Multi-wavelength Fluorescence Research Articles

Rare-earth dopant distribution measurement of optical fibers based on filtered back projection

We present a nondestructive measurement based on filtered back projection (FBP) for the distribution of rare-earth dopant in optical fibers. Based on the approach, a measurement system is established, where the single-mode pumped laser illuminates the fiber under test from the side, generating multi-wavelength fluorescence through photoluminescence (PL). The distribution of rare-earth doped particles is proportional to the distribution of fluorescence intensity. The projection images are collected by the CCD camera at 90° relative to the excitation arm. According to the computed tomography (CT), the rotation of the optical fiber enables the experimental measurements of intensity information extracted from multi-angle projections. The rare-earth dopant distribution of the fiber is reconstructed by the improved filtered back projection algorithm, which improves the quality of the reconstructed result by incorporating total variation (TV) algorithm. To evaluate the proposed approach, rare-earth dopant distribution is compared with results measured by EPMA, exhibiting a correlation coefficient above 0.99, and the relationship between the dopant distribution and the refractive index is analyzed. Measurements on doped particle distributions at various wavelengths are conducted, and the fluorescence spectrum are constructed to further analyze the fluorescence characteristics of rare earth doped fiber. Measurement of RE dopant distribution will be useful for quality evaluation and optimization of partial active optical fibers.

Multi-wavelength Fluorescence Monitoring of Faecal Contamination in Waters: A Laboratory-based Quantification

Near real-time monitoring of faecal indicator bacteria (FIB) in waters is currently not feasible, and current monitoring methods require field sampling and laboratory testing that inhibits decision-making within a relevant timeframe. While recent studies identified the potential of using specific fluorescence regions for FIB monitoring, sufficient accuracy often requires site-specific calibration due to minor variations in fluorescence peak locations. In this study, a series of lab experiments were completed to address some of the selectivity issues. Specifically, the study explored correlations between wavelength-specific fluorescence signals acquired through fluorescence excitation-emission matrices (EEM) and the amount of E. coli K-12 (E. coli) and E. faecalis (enterococci) in exponential and stationary phase broth cultures. Subsequently, the experiments quantified how the addition of known concentrations of L-tryptophan amplifies an indole pulse, specifically its concentration and the corresponding fluorescence properties. Results show unique peak excitation/emission (λex/λem) wavelengths (± 5 nm) in EEMs for E. coli cell pellet and in M9 broth (~ 280/ ~ 327 nm), enterococci cell pellet (~ 276/ ~ 324 nm), L-tryptophan (~ 278/ ~ 343 nm and ~ 298/ ~ 344 nm), and indole (~ 232/ ~ 321 nm). The findings demonstrate that L-tryptophan concentrations in E. coli broth were reduced. At the same time, the indole content increased throughout the initiation phase to the stationary phase of the bacteria growth curve, with the peak indole pulse occurring approximately at the time of transition from the exponential to stationary phase. Such unique fluorescence signatures for not only FIB but also indole (whose pulse can be triggered by L-tryptophan) provide foundations for developing reliable and near real-time in situ FIB sensors.

Multi-wavelength excitation-dependent fluorescence with dynamic color gradients for information encryption and anti-counterfeiting

The unprecedented security labels have multi-excitation wavelength triggered triple-mode polychromatic and dynamic color-gradient features, offering a new development direction for future information encryption and anti-counterfeiting technologies.

Multiwavelength Fluorescence and Diffuse Reflectance Spectroscopy for an In Situ Analysis of Kidney Stones

This study explores the use of diffuse reflectance spectroscopy (DRS) and multiwavelength fluorescence spectroscopy for real-time kidney stone identification during laser lithotripsy. Traditional methods are not suitable for in situ analysis, so the research focuses on optical techniques that can be integrated with lithotripsy fibers. Experiments were conducted ex vivo, using DRS and multiwavelength fluorescence spectroscopy (emission–excitation matrix (EEM)) to distinguish between 48 urinary stones of three types: urate, oxalate and hydroxyapatite, with infrared spectroscopy as a reference. A classification model was developed based on EEM and DRS data. Initial classification relying solely on EEM data achieved an f1-score of 87%, which increased to 92% when DRS data were included. The findings suggest that optical spectroscopy can effectively determine stone composition during laser lithotripsy, potentially enhancing surgical outcomes via the real-time automatic optimization of laser radiation parameters.

Recycling of bacterial RNA polymerase by the Swi2/Snf2 ATPase RapA

Free-living bacteria have regulatory systems that can quickly reprogram gene transcription in response to changes in the cellular environment. The RapA ATPase, a prokaryotic homolog of the eukaryotic Swi2/Snf2 chromatin remodeling complex, may facilitate such reprogramming, but the mechanisms by which it does so are unclear. We used multiwavelength single-molecule fluorescence microscopy in vitro to examine RapA function in the Escherichia coli transcription cycle. In our experiments, RapA at <5 nM concentration did not appear to alter transcription initiation, elongation, or intrinsic termination. Instead, we directly observed a single RapA molecule bind specifically to the kinetically stable post termination complex (PTC)-consisting of core RNA polymerase (RNAP)-bound sequence nonspecifically to double-stranded DNA-and efficiently remove RNAP from DNA within seconds in an ATP-hydrolysis-dependent reaction. Kinetic analysis elucidates the process through which RapA locates the PTC and the key mechanistic intermediates that bind and hydrolyze ATP. This study defines how RapA participates in the transcription cycle between termination and initiation and suggests that RapA helps set the balance between global RNAP recycling and local transcription reinitiation in proteobacterial genomes.

Advancing 2D fluorescence online monitoring in microtiter plates by separating scattered light and fluorescence measurement, using a tunable emission monochromator.

Online fluorescence monitoring has become a key technology in modern bioprocess development, as it provides in-depth process knowledge at comparably low costs. In particular, the technology is widely established for high-throughput microbioreactor cultivation systems, due to its noninvasive character. For microtiter plates, previously also multi-wavelength 2D fluorescence monitoring was developed. To overcome an observed limitation of fluorescence sensitivity, this study presents a modified spectroscopic setup, including a tunable emission monochromator. The new optical component enables the separation of the scattered and fluorescent light measurements, which allows for the adjustment of integration times of the charge-coupled devicedetector. The resulting increased fluorescence sensitivity positively affected the performance of principal component analysisfor spectral data of Escherichiacoli batch cultivation experiments with varying sorbitol concentration supplementation. In direct comparison with spectral data recorded at short integration times, more biologically consistent signal dynamics were calculated. Furthermore, during partial least squareregression for E. coli cultivation experiments with varying glucose concentrations, improved modeling performance was observed. Especially, for the growth-uncoupled acetate concentration, a considerable improvement of the root-mean-square errorfrom 0.25to 0.17 g/L was achieved. In conclusion, the modified setup represents another important step in advancing 2D fluorescence monitoring in microtiter plates.

Design and construction of a microfluidics workstation for high-throughput multi-wavelength fluorescence and transmittance activated droplet analysis and sorting.

Droplet microfluidics has revolutionized quantitative high-throughput bioassays and screening, especially in the field of single-cell analysis where applications include cell characterization, antibody discovery and directed evolution. However, droplet microfluidic platforms capable of phenotypic, fluorescence-based readouts and sorting are still mostly found in specialized labs, because their setup is complex. Complementary to conventional FACS, microfluidic droplet sorters allow the screening of cell libraries for secreted factors, or even for the effects of secreted or surface-displayed factors on a second cell type. Furthermore, they also enable PCR-activated droplet sorting for the isolation of genetic material harboring specific markers. In this protocol, we provide a detailed step-by-step guide for the construction of a high-throughput droplet analyzer and sorter, which can be accomplished in ~45 working hours by nonspecialists. The resulting instrument is equipped with three lasers to excite the fluorophores in droplets and photosensors that acquire fluorescence signals in the blue (425-465 nm), green (505-545 nm) and red (580-630 nm) spectrum. This instrument also allows transmittance-activated droplet sorting by analyzing the brightfield light intensity transmitting through the droplets. The setup is validated by sorting droplets containing fluorescent beads at 200 Hz with 99.4% accuracy. We show results from an experiment where droplets hosting single cells were sorted on the basis of increased matrix metalloprotease activity as an application of our workstation in single-cell molecular biology, e.g., to analyze molecular determinants of cancer metastasis.

A validated eco-friendly HPLC-FLD for analysis of the first approved antiviral remdesivir with other potential add-on therapies for COVID-19 in human plasma and pharmaceuticals.

It is crucial to have a reliable and sensitive method for separating common drugs used in SARS-CoV-2 pneumonia treatment protocols for ongoing treatment and upcoming investigations. This study presents an HPLC-FLD approach to analyze three co-administered medicines - remdesivir (RDV), hydroxychloroquine sulphate (HCQ), and levofloxacin hemihydrate (LVX) - in their pure forms, pharmaceutical preparations, and spiked human plasma. The HPLC-FLD analysis was conducted using a Symmetry® C18 column (100 mm × 4.6 mm ID, 3.5 μm particle size) at 40 °C, with (A) an aqueous mixture of 0.02 M phosphate buffer and 0.2% heptane-1-sulphonic acid sodium solutions (50 : 50) adjusted to pH 3, (B) acetonitrile, and (C) methanol as the mobile phase. The injection volume was 10 μL, and the flow rate was 1.5 mL min-1. The detection was done using a multi-wavelength excitation and emission fluorescence detector, with individual optimization for each drug. The drug separation time was less than 10 minutes, and the method showed sensitive and wide linearity ranges for all medicines, with r2 values of more than 0.999. The impact of the mobile phase pH and flow rate on suitability parameters (retention time and number of theoretical plates) was studied. The method was found to be environmentally friendly based on GAPI and AGREE metrics. The validity of the method was evaluated following ICH and FDA guidelines.

Deep learning and multiwavelength fluorescence imaging for cleanliness assessment and disinfection in Food Services

Precise, reliable, and speedy contamination detection and disinfection is an ongoing challenge for the food-service industry. Contamination in food-related services can cause foodborne illness, endangering customers and jeopardizing provider reputations. Fluorescence imaging has been shown to be capable of identifying organic residues and biofilms that can host pathogens. We use new fluorescence imaging technology, applying Xception and DeepLabv3+ deep learning algorithms to identify and segment contaminated areas in images of equipment and surfaces. Deep learning models demonstrated a 98.78% accuracy for differentiation between clean and contaminated frames on various surfaces and resulted in an intersection over union (IoU) score of 95.13% for the segmentation of contamination. The portable imaging system’s intrinsic disinfection capability was evaluated on S. enterica, E. coli, and L. monocytogenes, resulting in up to 8-log reductions in under 5 s. Results showed that fluorescence imaging with deep learning algorithms could help assure safety and cleanliness in the food-service industry.

Online 2D Fluorescence Monitoring in Microtiter Plates Allows Prediction of Cultivation Parameters and Considerable Reduction in Sampling Efforts for Parallel Cultivations of Hansenula polymorpha

Multi-wavelength (2D) fluorescence spectroscopy represents an important step towards exploiting the monitoring potential of microtiter plates (MTPs) during early-stage bioprocess development. In combination with multivariate data analysis (MVDA), important process information can be obtained, while repetitive, cost-intensive sample analytics can be reduced. This study provides a comprehensive experimental dataset of online and offline measurements for batch cultures of Hansenula polymorpha. In the first step, principal component analysis (PCA) was used to assess spectral data quality. Secondly, partial least-squares (PLS) regression models were generated, based on spectral data of two cultivation conditions and offline samples for glycerol, cell dry weight, and pH value. Thereby, the time-wise resolution increased 12-fold compared to the offline sampling interval of 6 h. The PLS models were validated using offline samples of a shorter sampling interval. Very good model transferability was shown during the PLS model application to the spectral data of cultures with six varying initial cultivation conditions. For all the predicted variables, a relative root-mean-square error (RMSE) below 6% was obtained. Based on the findings, the initial experimental strategy was re-evaluated and a more practical approach with minimised sampling effort and elevated experimental throughput was proposed. In conclusion, the study underlines the high potential of multi-wavelength (2D) fluorescence spectroscopy and provides an evaluation workflow for PLS modelling in microtiter plates.

Monosaccharide-mediated rational synthesis of a universal plasmonic platform with broad spectral fluorescence enhancement for high-sensitivity cancer biomarker analysis

BackgroundEffective and accurate screening of oncological biomarkers in peripheral blood circulation plays an increasingly vital role in diagnosis and prognosis. High-sensitivity assays can effectively aid clinical decision-making and intervene in cancer in a localized status before they metastasize and become unmanageable. Meanwhile, it is equally pivotal to prevent overdiagnosis of non-life-threatening cancer by eliminating unnecessary treatment and repeated blood draws. Unfortunately, current clinical screening methodologies can hardly simultaneously attain sufficient sensitivity and specificity, especially under resource-restrained circumstances. To circumvent such limitations, particularly for cancer biomarkers from early-onset and recurrence, we aim to develop a universal plasmonic platform for clinical applications, which macroscopically amplifies multiplexed fluorescence signals in a broad spectral window and readily adapts to current assay setups without sophisticated accessories or expertise at low cost.MethodsThe plasmonic substrate was chemically synthesized in situ at the solid–liquid interface by rationally screening a panel of reducing monosaccharides and tuning the redox reactions at various catalyst densities and precursor concentrations. The redox properties were studied by Benedict’s assay and electrochemistry. We systemically characterized the morphologies and optical properties of the engineered plasmonic Ag structures by scanning electron microscopy (SEM) and spectroscopy. The structure-fluorescence enhancement correlation was explicitly explained by the finite-difference time-domain (FDTD) simulation and a computational model for gap distribution. Next, we established an enhanced fluoroimmunoassay (eFIA) using a model biomarker for prostate cancer (PCa) and validated it in healthy and PCa cohorts. Prognosis was explored in patients subject to surgical and hormonal interventions following recommended PCa guidelines.ResultsThe monosaccharide-mediated redox reaction yielded a broad category of Ag structures, including sparsely dispersed nanoparticles (NPs) of various sizes, semi-continuous nanoislands, and crackless continuous films. Optimal broad-spectral fluorescence enhancement from green to far-red was observed for the inhomogeneous, irregularly-shaped semi-continuous Ag nanoisland substrate (AgNIS), synthesized from a well-balanced redox reaction at a stable rate mediated by mannose. In addition, different local electric field intensity distributions in response to various incident excitations were observed at the nanoscale, elucidating the need for irregular and inhomogeneous structures. AgNIS enabled a maximized 54.7-fold macroscopically amplified fluorescence and long-lasting photostability. Point-of-care availability was fulfilled using a customized smartphone prototype with well-paired optics. The eFIA effectively detected the PCa marker in cell lines, xenograft tumors, and patient sera. The plasmonic platform rendered a diagnostic sensitivity of 86.0% and a specificity of 94.7% and capably staged high-grade PCa that the clinical gold standard test failed to stratify. Patient prognosis of robotic-assisted surgeries and hormone therapies was non-invasively monitored following efficient medical interventions. The assay time was significantly curtailed on the plasmonic platform upon microwave irradiation.ConclusionsBy investigating the effects of reducing monosaccharides on the seed-mediated chemical synthesis of plasmonic Ag structures, we deduced that potent multiplexed fluorescence enhancement originated from both an adequate reducing power and a steady reduction rate. Furthermore, the inhomogeneous structure with adequate medium gap distances afforded optimal multiwavelength fluorescence enhancement, thus empowering an effective eFIA for PCa. The clinically validated diagnostic and prognostic features, along with the low sample volume, point-of-care feasibility with a smartphone, and microwave-shortened assay time, warrant its potential clinical translation for widespread cancer biomarker analysis.Graphical

Bayesian machine learning analysis of single-molecule fluorescence colocalization images.

Multi-wavelength single-molecule fluorescence colocalization (CoSMoS) methods allow elucidation of complex biochemical reaction mechanisms. However, analysis of CoSMoS data is intrinsically challenging because of low image signal-to-noise ratios, non-specific surface binding of the fluorescent molecules, and analysis methods that require subjective inputs to achieve accurate results. Here, we use Bayesian probabilistic programming to implement Tapqir, an unsupervised machine learning method that incorporates a holistic, physics-based causal model of CoSMoS data. This method accounts for uncertainties in image analysis due to photon and camera noise, optical non-uniformities, non-specific binding, and spot detection. Rather than merely producing a binary 'spot/no spot' classification of unspecified reliability, Tapqir objectively assigns spot classification probabilities that allow accurate downstream analysis of molecular dynamics, thermodynamics, and kinetics. We both quantitatively validate Tapqir performance against simulated CoSMoS image data with known properties and also demonstrate that it implements fully objective, automated analysis of experiment-derived data sets with a wide range of signal, noise, and non-specific binding characteristics.

A Naked Eye-Invisible Ratiometric Fluorescent Microneedle Tattoo for Real-Time Monitoring of Inflammatory Skin Conditions.

The field of portable healthcare monitoring devices has an urgent need for the development of real-time, noninvasive sensing and detection methods for various physiological analytes. Currently, transdermal sensing techniques are severely limited in scope (i.e., measurement of heart rate or sweat composition), or else tend to be invasive, often needing to be performed in a clinical setting. This study proposes a minimally invasive alternative strategy, consisting of using dissolving polymeric microneedles to deliver naked eye-invisible functional fluorescent ratiometric microneedle tattoos directly to the skin for real-time monitoring and quantification of physiological and pathological parameters. Reactive oxygen species are overexpressed in the skin in association with various pathological conditions. Here, one demonstrates for the first time the microneedle-based delivery to the skin of active fluorescent sensors in the form of an invisible, ratiometric microneedle tattoo capable of sensing reactive oxygen species in a reconstructed human-based skin disease model, as well as an in vivo model of UV-induced dermal inflammation. One also elaborates a universal ratiometric quantification concept coupled with a custom-built, multiwavelength portable fluorescence detection system. Fully realized, this approach presents an opportunity for the minimally invasive monitoring of a broad range of physiological parameters through the skin.

Comprehensive identification and distribution pattern of 37 oxygenated heterocyclic compounds in commercially important citrus juices

Oxygenated heterocyclic compounds (OHCs) are among the most biologically active components in citrus fruits which exert health benefits but also involve juice-drug interactions. However, current knowledge of OHC distribution in citrus juices is incomplete. By analyzing 173 samples from orange, mandarin, grapefruit, pomelo, lemon, and lime juices, this study constructed a comprehensive OHC (10 methoxyflavones, 12 coumarins, 15 furanocoumarins) compositional pattern of the six commercially important species. OHCs were determined using a newly developed high-resolution HPLC separation with UV and fluorescence detection. Identifications were based on matching an innovative combination of full UV and fluorescence spectra, multiwavelength fluorescence emission peak ratios, and alkyl aryl ketone retention index values with those of standards. Forty-six previously unreported OHC occurrences (12 methoxyflavones, 27 coumarins and 9 furanocoumarins) were identified in the six juice-species. Unique OHC markers were identified in mandarin (5-demethyltangeretin), pomelo (meranzin), and lime (heraclenol) juices. Orange and mandarin juices are characterized by two and five methoxyflavones respectively; pomelo and grapefruit by 5-substituted furanocoumarins and 7,8-disubstituted coumarins; lemon and lime by byakangelicin, 8-geranyloxypsoralen, and 5-geranoxy-7-methoxycoumarin. The greatly expanded and detailed OHC distribution pattern provides additional information for future epidemiological work investigating the relationships between citrus juice OHCs intake and health outcomes.

Rapid Characterization and Quantification of Extracellular Vesicles by Fluorescence-Based Microfluidic Diffusion Sizing.

Extracellular vesicles (EVs) are emerging as promising diagnostic and therapeutic tools for a variety of diseases. The characterization of EVs requires a series of orthogonal techniques that are overall time- and material-consuming. Here, a microfluidic device is presented that exploits the combination of diffusion sizing and multiwavelength fluorescence detection to simultaneously provide information on EV size, concentration, and composition. The latter is achieved with the nonspecific staining of lipids and proteins combined with the specific staining of EV markers such as EV-associated tetraspanins via antibodies. The device can be operated as a single-step immunoassay thanks to the integrated separation and quantification of free and EV-bound fluorophores. This microfluidic technique is capable of detecting and quantifying components associated to EV subtypes and impurities and thus to measure EV purity in a time scale of minutes, requiring less than 5 µL of sample and minimal sample handling before the analysis. Moreover, the analysis is performed directly in solution without immobilization steps. Therefore, this method can accelerate screening of EV samples and aid the evaluation of sample reproducibility, representing an important complementary tool to the current array of biophysical methods for EV characterization, particularly valuable for instance for bioprocess development.

Iron availability allows sustained cyanobacterial blooms: a dual-lake case study

ABSTRACT Lakes dominated by cyanobacterial harmful algal blooms (CyanoHABs) are geographically widespread. These blooms are associated with fish kills and toxin production and have negative impacts on human health. There is compelling evidence that, in addition to nitrogen and phosphorus, iron (Fe) can also regulate CyanoHABs. Low Fe concentration (tens of ng L−1) has been documented to limit CyanoHABs in freshwaters, whereas high Fe concentration (hundreds of μg L−1) can promote CyanoHABs. However, the extent to which Fe influences CyanoHABs in lakes that lack an extensive anoxic zone and where the Fe concentration ranges from tens to <400 μg L−1 is unclear. The aim of this study was to assess Fe trends, measured as dissolved Fe (DFe), associated with cyanobacteria dynamics in lakes in northwestern Iowa. We utilised a multiwavelength fluorometer to monitor and classify phytoplankton based on their unique fluorescence spectra from chlorophyll a (Chl-a) and accessory pigments. Here we report a declining DFe trend (drawdown) potentially due to phytoplankton uptake in East Okoboji and its effect on CyanoHABs. Multiwavelength fluorescence data show cyanobacteria peaked (>50% cyanobacteria Chl-a) during a DFe minimum. The DFe is inversely correlated with phosphorus, suggesting decoupled Fe and phosphorus cycling. The strong positive correlation between phosphorus and cyanobacteria Chl-a supports a role for phosphorus in triggering CyanoHABs. This study suggests an underlying mechanistic role of Fe on CyanoHABs; the intermediate range of Fe concentrations investigated here may be sufficient to sustain CyanoHABs.

Integrated multi-wavelength microscope combining TIRFM and IRM modalities for imaging cellulases and other processive enzymes.

We describe a multimodal microscope for visualizing processive enzymes moving on immobilized substrates. The instrument combines interference reflection microscopy (IRM) with multi-wavelength total internal reflectance fluorescence microscopy (TIRFM). The microscope can localize quantum dots with a precision of 2.8 nm at 100 frames/s, and was used to image the dynamics of the cellulase, Cel7a interacting with surface-immobilized cellulose. The instrument, which was built with off-the-shelf components and is controlled by custom software, is suitable for tracking other degradative enzymes such as collagenases, as well as motor proteins moving along immobilized tracks.

Bayesian Classification and Modeling of Single Molecule Fluorescence Colocalization Images

Multi-wavelength single-molecule fluorescence colocalization methods allow elucidation of complex biochemical reaction mechanisms. However, analysis of colocalization data is an intrinsically challenging and time-consuming problem for multiple reasons. First, to minimize dye photobleaching, images frequently are collected at low signal-to-noise ratios, making it challenging to discriminate real fluorescent spots from noise. Second, transient non-specific interactions of the binder molecule with the surface of the microscope slide can give rise to both false-positive and false-negative detection.

Multi-wavelength fluorescence polarization immunoassays for simultaneous detection of amantadine and ribavirin in chicken and human serum

ABSTRACTIn this study, a homologous multi-wavelength fluorescence polarization immunoassays (MWFPIA) for rapid determination of amantadine (AMD) and ribavirin (RVB) simultaneously was established. The MWFPIA could quantify AMD and RVB concentration simultaneously in 1 min. After optimisation, long-wavelength tracers AEDA-AF647 with monoclonal antibody (Mab) 1A12 and RVB-CP-EDF with polyclonal antibody (Pab) G412 were chosen for the development of MWFPIA. The limit of detections (LODs) of AMD and RVB were 1.7 and 1.0 μg/kg in chickens, and 17.6 and 10.4 μg/L in human serum, respectively. Recoveries of AMD and RVB in chickens were 61.2–94.9% and 87.3–90.2%, respectively with coefficient of variations (CVs) lower than 16.7%. For human serum, recoveries were 60.2–84.3% and 63.5–74.2% for AMD and RVB, respectively, and CVs was lower than 15.3%. The results indicated that the MWFPIA could be used for the monitoring of antiviral drugs with short analysis time, great accuracy and high throughput.

Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription

In eukaryotes, RNA polymerase II (RNApII) transcribes messenger RNA from template DNA. Decades of experiments have identified the proteins needed for transcription activation, initiation complex assembly, and productive elongation. However, the dynamics of recruitment of these proteins to transcription complexes, and of the transitions between these steps, are poorly understood. We used multiwavelength single-molecule fluorescence microscopy to directly image and quantitate these dynamics in a budding yeast nuclear extract that reconstitutes activator-dependent transcription in vitro. A strong activator (Gal4-VP16) greatly stimulated reversible binding of individual RNApII molecules to template DNA. Binding of labeled elongation factor Spt4/5 to DNA typically followed RNApII binding, was NTP dependent, and was correlated with association of mRNA binding protein Hek2, demonstrating specificity of Spt4/5 binding to elongation complexes. Quantitative kinetic modeling shows that only a fraction of RNApII binding events are productive and implies a rate-limiting step, probably associated with recruitment of general transcription factors, needed to assemble a transcription-competent preinitiation complex at the promoter. Spt4/5 association with transcription complexes was slowly reversible, with DNA-bound RNApII molecules sometimes binding and releasing Spt4/5 multiple times. The average Spt4/5 residence time was of similar magnitude to the time required to transcribe an average length yeast gene. These dynamics suggest that a single Spt4/5 molecule remains associated during a typical transcription event, yet can dissociate from RNApII to allow disassembly of abnormally long-lived (i.e., stalled) elongation complexes.