Fluorescence Detection Excitation Research Articles

Pulsed-Interleaved-Excitation Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy.

We report on pulsed-interleaved-excitation two-dimensional fluorescence lifetime correlation spectroscopy (PIE 2D FLCS) to study biomolecular structural dynamics with high sensitivity and high time resolution using Förster resonance energy transfer (FRET). PIE 2D FLCS is an extension of 2D FLCS, which is a unique single-molecule fluorescence method that uses fluorescence lifetime information to distinguish different fluorescence species in equilibrium and resolves their interconversion dynamics with a submicrosecond time resolution. Because 2D FLCS has used only a single-color excitation so far, it was difficult to distinguish a very low-FRET (or zero-FRET) species from only donor-labeled species. We overcome this difficulty by implementing the PIE scheme (i.e., alternate excitation of the donor and acceptor dyes using two temporally interleaved excitations with different colors) to 2D FLCS, realizing two-color excitation and two-color fluorescence detection in 2D FLCS. After proof-of-principle PIE 2D FLCS analysis on the photon data synthesized with Monte Carlo simulation, we apply PIE 2D FLCS to a DNA-hairpin sample and show that this method readily distinguishes four fluorescent species, i.e., high-FRET, low-FRET, and two single-dye-labeled species. In addition, we show that PIE 2D FLCS can also quantitatively evaluate the contributions of the donor-acceptor spectral crosstalk, which often appears as artifacts in FRET studies and degrades the information obtained.

Two-photon fluorescence lifetime for label-free microfluidic droplet sorting

Microfluidic droplet sorting systems facilitate automated selective micromanipulation of compartmentalized micro- and nano-entities in a fluidic stream. Current state-of-the-art droplet sorting systems mainly rely on fluorescence detection in the visible range with the drawback that pre-labeling steps are required. This limits the application range significantly, and there is a high demand for alternative, label-free methods. Therefore, we introduce time-resolved two-photon excitation (TPE) fluorescence detection with excitation at 532 nm as a detection technique in droplet microfluidics. This enables label-free in-droplet detection of small aromatic compounds that only absorb in a deep-UV spectral region. Applying time-correlated single-photon counting, compounds with similar emission spectra can be distinguished due to their fluorescence lifetimes. This information is then used to trigger downstream dielectrophoretic droplet sorting. In this proof-of-concept study, we developed a polydimethylsiloxane-fused silica (FS) hybrid chip that simultaneously provides a very high optical transparency in the deep-UV range and suitable surface properties for droplet microfluidics. The herein developed system incorporating a 532-nm picosecond laser, time-correlated single-photon counting (TCSPC), and a chip-integrated dielectrophoretic pulsed actuator was exemplarily applied to sort droplets containing serotonin or propranolol. Furthermore, yeast cells were screened using the presented platform to show its applicability to study cells based on their protein autofluorescence via TPE fluorescence lifetime at 532 nm.Graphical abstract

Integration of fluidic channels and total internal reflection excitation with supercritical angle fluorescence detection

In many diagnostic applications, there is a growing demand for small cost effective portable devices that enable fast on-site analysis. For this target, we are developing an integrated approach of a previously reported surface-sensitive measurement system based on total internal reflection excitation and supercritical angle fluorescence detection. The integrated measurement platform includes a polystyrene substrate on which the fluidic channels for sample transport, waveguides for fluorescence excitation and the readout optics are fabricated. In addition, thin layers with dielectric properties differing from those of polystyrene may be needed on the fluidic channels for optimal sample transport or biomolecule immobilisation. Here, special attention is paid to the influence of the dielectric environment near the solid-liquid interface on the fluorescence distribution and to the design of the readout optics. The first version of the designed readout optics of the integrated measurement platform is presented.

The Preprotein Binding Domain of SecA Displays Intrinsic Rotational Dynamics

SecA converts ATP energy to protein translocation work. Together with the membrane-embedded SecY channel it forms the bacterial protein translocase. How secretory proteins bind to SecA and drive conformational cascades to promote their secretion remains unknown. To address this, we focus on the preprotein binding domain (PBD) of SecA. PBD crystalizes in three distinct states, swiveling around its narrow stem. Here, we examined whether PBD displays intrinsic dynamics in solution using single-molecule Förster resonance energy transfer (smFRET). Unique cysteinyl pairs on PBD and apposed domains were labeled with donor/acceptor dyes. Derivatives were analyzed using pulsed interleaved excitation and multi-parameter fluorescence detection. The PBD undergoes significant rotational motions, occupying atleast three distinct states in dimeric and four in monomeric soluble SecA. Nucleotides do not affect smFRET-detectable PBD dynamics. These findings lay the foundations for single-molecule dissection oftranslocase mechanics and suggest models for possible PBD involvement during catalysis.

Chiral capillary electrophoresis with UV-excited fluorescence detection for the enantioselective analysis of 9-fluorenylmethoxycarbonyl-derivatized amino acids

The potential of capillary electrophoresis (CE) with ultraviolet (UV)-excited fluorescence detection for sensitive chiral analysis of amino acids (AAs) was investigated. dl-AAs were derivatized with 9-fluorenylmethoxycarbonyl chloride (FMOC)-Cl to allow their fluorescence detection and enhance enantioseparation. Fluorescence detection was achieved employing optical fibers, leading UV excitation light (< 300 nm) from a Xe-Hg lamp to the capillary window, and fluorescence emission to a spectrograph equipped with a charge-coupled device (CCD). Signal averaging over time and emission wavelength intervals was carried out to improve the signal-to-noise ratio of the FMOC-AAs. A background electrolyte (BGE) of 40 mM sodium tetraborate (pH 9.5), containing 15% isopropanol (v/v), 30 mM sodium dodecyl sulfate (SDS), and 30 mM β-cyclodextrin (β-CD), was found optimal for AA chemo- and enantioseparation. Enantioresolutions of 1.0 or higher were achieved for 16 proteinogenic dl-AAs. Limits of detection (LODs) were in the 10–100-nM range (injected concentration) for the d-AA enantiomers, except for FMOC-d-tryptophan (536 nM) which showed intramolecular fluorescence quenching. Linearity (R2 > 0.997) and repeatability for peak height (relative standard deviations (RSDs) < 7.0%; n = 5) and electrophoretic mobility (RSDs < 0.6%; n = 5) of individual AA enantiomers were established for chiral analysis of dl-AA mixtures. The applicability of the method was investigated by the analysis of cerebrospinal fluid (CSF). Next to l-AAs, endogenous levels of d-glutamine and d-aspartic acid could be measured in CSF revealing enantiomeric ratios of 0.35 and 19.6%, respectively. This indicates the method’s potential for the analysis of low concentrations of d-AAs in presence of abundant l-AAs.

In Situ Determination of Illegal Drugs in Oral Fluid by Portable Capillary Electrophoresis with Deep UV Excited Fluorescence Detection.

The present study demonstrates the potential of a portable capillary electrophoresis (CE) instrument, coupled to deep UV fluorescence detector (FD) with a 230-255 nm excitation wavelength range, for the determination of the abuse of illegal drugs in oral fluids in situ. CE was introduced in this study due its exceptional power of separation and resolution, short analysis time, and ability for miniaturization for on-site assessment of different substances. The deep UV fluorescence detector was equipped with five interchangeable emission filters, in the emission wavelength range from 278 to 600 nm, and was successfully employed for determination of natively fluorescing illegal drugs, such as cocaine, cocaethylene, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxeamphetamine (MDA), 3,4-methylenedioxy- N-ethylamphetamine (MDEA), para-methoxyamphetamine (PMA), para-methoxy- N-methylamphetamine (PMMA), amphetamine (AMP), methamphetamine (METH), tetrahydrocannabinol (THC) and cannabidiol (CBD). The developed FD showed impressive sensitivity. The instrumental detection limit was 0.5 μg/L for MDMA. It also showed broad linearity, up to 50 mg/L for MDMA. The noise CV% was 1.1% for an empty capillary and 0.6% for a capillary filled with acetonitrile. The portable CE-FD with developed electrophoretic methodologies was successfully utilized for the determination of illegal abuse of drugs during "Weekend" 2016 and 2017 Music Festivals (Estonia). Moreover, CE-FD can be employed for detection of other natively fluorescing compounds in the proposed range (e.g., for different phenolic compounds, BTEX, naphthalene derivatives, and others), significantly widening the applicability of developed CE-FD instrument.

Continuous purification of reaction products by micro free-flow electrophoresis enabled by large area deep-UV fluorescence imaging.

Microreactors have gained increasing attention in their application toward continuous micro flow synthesis. An unsolved problem of continuous flow synthesis is the lack of techniques for continuous product purification. Herein, we present a micro free-flow electrophoresis device and accompanying setup that enables the continuous separation and purification of unlabeled organic synthesis products. The system is applied to the separation and purification of triarylmethanes. For imaging of the unlabeled analytes on-chip a novel setup for large area (3.6 cm2) deep ultra violet excitation fluorescence detection was developed. Suitable separation conditions based on low conductivity electrophoresis buffers were devised to purify the product. With the optimized conditions, starting materials and product of the synthesis were well separated (R > 1.2). The separation was found to be very stable with relative standard deviations of the peak positions smaller than 3.5% over 15 min. The stable conditions enabled collection of the separated compounds, and purity of the product fraction was confirmed using capillary electrophoresis and mass spectrometry. This result demonstrates the great potential of free-flow electrophoresis as a technique for product purification or continuous clean-up in flow synthesis. Graphical Abstract Micro free-flow electrophoresis (μFFE) allows continuous separation and purification of small organic synthesis products. Enabled by a novel deep-UV imaging setup starting materials and product of a recently developed synthesis for triarylmethanes could be purified. Thereby demonstrating the potential of μFFE as continuous purification technique for micro flow synthesis.

The potential of on- and off-resonant formaldehyde imaging combined with bootstrapping in diesel sprays

Formaldehyde (CH2O) is an important intermediate in spray combustion of diesel-like fuels. Experimentally, its spatial distribution is often assessed by means of planar Laser-Induced Fluorescence (PLIF), using a frequency-tripled Nd:YAG laser for excitation and spectrally-filtered fluorescence detection. Especially in soot-laden spray flames, however, considerable spectral interference cannot be avoided, which renders interpretation of the fluorescence distribution challenging. We introduce a more selective strategy, involving dye laser excitation and background subtraction by means of off-resonant excitation in combination with a bootstrap evaluation of sets of individual events. The latter extracts persistent features out of non-simultaneously recorded snapshots of stochastic events, thereby mitigating possible interpretation issues related to the turbulent nature of the process under study. Following this approach, recurrent patterns can be distinguished from random high-intensity events (allegedly due to turbulent fluctuations). We demonstrate our method on the Engine Combustion Network (ECN) “Spray A”-benchmark, with 15 vol-% and 21 vol-% ambient oxygen; we also present spatially well-delimited CH2O fluorescence distributions, and compare these to simultaneously recorded OH* chemiluminescence intensities.

Simultaneous monitoring of intra- and extracellular nitric oxide in living cells by means of dual-color fluorescence imaging

A dual-color fluorescence imaging method for simultaneous monitoring of intra- and extracellular nitric oxide (NO) was developed. Assisted by confocal laser scanning microscope, the intra- and extracellular NO can be successfully visualized by using two selected probes, 4,4-difluoro-8-(3,4-diaminophenyl)-3,5-bis(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene (p-MOPB) and disodium 2,6-disulfonate-1,3-dimethyl-5-hexadecyl-8-(3,4-diaminophenyl)-4,4’-difluoro-4-bora-3a,4a-diaza-s-indacene (DSDMHDAB), which display distinct membrane permeability and show different colors of fluorescence after reaction with NO. Results indicated that intra- and extracellular NO could be fluorometrically detected without mutual interference. The applicability of the proposed method was validated by dual-color imaging of NO on both sides of the plasma membrane in RAW 264.7 murine macrophages and human vascular endothelial (ECV-304) cells. This multi-labeling approach using multi-laser excitation and multi-color fluorescence detection holds great promise for simultaneous analysis of NO as well as other gasotransmitters in living cells with subcellular resolution.

The detection and subsequent volume optimization of biological nanocrystals.

Identifying and then optimizing initial crystallization conditions is a prerequisite for macromolecular structure determination by crystallography. Improved technologies enable data collection on crystals that are difficult if not impossible to detect using visible imaging. The application of second-order nonlinear imaging of chiral crystals and ultraviolet two-photon excited fluorescence detection is shown to be applicable in a high-throughput manner to rapidly verify the presence of nanocrystals in crystallization screening conditions. It is noted that the nanocrystals are rarely seen without also producing microcrystals from other chemical conditions. A crystal volume optimization method is described and associated with a phase diagram for crystallization.

Rapid Prototyping of Electrochromatography Chips for Improved Two-Photon Excited Fluorescence Detection

In the present study, we introduce two-photon excitation at 532 nm for label-free fluorescence detection in chip electrochromatography. Two-photon excitation at 532 nm offers a promising alternative to one-photon excitation at 266 nm, as it enables the use of economic chip materials instead of fused silica. In order to demonstrate these benefits, one-photon and two-photon induced fluorescence detection are compared in different chip layouts and materials with respect to the achievable sensitivity in the detection of polycyclic aromatic hydrocarbons (PAHs). Customized chromatography chips with cover or bottom slides of different material and thickness are produced by means of a rapid prototyping method based on liquid-phase lithography. The design of thin bottom chips (180 μm) enables the use of high-performance immersion objectives with low working distances, which allows one to exploit the full potential of two-photon excitation for a sensitive detection. The developed method is applied for label-free analysis of PAHs separated on a polymer monolith inside polymer glass sandwich chips made from fused silica or soda-lime glass. The obtained limits of detection range from 40 nM to 1.95 μM, with similar sensitivities in fused silica thin bottom chips for one-photon and two-photon excitation. In deep-UV non- or less-transparent devices two-photon excitation is mandatory for label-free detection of aromatics with high sensitivity.

High-throughput screening of colonization samples for methicillin-resistant Staphylococcus aureus

We present here the first application of 2-photon excited fluorescence detection (TPX) technology for the direct screening of clinical colonization samples for methicillin-resistant Staphylococcus aureus (MRSA). A total of 125 samples from 14 patients with previously identified MRSA carriage and 16 controls from low-prevalence settings were examined. The results were compared to those obtained by both standard phenotypic and molecular methods. In identifying MRSA carriers, i.e. persons with at least 1 MRSA positive colonization sample by standard methods, the sensitivity of the TPX technique was 100%, the specificity 78%, the positive predictive value 75%, and the negative predictive value 100%. The TPX assay sensitivity per colonization sample was 89%, the specificity 93%, the positive predictive value 84%, and the negative predictive value 95%. The median time for a true-positive test result was 3 h and 26 min; negative test results are available after 13 h. The assay capacity was 48 samples per test run. The TPX MRSA technique could provide early preliminary results for clinicians, while simultaneously functioning as a selective enrichment step for further conventional testing. Costs and workload associated with hospital infection control can be reduced using this high-throughput, point-of-care compatible methodology.

Label-Free Fluorescence Detection of Aromatic Compounds in Chip Electrophoresis Applying Two-Photon Excitation and Time-Correlated Single-Photon Counting

In this study, we introduce time-resolved fluorescence detection with two-photon excitation at 532 nm for label-free analyte determination in microchip electrophoresis. In the developed method, information about analyte fluorescence lifetimes is collected by time-correlated single-photon counting, improving reliable peak assignment in electrophoretic separations. The determined limits of detection for serotonin, propranolol, and tryptophan were 51, 37, and 280 nM, respectively, using microfluidic chips made of fused silica. Applying two-photon excitation microchip separations and label-free detection could also be performed in borosilicate glass chips demonstrating the potential for label-free fluorescence detection in non-UV-transparent devices. Microchip electrophoresis with two-photon excited fluorescence detection was then applied for analyses of active compounds in plant extracts. Harmala alkaloids present in methanolic plant extracts from Peganum harmala could be separated within seconds and detected with on-the-fly determination of fluorescence lifetimes.

Conformational Dynamics in Designer Cellulosomes Studied by Single-Pair Fret with Mfd-Pie

Cellulose is the most abundant biopolymer on earth and when broken down into oligosugars, it can be used for the production of bioethanol. As such, cellulose holds great potential as a novel energy source. The anaerobic thermophilic bacterium Clostridium thermocellum expresses an extracellular multi-enzyme complex that degrades cellulosose: the cellulosome. Cellulosomes, in general, consist of a scaffoldin domain harboring binding sites for multiple cellulases. These enzymes interact via their dockerin domain with complementary cohesin domain on the scaffoldin. An emerging research field aims at engineering ‘designer cellulosomes’ by genetically coupling different types of cohesins. These novel minimal cellulosomes should provide similar, if not better, enzymatic activity as the wild-type. Little is known about the exact role of the linker peptides between cohesin moieties. To address this, we have site-specifically labeled a designer cellulosome consisting of two cohesin subunits connected by either a natural or shortened peptide linker and performed single pair Forster resonance energy transfer experiments using pulsed interleaved excitation and multi-parameter fluorescence detection. We observe different conformational states of the cohesin dimer, suggesting the presence of conformational dynamics. Relating the conformational dynamics of such designer cellulosomes with their activity will be of great help for understanding and improving their function.

Evaluation of the TPX MRSA assay for the detection of methicillin-resistant Staphylococcus aureus

The aim of this study was to evaluate a new type of assay for the phenotypic detection of methicillin-resistant Staphylococcus aureus (MRSA). The assay is based on a point-of-care compatible two-photon excitation fluorescence detection technology (TPX). A collection of 243 epidemic MRSA isolates was tested in addition to 138 sporadic MRSA and 101 negative control strains. The assay proved to be both sensitive (97.9%) and specific (94.1%) in the identification of MRSA, with adequate positive (98.4%) and negative (92.2%) predictive values. The time required for obtaining a positive test result was less than 14 h for 99.0% of the MRSA true-positive samples. After a test run, the selectively enriched reaction mixtures may be recovered and further studied by molecular or standard phenotypic methods. The main benefits of the TPX methodology include a simple assay procedure, low reagent consumption, and a high-throughput capacity.

Applying a Surface-Sensitive Fluorescence Method to Fast on- Site Detection of Cocaine in Saliva

Fast and sensitive detection of cocaine in saliva is realized utilizing a surface-sensitive fluorescence measurement platform. The platform is based on a polystyrene parabolic lens that enables the simultaneous application of total internal reflection excitation (TIR) and supercritical angle fluorescence detection (SAF), which results in extreme surface sensitivity in the measurements. The molecular recognition takes place in a scheme, where cocaine molecules to be detected generate a displacement of the labelled anti-cocaine antibodies from the cocaine-BSA-conjugate molecules immobilized on a surface. The results with untreated saliva spiked with cocaine demonstrate that by monitoring the dissociation process in real time, cocaine concentrations down to 1 ng/mL can be detected within 60s.

Applying Total Internal Reflection Excitation and Super Critical Angle Fluorescence Detection to a Morphine Assay

A surface-sensitive fluorescence measurement platform is utilised in the detection of morphine. The platform is based on a polystyrene parabolic lens that enables the simultaneous application of total internal reflection excitation and supercritical angle fluorescence detection in the measurements. The molecular recognition of morphine is based on two antibodies, one against morphine and the other against the immune complex formed between the anti-morphine antibody and a morphine molecule. The antibodies are applied in a sandwich-like format in a one-step test, where the molecular binding onto a liquid-solid-interface is monitored in real time. Morphine concentrations between 0.6 and 18.2 ng/mL were reliably determined in 60 s, while concentrations down to 2.7 ng/mL were detected already in 20 s. With appropriate recognition molecules the technique is applicable also to other drugs and small analytes.

ATP Dependent Nucleosome Remodelling - Mechanistic Insights from Single Molecule Experiments

DNA in eukaryotes is highly compacted. However, it is essential that proteins can have controlled access to the DNA during a number of fundamental cellular processes such as replication, transcription or repair. A large family of protein complexes commonly referred to as Swi2/Snf2 complexes allows for the transient remodelling of the essential compaction unit, the nucleosome and thus assures the access to, or repression of certain segments of DNA. In spite of a large number of research efforts using bio-chemical, structural and theoretical approaches the molecular mechanism of the ATP dependent nucleosome remodelling is currently not well understood. We therefore performed single molecule FRET experiments aimed to unravel details of the remodelling kinetics and pathway.We present remodelling data obtained by the remodelling complex ACF from drosophila on mono-nucleosomal constructs that use the 601- localisation sequence in combination with a biochemically well characterised linker DNA. The remodelling data was obtained, both, on nucleosomes immobilised onto surfaces of micro-fluidic chambers as well as from solution measurements using pulsed interleaved excitation and multi-parameter fluorescence detection.

Methicillin-Resistant Staphylococcus aureus Screening by Online Immunometric Monitoring of Bacterial Growth under Selective Pressure

Rapid, high-throughput screening tools are needed to contain the spread of hospital-acquired methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) strains. Most techniques used in current clinical practice still require time-consuming culture for primary isolation of the microbe. We present a new phenotypic assay for MRSA screening. The technique employs a two-photon excited fluorescence (TPX) detection technology with S. aureus-specific antibodies that allows the online monitoring of bacterial growth in a single separation-free process. Different progressions of fluorescence signals are recorded for methicillin-susceptible and -resistant strains when the growth of S. aureus is monitored in the presence of cefoxitin. The performance of the new technique was evaluated with 20 MRSA strains, 6 methicillin-susceptible S. aureus strains, and 7 coagulase-negative staphylococcal strains and two different monoclonal S. aureus-specific antibodies. When either of these antibodies was used, the sensitivity and the specificity of the TPX assay were 100%. All strains were correctly classified within 8 to 12 h, and up to 70 samples were simultaneously analyzed on a single 96-well microtiter plate. As a phenotypic method, the TPX assay is suited for screening purposes. The final definition of methicillin resistance in any S. aureus strain should be based on the presence of the mecA gene. The main benefit afforded by the initial use of the TPX methodology lies in its low cost and applicability to high-throughput analysis.

A novel platform for highly surface-sensitive fluorescent measurements applying simultaneous total internal reflection excitation and super critical angle detection

A highly surface-sensitive fluorescent measurement platform for chemical and biochemical analysis is presented. The core component of the platform is a polystyrene parabolic lens that is utilised in simultaneous total internal reflection excitation and super critical angle fluorescence detection. Both the theoretical calculations and the experimental results of the immunological model assays show that the system is extremely sensitive to the surface bound fluorescence. The binding of analytes at concentrations down to 5 pmol/l has been detected in real time while the unbound fluorescent labels in the sample liquid did not show any response until at three decades higher concentrations.