Rapid, label-free antibiotic susceptibility determined directly from positive blood culture.

Bacterial bloodstream infections are a significant cause of global morbidity and mortality. Constrained by low bacterial burdens of 1-100 colony-forming-units per ml blood (CFU/ml), clinical diagnosis relies on lengthy culture amplification and isolation steps prior to identification and antibiotic susceptibility testing (AST). The resulting >60-h time to actionable treatment not only negatively impacts patient outcomes, but also increases the misuse and overuse of broad-spectrum antibiotics that accelerates the rise in multidrug resistant infections. Consequently, the development of novel technologies capable of rapidly recovering bacteria from blood-derived samples is crucial to human health. To address this need, we report a novel bacterial recovery technology from positive blood cultures that couples selective hemolysis with centrifugation through a sucrose cushion to perform rapid, background-free cytometric ASTs without long subculturing steps. Demonstrated on the most common bloodstream infection-causing bacteria: Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, near-pure bacteria are rapidly recovered (≤15 min) with minimal user intervention. Susceptibilities of recovered bacteria are readily performed via high throughput flow cytometry with excellent agreement with much slower, standard microbroth dilution assays. Altogether, this novel direct-from-positive blood culture AST technology enables susceptibility determinations within as little as 5 h, post blood culture positivity.

Facile autofluorescence suppression enabling tracking of single viruses in live cells.

Live cell fluorescence imaging is the method of choice for studying dynamic processes, such as nuclear transport, vesicular trafficking, and virus entry and egress. However, endogenous cellular autofluorescence masks a useful fluorescence signal, limiting the ability to reliably visualize low-abundance fluorescent proteins. Here, we employed synchronously amplified fluorescence image recovery (SAFIRe), which optically alters ground versus photophysical dark state populations within fluorescent proteins to modulate and selectively detect their background-free emission. Using a photoswitchable rsFastLime fluorescent protein combined with a simple illumination and image-processing scheme, we demonstrate the utility of this approach for suppressing undesirable, unmodulatable fluorescence background. Significantly, we adapted this technique to different commercial wide-field and spinning-disk confocal microscopes, obtaining >10-fold improvements in signal to background. SAFIRe allowed visualization of rsFastLime targeted to mitochondria by efficiently suppressing endogenous autofluorescence or overexpressed cytosolic unmodulatable EGFP. Suppression of the overlapping EGFP signal provided a means to perform multiplexed imaging of rsFastLime and spectrally overlapping fluorophores. Importantly, we used SAFIRe to reliably visualize and track single rsFastLime-labeled HIV-1 particles in living cells exhibiting high and uneven autofluorescence signals. Time-lapse SAFIRe imaging can be performed for an extended period of time to visualize HIV-1 entry into cells. SAFIRe should be broadly applicable for imaging live cell dynamics with commercial microscopes, even in strongly autofluorescent cells or cells expressing spectrally overlapping fluorescent proteins.

Triplet Shelving in Fluorescein and Its Derivatives Provides Delayed, Background-Free Fluorescence Detection.

Fluorescence from the xanthene dyes rose bengal, erythrosine B, eosin Y, and fluorescein is modulated by reversibly optically populating and depopulating their long-lived triplet states through coillumination with a second, long-wavelength laser. Here, we show that repumping the S1 state from the triplet generates strong, nanosecond-lived optically activated delayed fluorescence (OADF), microseconds to milliseconds after primary pulsed excitation. This time-delayed emission upon long-wavelength illumination generates fluorescence after triplet shelving and is a major contribution to fluorescence enhancement/modulation. The time-delayed and background-free OADF component is further increased using a >1 µs burst continuous wave excitation scheme to increase the steady-state triplet populations, yielding strong OADF even from strongly emissive fluorescein. Because emission is delayed long after the high-energy primary excitation, yellow-orange fluorescence is readily observed on zero background. As OADF generation depends on the triplet quantum yields and the reverse intersystem crossing rates, we directly probe the usually difficult-to-measure photophysics, create new zero-background detection schemes, and increase OADF through tailored excitation schemes, all improving sensitivity. The excellent match between experiments and simulations demonstrates the promise of these studies for OADF characterization, while enabling us to determine that OADF (in contrast to ground-state recovery and re-excitation) is the major component of fluorescence enhancement for xanthenes studied with triplet quantum yields exceeding 0.1.

Atomic Structure of a Fluorescent Ag8 Cluster Templated by a Multistranded DNA Scaffold.

Multinuclear silver clusters encapsulated by DNA exhibit size-tunable emission spectra and rich photophysics, but their atomic organization is poorly understood. Herein, we describe the structure of one such hybrid chromophore, a green-emitting Ag8 cluster arranged in a Big Dipper-shape bound to the oligonucleotide A2C4. Three 3' cytosine metallo-base pairs stabilize a parallel A-form-like duplex with a 5' adenine-rich pocket, which binds a metallic, trapezoidal-shaped Ag5 moiety via Ag-N bonds to endo- and exocyclic nitrogens of cytosine and adenine. The unique DNA configuration, constrained coordination environment, and templated Ag8 cluster arrangement highlight the reciprocity between the silvers and DNA in adopting this structure. These first atomic details of a DNA-encapsulated Ag cluster fluorophore illuminate many aspects of biological assembly, nanoscience, and metal cluster photophysics.

Optically Activated Delayed Fluorescence through Control of Cyanine Dye Photophysics.

Merocyanine 540 fluorescence can be enhanced by optically depopulating dark photoisomer states to regenerate the fluorescence-generating manifold of the all-trans isomer. Here, we utilize a competing modulation route, long-wavelength coexcitation of the trans triplet population to not only modulate fluorescence through enhanced ground-state recovery but also generate optically activated delayed fluorescence (OADF) with longer-wavelength co-illumination. Such OADF (∼580 nm) is directly observed with pulsed fluorescence excitation at 532 nm, followed by long-wavelength (637 nm) continuous wave depopulation of the photogenerated triplet by repopulating the emissive S1 state. Such reverse intersystem crossing (RISC) results in ns-lived fluorescence delayed by several microseconds after the initial primary excitation pulse and the prompt 1 ns-lived fluorescence that it induces. The dark state from which OADF is generated decays more rapidly with increased secondary laser intensity, as the optically induced RISC rate increases. This first OADF from organic dyes is observed, as the red secondary laser excites ∼580 nm, <1 ns-lived fluorescence from the previously optically prepared ∼1 µs-lived triplet state. This sequential two-photon, repumped fluorescence yields background-free collection with potential for new high-sensitivity fluorescence imaging schemes.

FAST: Rapid determinations of antibiotic susceptibility phenotypes using label-free cytometry.

Sepsis, a life-threatening immune response to blood infections (bacteremia), has a ∼30% mortality rate and is the 10th leading cause of US hospital deaths. The typical bacterial loads in adult septic patients are ≤100 bacterial cells (colony forming units, CFU) per ml blood, while pediatric patients exhibit only ∼1000 CFU/ml. Due to the low numbers, bacteria must be propagated through ∼24-hours blood cultures to generate sufficient CFUs for diagnosis and further analyses. Herein, we demonstrate that, unlike other rapid post-blood culture antibiotic susceptibility tests (ASTs), our phenotypic approach can drastically accelerate ASTs for the most common sepsis-causing gram-negative pathogens by circumventing long blood culture-based amplification. For all blood isolates of multi-drug resistant pathogens investigated (Escherichia coli, Klebsiella pneumoniae, and Acinetobacter nosocomialis), effective antibiotic(s) were readily identified within the equivalent of 8 hours from initial blood draw using <0.5 mL of adult blood per antibiotic. These methods should drastically improve patient outcomes by significantly reducing time to actionable treatment information and reduce the incidence of antibiotic resistance. © 2018 International Society for Advancement of Cytometry.

Optically Modulated Photoswitchable Fluorescent Proteins Yield Improved Biological Imaging Sensitivity.

Photoswitchable fluorescent proteins (PS-FPs) open grand new opportunities in biological imaging. Through optical manipulation of FP emission, we demonstrate that dual-laser modulated synchronously amplified fluorescence image recovery (DM-SAFIRe) improves signal contrast in high background through unambiguous demodulation and is linear in relative fluorophore abundance at different points in the cell. The unique bright-to-dark state interconversion rates of each PS-FP not only enables discrimination of different, yet spectrally indistinguishable FPs, but also allows signal rejection of diffusing relative to bound forms of the same PS-FP, rsFastLime. Adding to the sensitivity gains realized from rejecting non-modulatable background, the selective signal recovery of immobilized vs diffusing intracellular rsFastLime suggests that DM-SAFIRe can detect weak protein-protein interactions that are normally obscured by large fractions of unbound FPs.

Rapid cytometric antibiotic susceptibility testing utilizing adaptive multidimensional statistical metrics.

Flow cytometry holds promise to accelerate antibiotic susceptibility determinations; however, without robust multidimensional statistical analysis, general discrimination criteria have remained elusive. In this study, a new statistical method, probability binning signature quadratic form (PB-sQF), was developed and applied to analyze flow cytometric data of bacterial responses to antibiotic exposure. Both sensitive lab strains (Escherichia coli and Pseudomonas aeruginosa) and a multidrug resistant, clinically isolated strain (E. coli) were incubated with the bacteria-targeted dye, maltohexaose-conjugated IR786, and each of many bactericidal or bacteriostatic antibiotics to identify changes induced around corresponding minimum inhibition concentrations (MIC). The antibiotic-induced damages were monitored by flow cytometry after 1-h incubation through forward scatter, side scatter, and fluorescence channels. The 3-dimensional differences between the flow cytometric data of the no-antibiotic treated bacteria and the antibiotic-treated bacteria were characterized by PB-sQF into a 1-dimensional linear distance. A 99% confidence level was established by statistical bootstrapping for each antibiotic-bacteria pair. For the susceptible E. coli strain, statistically significant increments from this 99% confidence level were observed from 1/16x MIC to 1x MIC for all the antibiotics. The same increments were recorded for P. aeruginosa, which has been reported to cause difficulty in flow-based viability tests. For the multidrug resistant E. coli, significant distances from control samples were observed only when an effective antibiotic treatment was utilized. Our results suggest that a rapid and robust antimicrobial susceptibility test (AST) can be constructed by statistically characterizing the differences between sample and control flow cytometric populations, even in a label-free scheme with scattered light alone. These distances vs paired controls coupled with rigorous statistical confidence limits offer a new path toward investigating initial biological responses, screening for drugs, and shortening time to result in antimicrobial sensitivity testing.

Optically modulated fluorescence bioimaging: visualizing obscured fluorophores in high background.

Fluorescence microscopy and detection have become indispensible for understanding organization and dynamics in biological systems. Novel fluorophores with improved brightness, photostability, and biocompatibility continue to fuel further advances but often rely on having minimal background. The visualization of interactions in very high biological background, especially for proteins or bound complexes at very low copy numbers, remains a primary challenge. Instead of focusing on molecular brightness of fluorophores, we have adapted the principles of high-sensitivity absorption spectroscopy to improve the sensitivity and signal discrimination in fluorescence bioimaging. Utilizing very long wavelength transient absorptions of kinetically trapped dark states, we employ molecular modulation schemes that do not simultaneously modulate the background fluorescence. This improves the sensitivity and ease of implementation over high-energy photoswitch-based recovery schemes, as no internal dye reference or nanoparticle-based fluorophores are needed to separate the desired signals from background. In this Account, we describe the selection process for and identification of fluorophores that enable optically modulated fluorescence to decrease obscuring background. Differing from thermally stable photoswitches using higher-energy secondary lasers, coillumination at very low energies depopulates transient dark states, dynamically altering the fluorescence and giving characteristic modulation time scales for each modulatable emitter. This process is termed synchronously amplified fluorescence image recovery (SAFIRe) microscopy. By understanding and optically controlling the dye photophysics, we selectively modulate desired fluorophore signals independent of all autofluorescent background. This shifts the fluorescence of interest to unique detection frequencies with nearly shot-noise-limited detection, as no background signals are collected. Although the fluorescence brightness is improved slightly, SAFIRe yields up to 100-fold improved signal visibility by essentially removing obscuring, unmodulated background (Richards, C. I.; J. Am. Chem. Soc. 2009, 131, 4619). While SAFIRe exhibits a wide, linear dynamic range, we have demonstrated single-molecule signal recovery buried within 200 nM obscuring dye. In addition to enabling signal recovery through background reduction, each dye exhibits a characteristic modulation frequency indicative of its photophysical dynamics. Thus, these characteristic time scales offer opportunities not only to expand the dimensionality of fluorescence imaging by using dark-state lifetimes but also to distinguish the dynamics of subpopulations on the basis of photophysical versus diffusional time scales, even within modulatable populations. The continued development of modulation for signal recovery and observation of biological dynamics holds great promise for studying a range of transient biological phenomena in natural environments. Through the development of a wide range of fluorescent proteins, organic dyes, and inorganic emitters that exhibit significant dark-state populations under steady-state illumination, we can drastically expand the applicability of fluorescence imaging to probe lower-abundance complexes and their dynamics.

Optically modulatable blue fluorescent proteins.

Blue fluorescent proteins (BFPs) offer visualization of protein location and behavior, but often suffer from high autofluorescent background and poor signal discrimination. Through dual-laser excitation of bright and photoinduced dark states, mutations to the residues surrounding the BFP chromophore enable long-wavelength optical modulation of BFP emission. Such dark state engineering enables violet-excited blue emission to be increased upon lower energy, green coillumination. Turning this green coillumination on and off at a specific frequency dynamically modulates collected blue fluorescence without generating additional background. Interpreted as transient photoconversion between neutral cis and anionic trans chromophoric forms, mutations tune photoisomerization and ground state tautomerizations to enable long-wavelength depopulation of the millisecond-lived, spectrally shifted dark states. Single mutations to the tyrosine-based blue fluorescent protein T203V/S205V exhibit enhanced modulation depth and varied frequency. Importantly, analogous single point mutations in the nonmodulatable BFP, mKalama1, creates a modulatable variant. Building modulatable BFPs offers opportunities for improved BFP signal discrimination vs background, greatly enhancing their utility.

Modulated fluorophore signal recovery buried within tissue mimicking phantoms.

Optically modulated fluorescence from ∼140 nM Cy5 is visualized when embedded up to 6 mm within skin tissue mimicking phantoms, even in the presence of overwhelming background fluorescence and scatter. Experimental and finite element analysis (FEA)-based computational models yield excellent agreement in signal levels and predict biocompatible temperature changes. Using synchronously amplified fluorescence image recovery (SAFIRe), dual-laser excitation (primary laser: λ = 594 nm, 0.29 kW/cm(2); secondary laser: λ = 710 nm, 5.9 kW/cm(2), intensity-modulated at 100 Hz) simultaneously excites fluorescence and dynamically optically reverses the dark state buildup of primary laser-excited Cy5 molecules. As the modulated secondary laser both directly modulates Cy5 emission and is of lower energy than the collected Cy5 fluorescence, modulated Cy5 fluorescence in phantoms is free of obscuring background emission. The modulated fluorescence emission due to the secondary laser was recovered by Fourier transformation, yielding a specific and unique signature of the introduced fluorophores, with largely background-free detection, at excitation intensities close to the maximum permissible exposure (MPE) for skin. Experimental and computational models agree to within 8%, validating the computational model. As modulated fluorescence depends on the presence of both lasers, depth information as a function of focal position is also readily obtained from recovered modulated signal strength.

Developing luminescent silver nanodots for biological applications.

Though creation and characterization of water soluble luminescent silver nanodots were achieved only in the past decade, a large variety of emitters in diverse scaffolds have been reported. Photophysical properties approach those of semiconductor quantum dots, but relatively small sizes are retained. Because of these properties, silver nanodots are finding ever-expanding roles as probes and biolabels. In this critical review we revisit the studies on silver nanodots in inert environments and in aqueous solutions. The recent advances detailing their chemical and physical properties of silver nanodots are highlighted with an effort to decipher the relations between their chemical/photophysical properties and their structures. The primary results about their biological applications are discussed here as well, especially relating to their chemical and photophysical behaviours in biological environments (216 references).

Fluorescence-informed photoacoustic discrimination of multiple chromophores by lifetime mapping optically gated responses.

Synchronously Amplified Photoacoustic Image Recovery (SAPhIRe) offers improved background suppression using non-linear properties of modulatable contrast agents. Using SAPhIRe, multiple contrast agents in the same absorption window can be detected independently based on their unique triplet-state lifetimes. Here, we have demonstrated the unmixing of rose bengal and eosin Y signals from solution based on triplet-state lifetime mapping using both fluorescence and photoacoustics. Varying the pump-probe delay enables resolution and recovery of fast-decaying rose bengal and of slowly decaying eosin Y modulated photoacoustic signals, resulting from optically gated triplet state residence. Distinct images were reconstructed within tissue-mimicking phantom using the fitting coefficients of triplet-state lifetimes. Fluorescence was used to screen for modulation prior to photoacoustic imaging. The results suggest that lifetime unmixing can be utilized to simultaneously detect multiple pathologies with overlapping spectra using photoacoustic imaging.

Sequential Two-Photon Delayed Fluorescence Anisotropy for Macromolecular Size Determination.

Time-resolved fluorescence anisotropy (FA) uses the fluorophore depolarization rate to report on rotational diffusion, conformation changes, and intermolecular interactions in solution. Although FA is a rapid, sensitive, and nondestructive tool for biomolecular interaction studies, the short (∼ns) fluorescence lifetime of typical dyes largely prevents the application of FA on larger macromolecular species and complexes. By using triplet shelving and recovery of optical excitation, we introduce optically activated delayed fluorescence anisotropy (OADFA) measurements using sequential two-photon excitation, effectively stretching fluorescence anisotropy measurement times from the nanosecond scale to hundreds of microseconds. We demonstrate this scheme for measuring slow depolarization processes of large macromolecular complexes, derive a quantitative rate model, and perform Monte Carlo simulations to describe the depolarization process of OADFA at the molecular level. This setup has great potential to enable future biomacromolecular and colloidal studies.

Optical modulation and selective recovery of Cy5 fluorescence.

Fluorescence modulation offers the opportunity to detect low-concentration fluorophore signals within high background. Applicable from the single-molecule to bulk levels, we demonstrate long-wavelength optical depopulation of dark states that otherwise limit Cy5 fluorescence intensity. By modulated excitation of a long-wavelength Cy5 transient absorption, we dynamically modulate Cy5 emission. The frequency dependence enables specification of the dark-state timescales enabling optical-demodulation-based signal recovery from high background. These dual-laser illumination schemes for high-sensitivity fluorescence-signal recovery easily improve signal-to-noise ratios by well over an order of magnitude, largely by discrimination against background. Previously limited to very specialized dyes, our utilization of long-lived dark states in Cy5 enables selective detection of this very common single-molecule and bulk fluorophore. Although, in principle, the "dark state" can arise from any photoinduced process, we demonstrate that cis-trans photoisomerization, with its unique transient absorption and lifetime enables this sensitivity boosting, long-wavelength modulation to occur in Cy5. Such studies underscore the need for transient absorption studies on common fluorophores to extend the impact of fluorescence modulation for high-sensitivity fluorescence imaging in a much wider array of applications.

Generation of luminescent noble metal nanodots in cell matrices.

Cell matrices were used as rich libraries to screen proteins for the production of luminescent silver and gold nanodots. The study indicates that the proteins for silver and gold nanodot protection are quite different. The identification of such proteins in future may enrich the family of luminescent nanodots.

Tailoring silver nanodots for intracellular staining.

Through tailored oligonucleotide scaffolds, Ag nanocluster syntheses have yielded thermally and cell-culture medium stable silver cluster-based emitters. Optimizing ssDNA stability has enabled creation of highly concentrated and spectrally pure nanocluster emitters with strong intracellular emission. Both fixed and live-cell staining become possible, and intracellular delivery is demonstrated both through conjugation to cell-penetrating peptides and via microinjection.

Optically Modulated and Optically Activated Delayed Fluorescent Proteins through Dark State Engineering.

Modulating fluorescent protein emission holds great potential for increasing readout sensitivity for applications in biological imaging and detection. Here, we identify and engineer optically modulated yellow fluorescent proteins (EYFP, originally 10C, but renamed EYFP later, and mVenus) to yield new emitters with distinct modulation profiles and unique, optically gated, delayed fluorescence. The parent YFPs are individually modulatable through secondary illumination, depopulating a long-lived dark state to dynamically increase fluorescence. A single point mutation introduced near the chromophore in each of these YFPs provides access to a second, even longer-lived modulatable dark state, while a different double mutant renders EYFP unmodulatable. The naturally occurring dark state in the parent YFPs yields strong fluorescence modulation upon long-wavelength-induced dark state depopulation, allowing selective detection at the frequency at which the long wavelength secondary laser is intensity modulated. Distinct from photoswitches, however, this near IR secondary coexcitation repumps the emissive S1 level from the long-lived triplet state, resulting in optically activated delayed fluorescence (OADF). This OADF results from secondary laser-induced, reverse intersystem crossing (RISC), producing additional nanosecond-lived, visible fluorescence that is delayed by many microseconds after the primary excitation has turned off. Mutation of the parent chromophore environment opens an additional modulation pathway that avoids the OADF-producing triplet state, resulting in a second, much longer-lived, modulatable dark state. These Optically Modulated and Optically Activated Delayed Fluorescent Proteins (OMFPs and OADFPs) are thus excellent for background- and reference-free, high sensitivity cellular imaging, but time-gated OADF offers a second modality for true background-free detection. Our combined structural and spectroscopic data not only gives additional mechanistic details for designing optically modulated fluorescent proteins but also provides the opportunity to distinguish similarly emitting OMFPs through OADF and through their unique modulation spectra.

FRET-enabled optical modulation for high sensitivity fluorescence imaging.

Fluorescence resonance energy transfer is utilized to engineer donor photophysics for facile signal amplification and selective fluorescence recovery from high background. This is generalized such that many different fluorophores can be used in optical modulation schemes to drastically improve fluorescence imaging sensitivity. Dynamic, simultaneous, and direct excitation of the acceptor brightens and optically modulates higher energy donor emission. The externally imposed modulation waveform enables selective donor fluorescence extraction through demodulation. By incorporating an acceptor with significant, spectrally shifted, dark-state population, necessary excitation intensities are quite low and agree well with simulated enhancements. Enhancement versus modulation frequency directly yields dark-state lifetimes in a simple ensemble measurement. Using the long-lived Cy5 dark state in conjunction with Cy3 donors, we demonstrate image extraction from a large background to yield >>10-fold sensitivity improvements through synchronously amplified fluorescence image recovery (SAFIRe).

Transfection of living HeLa cells with fluorescent poly-cytosine encapsulated Ag nanoclusters.

The fluorescence of silver clusters encapsulated by single stranded oligo-DNA (24 cytosine base pairs, C(24):Ag(n)) was used to monitor the transfection of this new silver/DNA fluorophore inside living HeLa cells. For this, the C(24):Ag(n) molecules were complexed with a commercially available transfection reagent Lipofectamine and the internalization of C(24):Ag(n) was followed with confocal fluorescence microscopy. Bright near-infrared fluorescence was observed from inside the transfected HeLa cells, when exciting with 633 nm excitation, opening up the possibility for the use of these C(24):Ag(n) clusters for biological labelling and imaging of living cells and for monitoring the transfection process with limited harm to the living cells.