridging therapy to an implantable cardioverter defibrillator (ICD) for those at high risk, but are temporarily not suitable for an implantable device. Indications for use were explanted infected ICD, dilated cardiomyopathy, post partum cardiomyopathy, valvular heart disease and myocarditis. The default device settings were: ventricular tachycardia (VT) and ventricular fibrillation (VF) threshold of 150 bpm and 200 bpm respectively and response times were 60 secs for VT and 25 secs for VF. OUTCOME: WCD was used in eight patients. Duration of use ranged from five to 180 days with median of 77 days. Daily usage averaged 23.4±0.6hours. All except one were compliant with the device and none of our patients received shock or died during device usage. Four of the eight patients received ICD, two declined ICD, one was judged to no longer require ICD and one remains under assessment. CONCLUSION: WCD is easy to use, well tolerated and is effective for SCD prevention in patients who are temporarily not suitable for ICD. However patients need to be actively followed-up to reduce the duration of WCD usage and thereby be cost effective.

High-speed multiparameter photophysical analyses of fluorophore libraries.

There is a critical need for high-speed multiparameter photophysical measurements of large libraries of fluorescent probe variants for imaging and biosensor development. We present a microfluidic flow cytometer that rapidly assays 10(4)-10(5) member cell-based fluorophore libraries, simultaneously measuring fluorescence lifetime and photobleaching. Together, these photophysical characteristics determine imaging performance. We demonstrate the ability to resolve the diverse photophysical characteristics of different library types and the ability to identify rare populations.

Correlation of atomic structure and photoluminescence of the same quantum dot: pinpointing surface and internal defects that inhibit photoluminescence.

In a size regime where every atom counts, rational design and synthesis of optimal nanostructures demands direct interrogation of the effects of structural divergence of individuals on the ensemble-averaged property. To this end, we have explored the structure-function relationship of single quantum dots (QDs) via precise observation of the impact of atomic arrangement on QD fluorescence. Utilizing wide-field fluorescence microscopy and atomic number contrast scanning transmission electron microscopy (Z-STEM), we have achieved correlation of photoluminescence (PL) data and atomic-level structural information from individual colloidal QDs. This investigation of CdSe/CdS core/shell QDs has enabled exploration of the fine structural factors necessary to control QD PL. Additionally, we have identified specific morphological and structural anomalies, in the form of internal and surface defects, that consistently vitiate QD PL.

Microfluidics-based selection of red-fluorescent proteins with decreased rates of photobleaching.

Fluorescent proteins offer exceptional labeling specificity in living cells and organisms. Unfortunately, their photophysical properties remain far from ideal for long-term imaging of low-abundance cellular constituents, in large part because of their poor photostability. Despite widespread engineering efforts, improving the photostability of fluorescent proteins remains challenging due to lack of appropriate high-throughput selection methods. Here, we use molecular dynamics guided mutagenesis in conjunction with a recently developed microfluidic-based platform, which sorts cells based on their fluorescence photostability, to identify red fluorescent proteins with decreased photobleaching from a HeLa cell-based library. The identified mutant, named Kriek, has 2.5- and 4-fold higher photostability than its progenitor, mCherry, under widefield and confocal illumination, respectively. Furthermore, the results provide insight into mechanisms for enhancing photostability and their connections with other photophysical processes, thereby providing direction for ongoing development of fluorescent proteins with improved single-molecule and low-copy imaging capabilities.

Rapid, single-molecule assays in nano/micro-fluidic chips with arrays of closely spaced parallel channels fabricated by femtosecond laser machining.

Cost-effective pharmaceutical drug discovery depends on increasing assay throughput while reducing reagent needs. To this end, we are developing an ultrasensitive, fluorescence-based platform that incorporates a nano/micro-fluidic chip with an array of closely spaced channels for parallelized optical readout of single-molecule assays. Here we describe the use of direct femtosecond laser machining to fabricate several hundred closely spaced channels on the surfaces of fused silica substrates. The channels are sealed by bonding to a microscope cover slip spin-coated with a thin film of poly(dimethylsiloxane). Single-molecule detection experiments are conducted using a custom-built, wide-field microscope. The array of channels is epi-illuminated by a line-generating red diode laser, resulting in a line focus just a few microns thick across a 500 micron field of view. A dilute aqueous solution of fluorescently labeled biomolecules is loaded into the device and fluorescence is detected with an electron-multiplying CCD camera, allowing acquisition rates up to 7 kHz for each microchannel. Matched digital filtering based on experimental parameters is used to perform an initial, rapid assessment of detected fluorescence. More detailed analysis is obtained through fluorescence correlation spectroscopy. Simulated fluorescence data is shown to agree well with experimental values.

Three-dimensional tracking of a single fluorescent nanoparticle using four-focus excitation in a confocal microscope.

We report high sensitivity detection and tracking of a single fluorescent nanoparticle in solution by use of four alternately pulsed laser diodes for fluorescence excitation in a confocal microscope. Slight offsets between the centers of the overlapping laser foci together with time-resolved photon counting enable sub-micron precision position measurements. Real-time correction for diffusional motion with a xyz-piezo stage then enables tracking of a nanoparticle with diffusivity up to ~12 µm(2) s(-1). Fluorescence correlation spectroscopy and calibration measurements indicate a net fluorescence photon detection efficiency of ~6-9%, comparable to that of an optimized single-molecule microscope.

Microfluidic cell sorter for use in developing red fluorescent proteins with improved photostability.

This paper presents a novel microfluidic cytometer for mammalian cells that rapidly measures the irreversible photobleaching of red fluorescent proteins expressed within each cell and achieves high purity (>99%) selection of individual cells based on these measurements. The selection is achieved by using sub-millisecond timed control of a piezo-tilt mirror to steer a focused 1064-nm laser spot for optical gradient force switching following analysis of the fluorescence signals from passage of the cell through a series of 532-nm laser beams. In transit through each beam, the fluorescent proteins within the cell undergo conversion to dark states, but the microfluidic chip enables the cell to pass sufficiently slowly that recovery from reversible dark states occurs between beams, thereby enabling irreversible photobleaching to be quantified separately from the reversible dark-state conversion. The microfluidic platform achieves sorting of samples down to sub-millilitre volumes with minimal loss, wherein collected cells remain alive and can subsequently proliferate. The instrument provides a unique first tool for rapid selection of individual mammalian cells on the merits of photostability and is likely to form the basis of subsequent lab-on-a-chip platforms that combine photobleaching with other spectroscopic measurements for on-going research to develop advanced red fluorescent proteins by screening of genetic libraries.

Cardiac implantable electronic device therapy for bradyarrhythmias and tachyarrhythmias.

Pacemakers originally were developed for patients with profound bradycardia and complete heart block who, without them, usually suffered from syncope, heart failure and an early demise. Since that time, devices have evolved to include pacing and shock therapies for the management of tachyarrhythmias and heart failure with the aim of improving quality, and if possible, length of life. Whether to insert a device depends on a balance between the potential benefits of device therapy and its risks, which are not inconsiderable. We discuss current agreed indications for pacemakers and implantable defibrillators and some current controversies surrounding their use.

Single-nanocrystal spectroscopy of white-light-emitting CdSe nanocrystals.

We report the observation of broad-spectrum fluorescence from single CdSe nanocrystals. Individual semiconductor nanocrystals typically have a narrower emission spectrum than that of an ensemble. However, our experiments show that the ensemble white-light emission observed in ultrasmall CdSe nanocrystals is the result of many single CdSe nanocrystals, each emitting over the entire visible spectrum. These results indicate that each white-light-emitting CdSe nanocrystal contains all the trap states that give rise to the observed white-light emission.

Ligase detection reaction generation of reverse molecular beacons for near real-time analysis of bacterial pathogens using single-pair fluorescence resonance energy transfer and a cyclic olefin copolymer microfluidic chip.

Detection of pathogenic bacteria and viruses require strategies that can signal the presence of these targets in near real-time due to the potential threats created by rapid dissemination into water and/or food supplies. In this paper, we report an innovative strategy that can rapidly detect bacterial pathogens using reporter sequences found in their genome without requiring polymerase chain reaction (PCR). A pair of strain-specific primers was designed based on the 16S rRNA gene and were end-labeled with a donor (Cy5) or acceptor (Cy5.5) dye. In the presence of the target bacterium, the primers were joined using a ligase detection reaction (LDR) only when the primers were completely complementary to the target sequence to form a reverse molecular beacon (rMB), thus bringing Cy5 (donor) and Cy5.5 (acceptor) into close proximity to allow fluorescence resonance energy transfer (FRET) to occur. These rMBs were subsequently analyzed using single-molecule detection of the FRET pairs (single-pair FRET; spFRET). The LDR was performed using a continuous flow thermal cycling process configured in a cyclic olefin copolymer (COC) microfluidic device using either 2 or 20 thermal cycles. Single-molecule photon bursts from the resulting rMBs were detected on-chip and registered using a simple laser-induced fluorescence (LIF) instrument. The spFRET signatures from the target pathogens were reported in as little as 2.6 min using spFRET.

Simulation of single-molecule trapping in a nanochannel.

The detection and trapping of single fluorescent molecules in solution within a nanochannel is studied using numerical simulations. As optical forces are insufficient for trapping molecules much smaller than the optical wavelength, a means for sensing a molecule's position along the nanochannel and adjusting electrokinetic motion to compensate diffusion is assessed. Fluorescence excitation is provided by two adjacently focused laser beams containing temporally interleaved laser pulses. Photon detection is time-gated, and the displacement of the molecule from the middle of the two foci alters the count rates collected in the two detection channels. An algorithm for feedback control of the electrokinetic motion in response to the timing of photons, to reposition the molecule back toward the middle for trapping and to rapidly reload the trap after a molecule photobleaches or escapes, is evaluated. While accommodating the limited electrokinetic speed and the finite latency of feedback imposed by experimental hardware, the algorithm is shown to be effective for trapping fast-diffusing single-chromophore molecules within a micron-sized confocal region. Studies show that there is an optimum laser power for which loss of molecules from the trap due to either photobleaching or shot-noise fluctuations is minimized.

Cross-talk-free dual-color fluorescence cross-correlation spectroscopy for the study of enzyme activity.

We have developed an instrument for spectral cross-talk-free dual-color fluorescence cross-correlation spectroscopy (FCCS), which provides a readout modality for the study of enzyme activity in application areas such as high-throughput screening. Two spectrally distinct (approximately 250 nm) fluorophores, Cy3 and IRD800, were excited simultaneously using two different excitation sources: one poised at 532 nm and the other at 780 nm. The fluorescence information was processed on two different color channels monitored with single-photon avalanche diodes (SPADs) that could transduce events at the single-molecule level. The system provided no color cross-talk (cross-excitation and/or cross-emission) and/or fluorescence resonance energy transfer (FRET), significantly improving data quality. To provide evidence of cross-talk-free operation, the system was evaluated using bright microspheres (lambda(abs) = 532 nm, lambda(em) = 560 nm) and quantum dots (lambda(abs) = 532 nm, lambda(em) = 810 nm). Experimental results indicated that no color leakage from the microspheres or quantum dots into inappropriate color channels was observed. To demonstrate the utility of the system, the enzymatic activity of APE1, which is responsible for nicking the phosphodiester backbone in DNA on the 5' side of an apurinic/apyrimidinic site, was monitored by FCCS using a double-stranded DNA substrate dual labeled with Cy3 and IRD800. Activity of APE1 was also monitored in the presence of an inhibitor (7-nitroindole-2-carboxylic acid) of the enzyme using this cross-talk-free FCCS platform. In all cases, no spectral leakage from single-molecule events into inappropriate color channels was observed.

Single-pair fluorescence resonance energy transfer (spFRET) for the high sensitivity analysis of low-abundance proteins using aptamers as molecular recognition elements.

We have developed a strategy for the detection of single protein molecules, which uses single-pair fluorescence resonance energy transfer (spFRET) as the readout modality and provides exquisite analytical sensitivity and reduced assay turn-around-time by eliminating various sample pre-processing steps. The single-protein detection assay uses two independent aptamer recognition events to form an assembly conducive to intramolecular hybridization of oligonucleotide complements that are tethered to the aptamers. This hybridization brings a donor-acceptor pair within the Förster distance to create a fluorescence signature indicative of the presence of the protein-aptamer(s) association complex. As an example of spFRET, we demonstrate the technique for the analysis of serum thrombin. The assay requires co-association of two distinct epitope-binding aptamers, each of which is labeled with a donor or acceptor fluorescent dye (Cy3 or Cy5, respectively) to produce a FRET response. The FRET response between Cy3 and Cy5 was monitored by single-molecule photon-burst detection, which provides high analytical sensitivity when the number of single-molecule events is plotted versus the target concentration. We are able to identify thrombin with high efficiency based on photon burst events transduced in the Cy5 detection channel. We also demonstrate that the technique can discriminate thrombin molecules from its analogue prothrombin. The analytical sensitivity was >200-fold better than an ensemble measurement.

Single molecule diffusion coefficient estimation by image analysis of simulated CCD images to aid high-throughput screening.

Extension of one-dimensional signal analysis to two-dimensional image analysis could accelerate conventional methods of high-throughput screening in the discovery of new pharmaceutical agents. This work describes a first step taken towards this goal - the evaluation of image-analysis based estimation strategies of the diffusion coefficient of a single molecule transported within a microfabricated flowcell. A computer simulation of single-molecule imaging by a charge-coupled device (CCD) camera is used to determine if it is possible to distinguish three different types of molecules with different diffusion coefficients. The Gaussian fitting algorithm finds the variance of the transverse trajectory, which increases linearly with the diffusion coefficient; the path analysis algorithm determines the diffusion coefficient from cumulative summation of the squared displacement along the imaged path; the detector area analysis algorithm determines the number of resolvable positions or pixels in the imaged trajectory. Of the three methods, the path analysis strategy appears to provide the most reliable measure of diffusion coefficient with relative error of 13.6% and 6.4% between single molecules with diffusion coefficients of 2.85e-7 and 1.425e-7 cm(2)/s. The detector area analysis method can statistically distinguish between single molecules with diffusion coefficients of 5.7e-7 and 1.425e-7 cm(2)/s at the p(0.05) level.

Triplet-state investigations of fluorescent dyes at dielectric interfaces using total internal reflection fluorescence correlation spectroscopy.

The triplet-state kinetics of several fluorescent dyes used in ultrasensitive fluorescence microscopy are investigated using total internal reflection fluorescence correlation spectroscopy (TIR-FCS). A theoretical outline of the correlation analysis and the physical aspects of evanescent excitation and fluorescence emission at dielectric interfaces are given. From this analysis, the rates of intersystem crossing and triplet decay are deduced for fluorescein, ATTO 488, rhodamine 110, rhodamine 123, and rhodamine 6G in aqueous buffer solutions. All investigated dyes show slightly higher triplet rates at the dielectric interface compared to bulk solution measurements. We attribute this enhancement to possible modifications of the dyes' photophysical properties near a dielectric interface. In the case of rhodamine 6G, the impact of changes in the dye concentration, ionic strength of the solvent, and potassium iodide concentration are also investigated. This leads to a better understanding of the influences of dye-dye, dye-solvent, and dye-surface interactions on the increased triplet intersystem crossing and triplet decay rates. The study shows that analysis of triplet-state kinetics by TIR-FCS not only results in a better understanding of how the photophysical properties of the dyes are affected by the presence of an interface, but also provides a means for probing the microenvironment near dielectric interfaces.

Single-pulse ultrafast-laser machining of high aspect nano-holes at the surface of SiO2.

Use of high numerical aperture focusing with negative longitudinal spherical aberration is shown to enable deep (> microm), high aspect ratio, nano-scale-width holes to be machined into the surface of a fused-silica (SiO(2)) substrate with single pulses from a 200 fs, 4 microJ Ti-Sapphire laser source. The depths of the nano-holes are characterized by use of a non-destructive acetate replication technique and are confirmed by imaging of sectioned samples with a dual focused ion beam/scanning electron microscope.

Engineering the collected field for single-molecule orientation determination.

We theoretically investigate the use of spatial light modulators (SLMs) for transformation of the collected fluorescence field in a high numerical aperture confocal microscope, for improved molecular orientation determination in single-molecule spectroscopy. The electric vector field in the back aperture of the microscope objective is calculated using the Weyl representation and taking into account components emitted at angles above the critical angle of the coverglass-immersion fluid interface. The coherently imaged fluorescence undergoes spatially-dependent phase and polarization transformation by the SLMs, before it passes to a polarization beamsplitter, and is subsequently focused onto two pinholes and single-photon detectors.