Platinum plasmonic nanostructure arrays for massively parallel single-molecule detection based on enhanced fluorescence measurements

We fabricated platinum bowtie nanostructure arrays producing fluorescence enhancement and evaluated their performance using two-photon photoluminescence and single-molecule fluorescence measurements. A comprehensive selection of suitable materials was explored by electromagnetic simulation and Pt was chosen as the plasmonic material for visible light excitation near 500 nm, which is preferable for multicolor dye-labeling applications like DNA sequencing. The observation of bright photoluminescence (λ = 500-600 nm) from each Pt nanostructure, induced by irradiation at 800 nm with a femtosecond laser pulse, clearly indicates that a highly enhanced local field is created near the Pt nanostructure. The attachment of a single dye molecule was attempted between the Pt triangles of each nanostructure by using selective immobilization chemistry. The fluorescence intensities of the single dye molecule localized on the nanostructures were measured. A highly enhanced fluorescence, which was increased by a factor of 30, was observed. The two-photon photoluminescence intensity and fluorescence intensity showed qualitatively consistent gap size dependence. However, the average fluorescence enhancement factor was rather repressed even in the nanostructure with the smallest gap size compared to the large growth of photoluminescence. The variation of the position of the dye molecule attached to the nanostructure may influence the wide distribution of the fluorescence enhancement factor and cause the rather small average value of the fluorescence enhancement factor.     

Efficient detection of single DNA fragments in flowing sample streams by two-photon fluorescence excitation

This paper reports the demonstration of efficient single molecule detection in flow cytometry by two-photon fluorescence excitation. We have used two-photon excitation (TPE) to detect single DNA fragments as small as 383 base pairs (bp) labeled with the intercalating dye, POPO-1, at a dye:nucleotide ratio of 1:5. TPE of the dye-DNA complexes was accomplished using a mode-locked, 120 fs pulse width Ti:sapphire laser operating at 810 nm. POPO-1 labeled DNA fragments of 1.1 kilobase pairs (kbp) and larger were sequentially detected in our flow cytometry system with a detection efficiency of nearly 100%. The detection efficiency for the 383 bp DNA fragments was approximately 75%. We also demonstrate the ability to distinguish between different sized DNA fragments in a mixture by their individual fluorescence burst sizes by TPE. These studies indicate that using TPE for single molecule flow cytometry experiments lowers the intensity of the background radiation by approximately an order of magnitude compared to one-photon excitation, due to the large separation between the excitation and emission wavelengths in TPE.     

可见激光立体光造型树脂协同引发体系引发机理的研究

用紫外／可见光谱及荧光／磷光光谱研究了在514nm波长下能引发丙烯酸－环氧树脂聚合的由染料，紫外光引发剂，叔胺组成的协同引发体系的引发机理，染料吸收可见光被激发，其激发三线态与叔胺形成激基复合物，并由此生成叔胺自由基，叔胺自由基与紫外引发剂发生电子转移作用生成活性更大的自由基引发聚合。     

Multimode laser emission from dye doped polymer optical fiber

Multimode laser emission is observed in a polymer optical fiber doped with a mixture of Rhodamine 6G (Rh 6G) and Rhodamine B (Rh B) dyes. Tuning of laser emission is achieved by using the mixture of dyes due to the energy transfer occurring from donor molecule (Rh 6G) to acceptor molecule (Rh B). The dye doped poly(methyl methacrylate)-based polymer optical fiber is pumped axially at one end of the fiber using a 532 nm pulsed laser beam from a Nd:YAG laser and the fluorescence emission is collected from the other end. At low pump energy levels, fluorescence emission is observed. When the energy is increased beyond a threshold value, laser emission occurs with a multimode structure. The optical feedback for the gain medium is provided by the cylindrical surface of the optical fiber, which acts as a cavity. This fact is confirmed by the mode spacing dependence on the diameter of the fiber.     

Charge-coupled device operated in a time-delayed integration mode as an approach to high-throughput flow-based single molecule analysis

Single molecule detection (SMD) readouts are particularly attractive for assays geared toward high-throughput processing, because they can potentially reduce assay time by eliminating various processing steps. Unfortunately, most flow-based SMD experiments have generated low throughputs due primarily to the fact that they are configured in single assay formats. The use of a charge-coupled device (CCD) with flow-based SMD can image multiple single molecule assays simultaneously to realize high-throughput processing capabilities. We present, for the first time, the ability to simultaneously track and detect single molecules in multiple microfluidic channels by employing a CCD camera operated in time-delayed integration (TDI) mode as a means for increasing the throughput of any single molecule measurement. As an example of the technology, we have configured a CCD to operate in a TDI mode to detect single double-stranded DNA molecules (lambda and pBR322) labeled with an intercalating dye (TOTO-3) in a series of microfluidic channels poised on a poly(methyl methacrylate), PMMA, chip. A laser beam was launched into the side of the chip, which irradiated a series of fluidic channels (eight) with the resulting fluorescence imaged onto a CCD. Using this system, we were able to identify single DNA molecules based on the fluorescence burst intensity arising from differences in the extent of dye labeling associated with the DNA molecule length. The CCD/TDI approach allowed increasing sample throughput by a factor of 8 compared to a single-assay SMD experiment. A sampling throughput of 276 molecules s (-1) per channel and 2208 molecules s (-1) for an eight channel microfluidic system was demonstrated. Operated in its full capacity, this multichannel format was projected to yield a sample throughput of 1.7 x 10 (7) molecules s (-1), which represents a 170-fold improvement over previously reported single molecule sampling rates.     

Exploring fluorescence antibunching in solution to determine the stoichiometry of molecular complexes

Fluorescence antibunching is a well-known technique for determining the number of independent emitters per molecule or molecular complex. It was rarely applied to autofluorescent proteins due to the necessity of collecting large numbers of fluorescence photons from a single molecule, which is usually impossible to achieve with rather photolabile autofluorescent proteins. Here, we measure fluorescence antibunching on molecules in solution, allowing us to accumulate data over a large number of molecules. We use that method for determining an average stoichiometry of molecular complexes. The proposed method is absolute in the sense that it does not need any calibration or referencing. We develop the necessary theoretical background and check the method on pure dye solutions and on molecular complexes with known stoichiometry.     

Fluorescent Polymer Nanoparticles Based on Dyes: Seeking Brighter Tools for Bioimaging

Speed, resolution and sensitivity of today's fluorescence bioimaging can be drastically improved by fluorescent nanoparticles (NPs) that are many‐fold brighter than organic dyes and fluorescent proteins. While the field is currently dominated by inorganic NPs, notably quantum dots (QDs), fluorescent polymer NPs encapsulating large quantities of dyes (dye‐loaded NPs) have emerged recently as an attractive alternative. These new nanomaterials, inspired from the fields of polymeric drug delivery vehicles and advanced fluorophores, can combine superior brightness with biodegradability and low toxicity. Here, we describe the strategies for synthesis of dye‐loaded polymer NPs by emulsion polymerization and assembly of pre‐formed polymers. Superior brightness requires strong dye loading without aggregation‐caused quenching (ACQ). Only recently several strategies of dye design were proposed to overcome ACQ in polymer NPs: aggregation induced emission (AIE), dye modification with bulky side groups and use of bulky hydrophobic counterions. The resulting NPs now surpass the brightness of QDs by ≈10‐fold for a comparable size, and have started reaching the level of the brightest conjugated polymer NPs. Other properties, notably photostability, color, blinking, as well as particle size and surface chemistry are also systematically analyzed. Finally, major and emerging applications of dye‐loaded NPs for in vitro and in vivo imaging are reviewed.     

Enhancement of immunoassay's fluorescence and detection sensitivity using three-dimensional plasmonic nano-antenna-dots array

Protein detection is universal and vital in biological study and medical diagnosis (e.g., cancer detection). Fluorescent immunoassay is one of the most widely used and most sensitive methods in protein detection (Giljohann, D. A.; Mirkin, C. A. Nature2009, 462, 461-464; Yager, P.; et al. Nature2006, 442, 412-418). Improvements of such assays have many significant implications. Here, we report the use of a new plasmonic structure and a molecular spacer to enhance the average fluorescence of an immunoassay of Protein A and human immunoglobulin G (IgG) by over 7400-fold and the immunoassay's detection sensitivity by 3,000,000-fold (the limit of detection is reduced from 0.9 × 10(-9) to 0.3 × 10(-15) molar (i.e., from 0.9 nM to 300 aM), compared to identical assays performed on glass plates). Furthermore, the average fluorescence enhancement has a dynamic range of 8 orders of magnitude and is uniform over the entire large sample area with a spatial variation ±9%. Additionally, we observed that, when a single molecule fluorophore is placed at a "hot spot" of the plasmonic structure, its fluorescence is enhanced by 4 × 10(6)-fold, thus indicating the potential to further significantly increase the average fluorescence enhancement and the detection sensitivity. Together with good spatial uniformity, wide dynamic range, and ease to manufacture, the giant enhancement in immunoassay's fluorescence and detection sensitivity (orders of magnitude higher than previously reported) should open up broad applications in biology study, medical diagnosis, and others.     

Quantitative dosing of surfaces with fluorescent molecules: characterization of fractional monolayer coverages by counting single molecules.

Quantitative deposition of dye molecules onto a substrate has been achieved at very low surface concentrations, in the range of 5 x 10(-8) - 1 x 10(-6) monolayer, using the technique of controlled substrate withdrawal from solution. These small surface populations were determined with high (>96%) efficiency by single-molecule counting using an epi-illumination, fluorescence microscope with charge-coupled device detector. The fluorescence imaging resolution (3sigma) is 0.78 microm; over a uniform excitation area of 67 x 67 microm2, a large number (>7,500) of spatially resolved channels are available for counting individual molecules. At low coverages, the number density of fluorescence spots on the surface agrees with the expected surface concentration of molecules, based on the concentration of dye in solution and the solution film thickness predicted from theory. When the surface density of molecules is high enough that fluorescence spot overlap is likely to occur within the optical resolution of the instrument, the observed fewer number of spots can be corrected for overlap through a site occupation model based on Poisson statistics.     

Probes for detection of specific DNA sequences at the single-molecule level

A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide. The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 nm with a repetition rate of 64 MHz. Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe-target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules.     

Background correction by wavelength modulation for pulsed-laser-excited atomic fluorescence spectrometry.

Instrumentation was constructed to modulate the dye laser wavelength for background correction in laser-excited atomic fluorescence spectrometry (LEAFS). To achieve wavelength modulation a piezoelectric pusher was used to drive the wavelength tuning mirror in a laboratory-constructed grazing incidence dye laser. The laser pulses were synchronized with the piezoelectric pusher movement so that alternate laser pulses measured the atomic fluorescence signal at the analytical atomic spectral line (on-line) and the background signal at a wavelength displaced to one side of the atomic line (off-line). The background-corrected signal was obtained by subtracting the off-line "background" from the on-line "signal plus background". The spectral line width (fwhm) of the dye laser was 0.003 nm, while the wavelength modulation interval was controllable over the range from 0 to 0.2 nm with a spectral resolution limited only by the spectral line width of the laser. This type of background correction could, in principle, be applied to other types of tunable lasers such as pulsed Ti: sapphire lasers. The performance of background correction by wavelength modulation (WM) was demonstrated by measurement of sodium resonance fluorescence in an air-acetylene flame and by thallium nonresonance fluorescence in a graphite furnace. The experimental data indicated that the wavelength modulation corrected, effectively and quantitatively, for flame background, blackbody emission from a graphite furnace, and scatter of laser radiation off aluminum chloride (1 mg/mL as AI) matrix particles in both the furnace and the flame. Analytical results were in good agreement with certified values for the determination of sodium in standard reference materials by the use of modulated LEAFS.     

Laser-induced predissociative fluorescence: dynamics and polarization and the effect of lower-state rotational energy transfer on quantitative diagnostics

Laser-induced predissociative fluorescence is often used for diagnostics because its short-lived upper states are minimally disturbed by collisions. We discuss the effects of lower-state collisions with parameters relevant to our atmospheric H(2)-O(2) flame. A pulse of tunable KrF excimer-laser light induces the A ? X, Q(1)(11), 3 ? 0 transition in OH. We measure the intensity and the polarization of the resulting A ? X, Q(1)(11), 3 ? 2 fluorescence as a function of laser brightness. A simple model that uses no adjustable parameters produces a reasonable fit to the data. It predicts that, even at very modest laser energies, the fluorescence intensity is almost directly proportional to the rate constant for rotational energy transfer (RET) within the lower vibrational state. That rate constant can be a strong function of local conditions. Furthermore, under typical operating conditions the excimer will pump an amount of OH out of the lower state that is many times as large as that originally present. This occurs because RET within the X-state continuously replenishes the lower state during the laser pulse. Even when this occurs, the signal may still vary linearly with laser intensity, and the polarization may be nearly that expected for weak pumping. At the higher laser intensities, a significant fraction of the measured OH arises from two-photon photodissociation of the water from the flame reaction.     

Detection of flowing fluorescent particles in a microcapillary using fluorescence correlation spectroscopy

Capillary flow experiments are described with fluorescent molecules, bacteria, and microspheres using fluorescence correlation spectroscopy as an analytical tool. The flow velocity in the microcapillary is determined by fitting autocorrelation traces with a model containing parameters related to diffusion and flow. The flow profile of pressure-driven flow inside a microcapillary is determined by using the fluorescence fluctuations of a small dye molecule. It was found that bacteria and microspheres are retarded in their flow by optical forces produced by the laser beam.     

Photon-avalanche upconversion in thulium-doped lutetium aluminum garnet.

Strong blue fluorescence at 487 nm corresponding to the (1)G(4) ? (3)H(6) transition was generated from Tm(3+)-doped lutetium aluminum garnet on excitation with a 618-nm dye laser as a result of a photon-avalanche upconversion mechanism.     

Wide area polycrystalline diamond coating and stress control by sp hot filament CVD reactor

This paper discusses large area uniform diamond coatings deposited in the sp³ Inc. Model 600 hot filament diamond deposition system (made by sp³ Inc., California, USA). This model combines proven hot filament thermal reactor technology with advanced controls to produce high quality polycrystalline diamond films over a maximum square area of 380 mm × 380 mm on a wide variety of substrate materials such as carbide-based cutting tools, wear surfaces, Si wafers, etc. The reactor is characterized using instrumented 300 mm Si wafers and modified, accordingly, to optimize performance on 300 mm diameter wafers or multiple 100 mm diameter wafers. Roles of temperature and other process parameters in stress formation and development in the diamond thin films, grown in a wide area hot filament deposition system, are discussed along with some of the ways of controlling these stresses on a production basis.     

Side illumination fluorescence emission characteristics from a dye doped polymer optical fiber under two-photon excitation

Two-photon excited (TPE) side illumination fluorescence studies in a Rh6G-RhB dye mixture doped polymer optical fiber (POF) and the effect of energy transfer on the attenuation coefficient is reported. The dye doped POF is pumped sideways using 800 nm, 70 fs laser pulses from a Ti:sapphire laser, and the TPE fluorescence emission is collected from the end of the fiber for different propagation distances. The fluorescence intensity of RhB doped POF is enhanced in the presence of Rh6G as a result of energy transfer from Rh6G to RhB. Because of the reabsorption and reemission process in dye molecules, an effective energy transfer is observed from the shorter wavelength part of the fluorescence spectrum to the longer wavelength part as the propagation distance is increased in dye doped POF. An energy transfer coefficient is found to be higher at shorter propagation distances compared to longer distances. A TPE fluorescence signal is used to characterize the optical attenuation coefficient in dye doped POF. The attenuation coefficient decreases at longer propagation distances due to the reabsorption and reemission process taking place within the dye doped fiber as the propagation distance is increased.     

Optical properties of titanium dioxide nanoparticles/3-(2-benzothiazolyl)-7-N,N-diethylaminocoumarin/polymethyl methacrylate composite films

3-(2-benzothiazolyl)-7-N,N-diethylaminocoumarin organic laser dye-polymethyl methacrylate (PMMA) composite films doped with inorganic titanium dioxide (TiO₂) particles are fabricated by spin-coating technique. TiO₂ nanoparticles exhibit a strong influence on optical properties of the organic laser dye/PMMA composite films. The refractive index and absorbance (absorption intensity) of organic laser dye/PMMA composite film with micro- and nanoparticles of TiO₂ are reduced, compared to those without TiO₂ particles. The organic laser dye/PMMA composite film with TiO₂ nanoparticles has the lowest refractive index and absorbance values. Photoluminescence intensities of all systems exhibit a maximum peak around the excitation wavelength, close to that of the organic laser dye, at 450 nm and the minimum around the excitation wavelength of 350 nm. Photoluminescence intensity of the organic laser dye/PMMA composite film with TiO₂ microparticles is always the lowest at all excitation wavelengths. However, the photoluminescence intensity of the organic laser dye/PMMA composite film with TiO₂ nanoparticles has the highest value at excitation wavelengths of 330 and 380 nm, while the photoluminescence intensity of composite film without TiO₂ particles is more than that with nanoparticles at other excitation wavelengths.     

Highly sensitive NO2 optical detector with squarylium dye Langmuir-Blodgett film containing J aggregate

A highly sensitive optical gas detector based on the fluorescence quenching of a squarylium dye Langmuir-Blodgett film is reported. A squarylium dye Langmuir-Blodgett film containing J aggregate deposited on a substrate emits fluorescence from the J aggregate by excitations of He-Ne laser (632.8 nm) and laser diode (750 nm). This fluorescence was observed to be quenched quickly and reversibly by the presence of NO₂ in an air atmosphere. The change in fluorescence intensity can be easily detected by an NO₂ level of only parts per billion. High sensitivity was discussed in relation to the high mobility of excited states in the J aggregate.     

Spectroscopic properties and amplified spontaneous emission of fluorescein laser dye in ionic liquids as green media

The use of ionic liquids (ILs) as milieu materials for laser dyes is a promising field and quite competitive with volatile organic solvents and solid state-dye laser systems. This paper investigates some photo-physical parameters of fluorescein dye incorporated into ionic liquids; 1-Butyl-3-methylimidazolium chloride (BMIM Cl), 1-Butyl-3-methylimidazolium tetrachloroaluminate (BMIM AlCl₄) and 1-Butyl-3-methylimidazolium tetrafluoroborate (BMIM BF₄) as promising host matrix in addition to ethanol as reference. These parameters are: absorption and emission cross-sections, fluorescence lifetime and quantum yield, in addition to the transition dipole moment, the attenuation length and oscillator strength were also investigated. Lasing characteristics such as amplified spontaneous emission (ASE), the gain, and the photostability of fluorescein laser dye dissolved in different host materials were assessed. The composition and properties of the matrix of ILs were found that it has great interest in optimizing the laser performance and photostability of the investigated laser dye. Under transverse pumping of fluorescein dye by blue laser diode (450 nm) of (400 mW), the initial ASE for dye dissolved in BMIM AlCl₄ and ethanol were decreased to 39% and 36% respectively as time progressed 132 min. Relatively high efficiency and high fluorescence quantum yield (11.8% and 0.82% respectively) were obtained with good photostability in case of fluorescein in BMIM BF₄ that was decreased to ∼56% of the initial ASE after continuously pumping with 400 mW for 132 min.     

Picomolar assay of native proteins by capillary electrophoresis precolumn labeling, submicellar separation, and laser-induced fluorescence detection

We report a method for the assay of proteins at concentrations lower than 10(-)(10) M with as little as 200 amol of protein. High sensitivity is accomplished by derivatizing the ε-amino group of the protein's lysine residues with the fluorogenic dye 5-furoylquinoline-3-carboxaldehyde and use of a sheath flow cuvette fluorescence detector. Most proteins have a large number of lysine residues; therefore, a large number of fluorescent molecules can be attached to each protein molecule. In general, precolumn labeling improves sensitivity but degrades resolution due to the inhomogeneity of the reaction products from multiple labeling. However, we demonstrate that, through careful manipulation of the separation and reaction conditions, high sensitivity can be obtained without excessive loss in separation efficiency. Over 190?000 theoretical plates are obtained for fluorescently labeled ovalbumin.