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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 S

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.

Silver molecules are chromophores with diverse spectra and rich photophysics, and DNA strands act as ligands that develop specific molecular silver species. For example, C4AC4T*C3GT4 selectively yi...

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 S

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 Ag(8) cluster arranged in a Big Dipper shape bound to the oligonucleotide A(2)C(4). 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 Ag(5) moiety via Ag-N bonds to endo- and exo-cyclic nitrogens of cytosine and adenine. The unique DNA configuration, constrained coordination environment, and templated Ag(8) 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.

Silver clusters consisting of ∼10 atoms are readily bound by and encapsulated within DNA strands to yield strong absorption and emission. The coordination environments, however, are poorly understood, so cluster adducts can only be empirically tuned. This work describes the C4AC4TC3G strand that templates a particular cluster adduct. Its sequence has three types of nucleobases with distinct roles—tracts of cytosines that collectively coordinate the cluster, thymine acting as a junction in the overall strand, and the adenine/guanine pair that exclusively forms the cluster. In relation to the native oligonucleotide, the DNA–silver cluster complex diffuses faster and is more compact, thus suggesting that the strands fold because of the cluster. The Ag106+ adduct emits with λex/λem = 490/540 nm, a 19% quantum yield, and a biexponential 1.1/2.1 ns lifetime. The electronic environment for the cluster is controlled by the heteroatoms in the adenine and guanine. Most significantly, the N7 and the N2 in the guanin...

We harness the photophysics of few-atom silver nanoclusters to create the first fluorophores capable of Optically Activated Delayed Fluorescence (OADF). In analogy with thermally activated delayed fluorescence, often resulting from oxygen- or collision-activated reverse intersystem crossing from triplet levels, this optically controllable/reactivated visible emission occurs with the same 2.2 ns fluorescence lifetime as produced with primary excitation alone, but is excited with near infrared light from either of two distinct, long-lived photopopulated dark states. In addition to faster ground state recovery under long-wavelength co-illumination, this “repumped” visible fluorescence occurs many microsceconds after visible excitation, and only when gated by secondary near IR excitation of ~1–100 microsecond lived dark excited states. By deciphering the Ag nanocluster photophysics, we demonstrate that OADF improves upon previous optical modulation schemes for near complete background rejection in fluorescence detection. Likely extensible to other fluorophores with photopopulatable excited dark states, OADF holds potential for drastically improving fluorescence signal recovery from high backgrounds.

Modulation and optical control of photoswitchable fluorescent protein (PS-FP) dark state lifetimes drastically improves sensitivity and selectivity in fluorescence imaging. The dark state population of PS-FPs generates an out-of-phase fluorescence component relative to the sinusoidally modulated 488 nm laser excitation. Because this apparent phase advanced emission results from slow recovery to the fluorescent manifold, we hasten recovery and, therefore, modulation frequency by varying coillumination intensity at 405 nm. As 405 nm illumination regenerates the fluorescent ground state more rapidly than via thermal recovery, we experimentally demonstrate that secondary illumination can control PS-FPs dark state lifetime to act as an additional dimension for discriminating spatially and spectrally overlapping emitters. This experimental combination of out of phase imaging after optical modulation (OPIOM) and synchronously amplified fluorescence image recovery (SAFIRe) optically controls the fluorescent protein dark state lifetimes for improved time resolution, with the resulting modulation-based selective signal recovery being quantitatively modeled. The combined experimental results and quantitative numerical simulations further demonstrate the potential of SAFIRe-OPIOM for wide-field biological imaging with improved speed, sensitivity, and optical resolution over other modulation-based fluorescence microscopies.

Excitation of few-atom Ag cluster fluorescence produces significant steady-state dark state populations that can be dynamically optically depopulated with long wavelength co-illumination. Modulating this secondary illumination dynamically repopulates the ground state, thereby directly modulating nanodot fluorescence without modulating background. Both fast and slow modulation enable unmodulated background to be quantitatively removed in Fluorescence Correlation Spectroscopy (FCS) through simple correlation-based averaging. Such modulated dual-laser FCS enables recovery of pure Ag nanodot fluorescence correlations even in the presence of strong, spectrally overlapping background emission. Fluorescence recovery is linear with Fourier amplitude of the modulated fluorescence, providing a complementary approach to background-free quantitation of modulatable emitter concentration in high background environments. Using the expanding range of modulatable fluorophores, such methodologies should facilitate biologically relevant studies in both complex autofluorescent environments and multiplexed assays.

Conspectus 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 (RichardsC. I.; J. Am. Chem. Soc.2009, 131, 461919284790). 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 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.

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.

Photostability, inherent fluorescence brightness, and optical modulation of fluorescence are key attributes distinguishing silver nanoclusters as fluorophores. DNA plays a central role both by protecting the clusters in aqueous environments and by directing their formation. Herein, we characterize a new near-infrared-emitting cluster with excitation and emission maxima at 750 and 810 nm, respectively, that is stabilized within C3AC3AC3TC3A. Following chromatographic resolution of the near-infrared species, a stoichiometry of 10 Ag/oligonucleotide was determined. Combined with excellent photostability, the cluster’s 30% fluorescence quantum yield and 180 000 M−1 cm−1 extinction coefficient give it a fluorescence brightness that significantly improves on that of the organic dye Cy7. Fluorescence correlation analysis shows an optically accessible dark state that can be directly depopulated with longer wavelength coillumination. The coupled increase in total fluorescence demonstrates that enhanced sensitivity...

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.

Fluorescence intermittency severely limits brightness in both single molecule and bulk fluorescence. Herein, we demonstrate that optical depopulation of organic fluorophore triplet states opens a path to significantly increased sensitivity by simultaneously increasing brightness and greatly reducing background through synchronously detected fluorescence modulation. Image recovery is achieved through selective fluorescence enhancement via modulating a secondary laser excitation at much lower energy than the observed emission in order to depopulate the long-lived triplet states. A series of xanthene dyes that exhibit efficient triplet-state formation demonstrate that this method of selective signal extraction can be achieved at moderate primary and secondary excitation intensities through tailoring dye photophysics and imaging conditions. Up to 5-fold increases in solution-based fluorescence over primary laser excitation alone was achieved upon secondary laser excitation, and dynamic control of signal modulation was demonstrated over a wide time range simply by varying the modulation frequency of the laser used for depopulation of the triplet state. We identify the photophysical characteristics that enable existing or to-be-designed fluorophores to be used in synchronously amplified fluorescence image recovery (SAFIRe) microscopy.

Dongwon Lee1, Venkata R Erigala1,3, Madhuri Dasari1, Junhua Yu2, Robert M Dickson2, Niren Murthy11The Wallace H. Coulter Department of Biomedical Engineering; 2Department of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA; 3The Scripps Research Institute, La Jolla, CA, USAAbstract: The overproduction of hydrogen peroxide is implicated in the progress of numerous life-threatening diseases and there is a great need for the development of contrast agents that can detect hydrogen peroxide in vivo. In this communication, we present a new contrast agent for hydrogen peroxide, termed peroxalate micelles, which detect hydrogen peroxide through chemiluminescence, and have the physical/chemical properties needed for in vivo imaging applications. The peroxalate micelles are composed of amphiphilic peroxalate based copolymers and the fluorescent dye rubrene, they have a ‘stealth’ polyethylene glycol (PEG) corona to evade macrophage phagocytosis, and a diameter of 33 nm to enhance extravasation into permeable tissues. The peroxalate micelles can detect nanomolar concentrations of hydrogen peroxide (>50 nM) and thus have the sensitivity needed to detect physiological concentrations of hydrogen peroxide. We anticipate numerous applications of the peroxalate micelles for in vivo imaging of hydrogen peroxide, given their high sensitivity, small size, and biocompatible PEG corona.Keywords: hydrogen peroixde, chemiluminescence, micelles, amphiphilic copolymer

The water-soluble, near-IR-emitting DNA-encapsulated silver nanocluster presented herein exhibits extremely bright and photostable emission on the single-molecule and bulk levels. The photophysics have been elucidated by intensity-dependent correlation analysis and suggest a heavy atom effect of silver that rapidly depopulates an excited dark level before quenching by oxygen, thereby conferring great photostability, very high single-molecule emission rates, and essentially no blinking on experimentally relevant time scales (0.1 to >1,000 ms). Strong antibunching is observed from these biocompatible species, which emit >10 9 photons before photobleaching. The significant dark-state quantum yield even enables bunching from the emissive state to be observed as a dip in the autocorrelation curve with only a single detector as the dark state precludes emission from the emissive level. These species represent significant improvements over existing dyes, and the nonpower law blinking kinetics suggest that these very small species may be alternatives to much larger and strongly intermittent semiconductor quantum dots.

Highly fluorescent, water-soluble, few-atom noble-metal quantum dots have been created that behave as multielectron artificial atoms with discrete, size-tunable electronic transitions throughout the visible and near infrared. These molecular metals exhibit highly polarizable transitions and scale in size according to the simple relation EFermi/N1/3, predicted by the free-electron model of metallic behavior. This simple scaling indicates that fluorescence arises from intraband transitions of free electrons, and these conduction-electron transitions are the low-number limit of the plasmon—the collective dipole oscillations occurring when a continuous density of states is reached. Providing the missing link between atomic and nanoparticle behavior in noble metals, these emissive, water-soluble Au nanoclusters open new opportunities for biological labels, energy-transfer pairs, and light-emitting sources in nanoscale optoelectronics.

Cyanine dyes are well-known for their bright fluorescence and utility in biological imaging. However, cyanines also readily photoisomerize to produce nonemissive dark states. Co-illumination with a secondary, red-shifted light source on-resonance with the longer wavelength absorbing dark state reverses the photoisomerization and returns the cyanine dye to the fluorescent manifold, increasing steady-state fluorescence intensity. Modulation of this secondary light source dynamically alters emission intensity, drastically improving detection sensitivity and facilitating fluorescence signals to be recovered from an otherwise overwhelming background. Red and near-IR emitting cyanine derivatives have been synthesized with varying alkyl chain lengths and halogen substituents to alter dual-laser fluorescence enhancement. Photophysical properties and enhancement with dual laser modulation were coupled with density functional calculations to characterize substituent effects on dark state photophysics, potentially improving detection in high background biological environments.

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.

The reduction of silver cations bound to the oligonucleotide dC(12) was used to form silver nanoclusters. Mass spectra show that the oligonucleotides have 2-7 silver atoms that form multiple species, as evident from the number of transitions in the fluorescence and absorption spectra. The variations in the concentrations of the nanoclusters with time are attributed to the changing reducing capacity of the solution, and the formation of oxidized nanoclusters is proposed. Via mass spectrometry and circular dichroism spectroscopy, double-stranded structures with Ag(+)-mediated interactions between the bases are observed, but these structures are not maintained with the reduced nanoclusters. Through variations in the pH, the nanoclusters are shown to bind with the N3 of cytosine.

The high affinity of Ag+ for DNA bases has enabled creation of short oligonucleotide-encapsulated Ag nanoclusters without formation of large nanoparticles. Time-dependent formation of cluster sizes ranging from Ag1 to Ag4/oligonucleotide were observed with strong, characteristic electronic transitions between 400 and 600 nm. The slow nanocluster formation kinetics enables observation of specific aqueous nanocluster absorptions that evolve over a period of 12 h. Induced circular dichroism bands confirm that the nanoclusters are associated with the chiral ss-DNA template. Fluorescence, absorption, mass, and NMR spectra all indicate that multiple species are present, but that their creation is both nucleotide- and time-dependent.

Electrically written silver oxide nanoscale break junctions clearly show asymmetric, wavelength-dependent photoconductivity, but only when either the anode oxide or the silver nanoclusters spanning the junction are illuminated. The higher oxygen content in the cathode increases the cathode oxide band gap and inhibits production of photoinjecting Ag nanoclusters. The optical and electronic properties of silver and its oxides suggest that useful nanoscale optoelectronic components can be created through a very simple one-step electromigration process.

Individual, strongly electroluminescent Ag n molecules ( n = 2∼8 atoms) have been electrically written within otherwise nonemissive silver oxide films. Exhibiting characteristic single-molecule behavior, these individual room-temperature molecules exhibit extreme electroluminescence enhancements (>10 4 vs. bulk and dc excitation on a per molecule basis) when excited with specific ac frequencies. Occurring through field extraction of electrons with subsequent reinjection and radiative recombination, single-molecule electroluminescence is enhanced by a general mechanism that avoids slow bulk material response. Thus, while we detail strong electroluminescence from single, highly fluorescent Ag n molecules, this mechanism also yields strong ac-excited electroluminescence from similarly prepared, but otherwise nonemissive, individual Cu nanoclusters.

Thin AgO films are readily photoactivated at room temperature to produce dynamic, multicolored fluorescent Agn nanoclusters. Writing efficiencies were separately examined as functions of excitation wavelength, intensity, and environmental interactions. The effects of external conditions on written nanocluster fluorescence are interpreted in terms of a mechanistic understanding of fluorescent Agn nanocluster creation and subsequent spectral dynamics, illustrating the equilibrium between nanocluster creation and destruction.

We report results of emission pattern imaging experiments from single Eu3+:Y2O3 nanocrystals (3–12 nm size) designed to provide new insight on the luminescence dynamics of isolated rare-earth doped nano-phosphors. We observe dipolar emission patterns that are characteristic of single quantum emitters whose orientation appears fixed on the measurement time scale. We also show that the luminescence from single nanoparticles is linearly polarized, also characteristic of single quantum system behavior. Taken in combination with dynamical observations of blinking and discrete photobleaching, these experiments provide strong evidence for single ion luminescence, and confirm the dipolar nature of the optical transitions of Eu3+ in inorganic crystals.

We present experimental evidence for nuclear spin selection rules in chemical reactions that have been theoretically anticipated by Quack [M. Quack, Mol. Phys. 34, 477 (1997)]. The abundance ratio of ortho-H3+ (I=3/2) and para-H3+ (I=1/2), R=[o-H3+]/[p-H3+], has been measured from relative intensities of their infrared spectral lines in hydrogen plasmas using para-H2 and normal-H2 (75% o-H2 and 25% p-H2). The observed clear differences in the value of R between the p-H2 and n-H2 plasmas demonstrate the spin memory of protons even after ion-neutral reactions, and thus the existence of selection rules for spin modifications. Both positive column discharges and hollow cathode discharges have been used to demonstrate the effect. Experiments using pulsed plasmas have been conducted in the hollow cathode to minimize the uncertainty due to long-term conversion between p-H2 and o-H2 and to study the time dependence of the o-H3+ to p-H3+ ratio. The observed R(t) has been analyzed using simultaneous rate equations ...

Most molecular/cellular labeling utilizes organic dyes conjugated to bioactive molecules through general organic/inorganic chemistry. Though shortcomings of organic dyes such as poor photostability[1] and low brightness[2] limit observable copy numbers, lack of selectivity in labeling often more seriously limits their application in live cell imaging and single molecule studies[3]. Specificity can be addressed through antibodies and other strong affinity pairs such as avidin-biotin[4, 5], but direct covalent technologies are crucial at sub-pM concentrations, as this is beyond the binding limits of antibody-based affinities. Genetically encoded fluorescent proteins are an excellent solution for specific fluorescent labeling in live cells, but disadvantageous organic dye photophysical instabilities (blinking and bleaching) remain, coupled with the potential perturbation due to large label size[6]. Attempts to overcome photoinstabilities of organic labels have produced much brighter quantum dot labels[7], but, while providing excellent signals, these emitters introduce additional problems such as large physical size, aggregation, toxicity, polyvalency, and strong fluorescence intermittency[8–10]. Simultaneously addressing concerns of brightness, photostability, monovalency, size, and fluorescence intermittency, newly emerging silver cluster-based labels offer excellent potential as molecular labeling agents[11–15]. Spectrally-pure emitters have been produced ranging from the blue to the near IR, with fluorescence quantum yields (ΦF) up to 40% and hydrodynamic radii of the fully assembled ssDNA-encapsulated SCs of ~2.5 nm[16]. Moreover, at bulk and single molecule levels, SCs also show both excellent brightness and photostability[17].

Providing previously obscured positional and orientational information, a novel wide-field optical microscope capable of visualizing three-dimensional orientational dynamics of individual room temperature molecules has been developed. Utilizing a single detector, this facile method enables simultaneous observation of all molecular orientations without polarization optics. Such methods not only provide conclusive, nondestructive evidence of single molecule observation, but will also make single molecule orientational studies accessible to a wide range of researchers. Analysis of observed molecular emission patterns not only directly and noninvasively reveals true 3-D orientational dynamics of individual molecules, but also demonstrates that perceived molecular position is strongly dependent on orientation.

Utilizing noninvasive far- and near-field microscopy techniques, the dipole emission patterns of individual molecules have been directly visualized. By accurately modeling the imaging system upon introduction of specific aberrations, fits of simulated to observed molecular images have enabled true three-dimensional single-molecule orientation determinations at room temperature. Following emission pattern changes in time, this new imaging technique has enabled observation of individual molecule rotational motions within polymeric hosts. Such single-molecule orientational diffusion measurements cannot only probe nanoscale polymer dynamics, but should also lead to enhanced understandings of many materials and biological systems.

Fluorescence microscopy is a widely used non-invasive tool for investigating intracellular structure and processes. Although the palette of fluorescent proteins has revolutionized detection and dynamics in molecular and cellular biology, the limited brightness, low spectral discrimination, and high cellular autofluorescence continue to limit applications. In contrast to photoswitch-based methods, we have developed a spectroscopic method to recover fluorescence from photoaccessible dark states via long-wavelength secondary laser co-illumination. We selectively recover the higher energy fluorescence by modulating this secondary laser without modulating background autofluorescence. Using hypothesized photoreversible isomerizations of the chromophore, we have identified specific fluorescent protein mutants, ranging from blue to red, capable of optical modulation. Employing these methods, we have demonstrated the ability to recover the modulated signal, with >10-fold improvement, from background in live cells. Such modulation schemes enable new imaging modalities for probing intracellular kinetics and equilibria of low-abundance proteins.

Optical studies of individual molecules at low and room temperature can provide information about the dynamics of local environments in solids, liquids and biological systems unobscured by ensemble averaging. Here we present a study of the photophysical behaviour of single molecules of the green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria. Wild-type GFP and its mutant have attracted interest as fluorescent biological labels because the fluorophore may be formed in vivo. GFP mutants immobilized in aereated aqueous polymer gels and excited by 488-nm light undergo repeated cycles of fluorescent emission ('blinking') on a timescale of several seconds-behaviour that would be unobservable in bulk studies. Eventually the individual GFP molecules reach a long-lasting dark state, from which they can be switched back to the original emissive state by irradiation at 405 nm. This suggests the possibility of using these GFPs as fluorescent markers for time-dependent cell processes, and as molecular photonic switches or optical storage elements, addressable on the single-molecule level.

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 co-illumination. Turning this green co-illumination 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 non-modulatable BFP, mKalama1, creates a modulatable variant. Building modulatable BFPs offers opportunities for improved BFP signal discrimination vs. background, greatly enhancing their utility.

The crystal field splitting of single J=1 impurity orthohydrogen molecules in an otherwise pure J=0 parahydrogen crystal has been studied experimentally and theoretically over many years. We have measured this quantity directly from the fine structure of the Q3 (0) (v = 3← 0, J = 0← 0) overtone transition induced by J=1 hydrogen, in the near infrared using a Ti:sapphire laser. From our observed eight pairs of split spectral lines, we have determined the value Δ/k = [E(M=±1) − E(M=0)]/k = 0.0102 ± 0.0003 K including its sign. A straightforward numerical calculation based on ab initio calculations of the H2-H2 potential reproted by Mulder, van der Avoird, and Wormer gives Δ/k = 0.0116 K which is close to the experimental value. This suggests that the static and dynamic effects of phonons on the splitting may have been overestimated in the past.

We show that oriented nanostructures from single molecules of a conducting polymer act as highly robust room-temperature single-photon sources. Individual z-oriented polymer nanostructures show high-contrast photon antibunching with a modulation depth exceeding 90%. These results suggest the feasibility of a “push-button” technology for polymer-based single-photon sources in photonic-based quantum information processing applications.

Formed from pure, single-component silver oxide films, nanoscale heterojunctions are electrically written through electromigration. Instantly formed through applying dc current, the nanogap junctions have different oxygen contents at either electrode. This direct writing of junction asymmetry yields diode behavior with a forward to reverse bias current ratio of 71. Two different, electrically written diode logic gates were implemented based on these in situ formed heterojunctions. Together with the photoreduction of silver oxides to silver nanoclusters, this easy fabrication method of writing nanoscale wires and heterojunctions may provide useful instant device fabrication schemes utilizing only light and electricity.

Fluorescent proteins (FPs) have revolutionized molecular and cellular biology; yet, discrimination over cellular autofluorescence, spectral deconvolution, or detection at low concentrations remain challenging problems in many biological applications. By optically depopulating a photoinduced dark state with orange secondary laser co-excitation, the higher-energy green AcGFP fluorescence is dynamically increased. Modulating this secondary laser then modulates the higher-energy, collected fluorescence; enabling its selective detection by removing heterogeneous background from other FPs. Order-of-magnitude reduction in obscuring fluorophore background emission has been achieved in both fixed and live cells. This longwavelength modulation expands the dimensionality to discriminate FP emitters based on dark state lifetimes and enables signal of interest to be recovered by removing heterogeneous background emitter signals. Thus, AcGFP is not only useful for extracting weak signals from systems plagued by high background, but it is a springboard for further FP optimization and utilization for improving sensitivity and selectivity in biological fluorescence imaging.

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 modulation for selective recovery of desired fluorescence signals has to date required careful fluorophore selection combined with repeated optical recovery from long-lived photoinduced dark states. Adapting an all-optical scheme, modulated stimulated emission depletion (STED) generalizes such modulation schemes by eliminating the need for dark state residence by directly optically depopulating the emissive state at any externally applied frequency. Using two overlapped Gaussian laser spots with the depletion beam being intensity-modulated, fluorescence modulation is readily achieved with a depletion ratio governed by the intensity of the depleting laser. Selective image recovery of otherwise unmodulatable fluorophore signals is directly achieved through this all-optical modulation, and common STED-degrading multiphoton-excited background is readily discriminated against. Both beads and dyes in solution, as well as fluorophores bound within fixed cells are readily imaged in this manner.

A sharp spectral line has been observed in γ‐ray irradiated para‐H2 crystals and assigned to the pure vibrational Q1(0)(v=1←0, J=0←0) transition induced via the Condon effect by charges distributed in the crystal. The remarkable sharpness and stability of the signal is reported and discussed.

Few-atom silver clusters harbored by DNA are promising fluorophores due to their high molecular brightness along with their long- and short-term photostability. Furthermore, their emission rate can be enhanced when co-illuminated with low-energy light that optically depopulates the fluorescence-limiting dark state. The photophysical basis for this effect is evaluated for two near-infrared-emitting clusters. Clusters emitting at ∼800 nm form with C(3)AC(3)AC(3)TC(3)A and C(3)AC(3)AC(3)GC(3)A, and both exhibit a trap state with λ(max) ∼ 840 nm and an absorption cross section of (5-6) × 10(-16) cm(2)/molecule that can be optically depopulated. Transient absorption spectra, complemented by fluorescence correlation spectroscopy studies, show that the dark state has an inherent lifetime of 3-4 μs and that absorption from this state is accompanied by photoinduced crossover back to the emissive manifold of states with an action cross section of ∼2 × 10(-18) cm(2)/molecule. Relative to C(3)AC(3)AC(3)TC(3)A, C(3)AC(3)AC(3)GC(3)A produces a longer-lived trap state and permits more facile passage back to the emissive manifold. With the C(3)AC(3)AC(3)AC(3)G template, a spectrally distinct cluster forms having emission at ∼900 nm, and its trap state has a ∼4-fold shorter lifetime. These studies of optically gated fluorescence bolster the critical role of the nucleobases in both the formation and excited state dynamics of these highly emissive metallic clusters.

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.

The electron transfer (ET) dynamics from core/multi-shell (CdSe/CdS(3ML)ZnCdS(2ML)ZnS(2ML)) quantum dots (QDs) to adsorbed Fluorescein (F27) molecules have been studied by single particle spectroscopy to probe the relationship between single QD interfacial electron transfer and blinking dynamics. Electron transfer from the QD to F27 and the subsequent recombination were directly observed by ensemble-averaged transient absorption spectroscopy. Single QD-F27 complexes show correlated fluctuation of fluorescence intensity and lifetime, similar to those observed in free QDs. With increasing ET rate (controlled by F27-to-QD ratio), the lifetime of on states decreases and relative contribution of off states increases. It was shown that ET is active for QDs in on states, the excited state lifetime of which reflects the ET rate, whereas in the off state QD excitons decay by Auger relaxation and ET is not a competitive quenching pathway. Thus, the blinking dynamics of single QDs modulate their interfacial ET activity. Furthermore, interfacial ET provides an additional pathway for generating off states, leading to correlated single QD interfacial ET and blinking dynamics in QD-acceptor complexes. Because blinking is a general phenomenon of single QDs, it appears that the correlated interfacial ET and blinking and the resulting intermittent ET activity are general phenomena for single QDs.

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 yield10-fold sensitivity improvements through synchronously amplified fluorescence image recovery (SAFIRe).

Widefield cross-correlation microscopy enables large area measurements of flow vectors in microfluidic devices. Extending fluorescence cross-correlation spectroscopy, all points within each image give flow maps from image stacks acquired on a spinning-disk confocal microscope. The greater noise tolerance and efficient data collection for large volumes of interest enable even motion of single antibodies to be measured. A three-dimensional flow map for a typical microfluidic channel is evaluated, yielding full flow vector information. The method presented is general towards the input data, so applications concerning not only flow in microstructures, but biological transport and other microscopic motion phenomena are possible.

Fluorescence imaging in biological sciences is hindered by significant depth-dependent signal attenuation and highly fluorescent backgrounds. We have developed optically modulated near-IR-emitting few-atom Ag nanodots that are selectively and dynamically photobrightened upon simultaneous excitation with a secondary laser, enabling high-sensitivity image extraction to reveal only the demodulated fluorophores. Image demodulation is demonstrated in high-background environments to extract weak signals from completely obscuring background emission.

DNA-encapsulated silver clusters are readily conjugated to proteins and serve as alternatives to organic dyes and semiconductor quantum dots. Stable and bright on the bulk and single molecule levels, Ag nanocluster fluorescence is readily observed when staining live cell surfaces. Being significantly brighter and more photostable than organics and much smaller than quantum dots with a single point of attachment, these nanomaterials offer promising new approaches for bulk and single molecule biolabeling.

Water-soluble ssDNA-encapsulated Ag clusters exhibit large two-photon cross sections reaching 50 000 GM, with high quantum yields in the red and near-IR. Three distinct, spectrally pure, several atom clusters emitting at 660, 680, or 710 nm have been created with two-photon cross sections rivaling those of much larger water-soluble semiconductor quantum dots. Their stability, biocompatibility, and small size offer the promise of nontoxic, sensitive high-resolution biological imaging.