Laser-induced fluorescence of trapped gas-phase molecular ions generated by internal source matrix-assisted laser desorption/ionization in a Fourier transform ion cyclotron resonance mass spectrometer

The combination of laser-induced fluorescence with mass spectrometry opens up new possibilities both for detection purposes and for structural studies of trapped biomolecular ions in the gas phase. However, this approach is experimentally very challenging, and only a handful of studies have been reported so far. In this contribution, a novel scheme for laser-induced fluorescence measurements of ions trapped inside a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer will be introduced. It is based on an open FT-ICR cell design, continuous wave axial excitation of the fluorescence, orthogonal photon collection by fiber optics, and single photon counting detection. Rhodamine 6G ions generated by an internal matrix-assisted laser desorption/ionization source were used to develop and test the set-up. Due to photobleaching processes, the excitation laser power and the observation time window have to be carefully optimized. An ion tomography method was used to align the excitation laser. Potential applications for studying the gas-phase structure of fluorescent biomolecular ions and for investigating fluorescence resonance energy transfer of donor-acceptor pairs will be presented.     

A compactly integrated laser-induced fluorescence detector for microchip electrophoresis

A simple and easy-to-use integrated laser-induced fluorescence detector for microchip electrophoresis was constructed and evaluated. The fluid channels and optical fiber channels in the glass microchip were fabricated using standard photolithographic techniques and wet chemical etching. A 473 nm diode-pumped laser was used as the excitation source, and the collimation and collection optics and mirrors were discarded by using a multimode optical fiber to couple the excitation light straight into the microchannel and placing the microchip directly on the top of the photomultiplier tube. A combination of filter systems was incorporated into a poly(dimethylsiloxane) layer, which was reversibly sealed to the bottom of the microchip to eliminate the scattering excitation light reaching to the photomultiplier tube. Fluorescein/calcein samples were taken as model analytes to evaluate the performance with respect to design factors. The detection limits were 0.05 microM for fluorescein and 0.18 microM for calcein, respectively. The suitability of this simple detector for fluorescence detection was demonstrated by baseline separation of fluorescein isothiocyanate (FITC)-labeled arginine, phenylalanine, and glycine and FITC within 30 s at separation length of 3.8 cm and electrical field strength of 600 V/cm.     

An integrated optics microfluidic device for detecting single DNA molecules

A fluorescence-based integrated optics microfluidic device is presented, capable of detecting single DNA molecules in a high throughput and reproducible manner. The device integrates microfluidics for DNA stretching with two optical elements for single molecule detection (SMD): a plano-aspheric refractive lens for fluorescence excitation (illuminator) and a solid parabolic reflective mirror for fluorescence collection (collector). Although miniaturized in size, both optical components were produced and assembled onto the microfluidic device by readily manufacturable fabrication techniques. The optical resolution of the device is determined by the small and relatively low numerical aperture (NA) illuminator lens (0.10 effective NA, 4.0 mm diameter) that delivers excitation light to a diffraction limited 2.0 microm diameter spot at full width half maximum within the microfluidic channel. The collector (0.82 annular NA, 15 mm diameter) reflects the fluorescence over a large collection angle, representing 71% of a hemisphere, toward a single photon counting module in an infinity-corrected scheme. As a proof-of-principle experiment for this simple integrated device, individual intercalated lambda-phage DNA molecules (48.5 kb) were stretched in a mixed elongational-shear microflow, detected, and sized with a fluorescence signal to noise ratio of 9.9 +/-1.0. We have demonstrated that SMD does not require traditional high numerical aperture objective lenses and sub-micron positioning systems conventionally used in many applications. Rather, standard manufacturing processes can be combined in a novel way that promises greater accessibility and affordability for microfluidic-based single molecule applications.     

Combining optical trapping and single-molecule fluorescence spectroscopy: enhanced photobleaching of fluorophores

To obtain high-resolution information on position or conformation of a molecule and at the same time apply forces to it, one can combine optical trapping with single-molecule fluorescence microscopy. The technical challenge in such an experiment is to discriminate a minute fluorescence signal from the much larger background signals caused by the trap and the fluorescence excitation laser light. We show here that this is feasible even when the fluorophore is directly attached to the trapped particle, by using optimized optical filters. We found, however, that the photostability of the fluorophores we tested suffered from the presence of the additional laser light used for trapping. We found that bleaching rates increased linearly with both the intensity of the trapping laser and the intensity of the fluorescence excitation light. Photobleaching rates were unaffected by the presence or absence of oxygen, but were significantly diminished in the presence of antioxidants. Our results indicate that the enhanced photobleaching is caused by the absorption of a visible photon followed by the excited-state absorption of a near-infrared photon. The higher excited singlet states generated in this way readily form nonfluorescent dye cations. We found that different dyes suffer to a different extent from the excited-state absorption, with Cy3 being worst and tetramethylrhodamine least affected.     

Strategies to improve photostabilities in ultrasensitive fluorescence spectroscopy

Given the particular importance of dye photostability for single-molecule and fluorescence fluctuation spectroscopy investigations, refined strategies were explored for how to chemically retard dye photobleaching. These strategies will be useful for fluorescence correlation spectroscopy (FCS), fluorescence-based confocal single-molecule detection (SMD) and related techniques. In particular, the effects on the addition of two main categories of antifading compounds, antioxidants (n-propyl gallate, nPG, ascorbic acid, AA) and triplet state quenchers (mercaptoethylamine, MEA, cyclo-octatetraene, COT), were investigated, and the relevant rate parameters involved were determined for the dye Rhodamine 6G. Addition of each of the compound categories resulted in significant improvements in the fluorescence brightness of the monitored fluorescent molecules in FCS measurements. For antioxidants, we identify the balance between reduction of photoionized fluorophores on the one hand and that of intact fluorophores on the other as an important guideline for what concentrations to be added for optimal fluorescence generation in FCS and SMD experiments. For nPG/AA, this optimal concentration was found to be in the lower micromolar range, which is considerably less than what has previously been suggested. Also, for MEA, which is a compound known as a triplet state quencher, it is eventually its antioxidative properties and the balance between reduction of fluorophore cation radicals and that of intact fluorophores that defines the optimal added concentration. Interestingly, in this optimal concentration range the triplet state quenching is still far from sufficient to fully minimize the triplet populations. We identify photoionization as the main mechanism of photobleaching within typical transit times of fluorescent molecules through the detection volume in a confocal FCS or SMD instrument (<1-20 ms), and demonstrate its generation via both one- and multistep excitation processes. Apart from reflecting a major pathway for photobleaching, our results also suggest the exploitation of the photoinduced ionization and the subsequent reduction by antioxidants for biomolecular monitoring purposes and as a possible switching mechanism with applications in high-resolution microscopy.     

Single carbon nanotube optical spectroscopy.

This Minireview discusses novel insights into the electronic structure of carbon nanotubes obtained using single-molecule fluorescence spectroscopy. Fluorescence spectra from single nanotubes are well described by a single, Lorentzian lineshape. Nanotubes with identical structures fluoresce with different energies due to local electronic perturbations. Carbon nanotube fluorescence unexpectedly does not-show any intensity or spectral fluctuations at 300 K The lack of intensity blinking or bleaching demonstrates that carbon nanotubes have the potential to provide a stable, single-molecule infrared photon source, allowing for the exciting possibility of applications in quantum optics and biophotonics.     

X-ray fluorescence imaging with polycapillary X-ray optics

X-ray fluorescence spectrometry imaging is a powerful tool to provide information about the chemical composition and elemental distribution of a specimen. X-ray fluorescence spectrometry images were conventionally obtained by using a μ-X-ray fluorescence spectrometry spectrometer, which requires scanning a sample. Faster X-ray fluorescence spectrometry imaging would be achieved by eliminating the process of sample scanning. Thus, we developed an X-ray fluorescence spectrometry imaging instrument without sample scanning by using polycapillary X-ray optics, which had energy filter characteristics caused by the energy dependence of the total reflection phenomenon. In the present paper, we show that two independent straight polycapillary X-ray optics could be used as an energy filter of X-rays for X-ray fluorescence. Only low energy X-rays were detected when the angle between the two optical axes was increased slightly. Energy-selective X-ray fluorescence spectrometry images with projection mode were taken by using an X-ray CCD camera equipped with two polycapillary optics. It was shown that Fe Kα (6.40 keV) and Cu Kα (8.04 keV) could be discriminated for Fe and Cu foils.     

Photobleaching pathways in single-molecule FRET experiments

To acquire accurate structural and dynamical information on complex biomolecular machines using single-molecule fluorescence resonance energy transfer (sm-FRET), a large flux of donor and acceptor photons is needed. To achieve such fluxes, one may use higher laser excitation intensity; however, this induces increased rates of photobleaching. Anti-oxidant additives have been extensively used for reducing acceptor's photobleaching. Here we focus on deciphering the initial step along the photobleaching pathway. Utilizing an array of recently developed single-molecule and ensemble spectroscopies and doubly labeled Acyl-CoA binding protein and double-stranded DNA as model systems, we study these photobleaching pathways, which place fundamental limitations on sm-FRET experiments. We find that: (i) acceptor photobleaching scales with FRET efficiency, (ii) acceptor photobleaching is enhanced under picosecond-pulsed (vs continuous-wave) excitation, and (iii) acceptor photobleaching scales with the intensity of only the short wavelength (donor) excitation laser. We infer from these findings that the main pathway for acceptor's photobleaching is through absorption of a short wavelength photon from the acceptor's first excited singlet state and that donor's photobleaching is usually not a concern. We conclude by suggesting the use of short pulses for donor excitation, among other possible remedies, for reducing acceptor's photobleaching in sm-FRET measurements.     

Single-photon atomic force microscopy

In the last few years, an array of novel technologies, especially the big family of scanning probe microscopy, now often integrated with other powerful imaging tools such as laser confocal microscopy and total internal reflection fluorescence microscopy, have been widely applied in the investigation of biomolecular interactions and dynamics. But it is still a great challenge to directly monitor the dynamics of biomolecular interactions with high spatial and temporal resolution in living cells. An innovative method termed “single-photon atomic force microscopy” (SP-AFM), superior to existing techniques in tracing biomolecular interactions and dynamics in vivo, was proposed on the basis of the combination of atomic force microscopy with the technologies of carbon nanotubes and single-photon detection. As a unique tool, SP-AFM, capable of simultaneous topography imaging and molecular identification at the subnanometer level by synchronous acquisitions and analyses of the surface topography and fluorescent optical signals while scanning the sample, could play a very important role in exploring biomolecular interactions and dynamics in living cells or in a complicated biomolecular background.     

Pulsed microchip laser induced fluorescence for in situ tracer experiments

A compact system for remote and non intrusive in situ analysis of fluorescent tracers using a newly developed pulsed microchip laser coupled to fiber optics was used for in situ rhodamine determinations. By using a crystal doubling in front of the microchip Nd-YAG laser, it is possible to obtain 532 nm at 5 kHz with an energy of 0.6 μJ in a 0.5 ns pulse. Using fiber optics and a passive optode, it was possible to analyze remotely the fluorescence of rhodamine with a compact detection system (monochromator and photomultiplier). Limits of detection down to 10^–10–10^–11 mol/L can be reached depending on the rhodamine studied. Such a laser can be directly implanted in the optode avoiding laser losses when exciting in the U.V. Received: 30 July 1997 / Revised: 6 October 1997 / Accepted: 10 October 1997     

Simultaneous High Throughput Single Molecule Electrophoresis and Single Molecule Spectroscopy

Single molecule detection (SMD) has developed rapidly in recent years, especially high-throughput single molecule detection. Such research facilitated several fundamental studies at the single molecule level. In the fixture, SMD may be successfully applied to biological, clinical and medical research for DNA sequencing and single-molecule scans for disease detection. Presently, single-molecule identification of DNA and proteins is performed using fluorescence intensity, mobility or hybridization with a selective probe. In some cases, such methods are insufficient for confident single-molecule identification. Therefore, we invented a high-throughput combination single-molecule spectroscopy/imaging technique for monitoring the spectroscopic differences of several different individual molecules while they migrate in solution. The technique can offer three-dimensional data for each molecule:mobility, fluorescence intensity and spectroscopy information. Two sample systems were selected as test cases. In the first case, λ DNA is labeled with YOYO-Ⅰ,POPO-Ⅲ and a combination of the two dyes. Many individual λ DNA molecules are simultaneously imaged and identified by their spectroscopic differences. In the second case, a biotinylated 2.1 kb PCR product (also labeled with YOYO-Ⅰ) was reacted with avidin-conjugated R-phycoerythrin. The individual reactants and products are also simultaneously imaged and identified by their spectroscopic differences. This technique can be used for high-throughput DNA screening, molecular identification and monitoring intermolecular interactions with a speed of over 2,000,000 molecules per second. The existing method is the highest and most powerful single-molecule screening method available to date. Such technology is expected to have a great impact on single-molecule diagnosis and monitoring molecular interaction at the single molecule level and will be beneficial to early detection and diagnosis of disease (e.g. cancer, HIV). Furthermore, this technique allows one to directly observe and evaluate the data without any complicated calculations.     

Pulsed microchip laser induced fluorescence for in situ tracer experiments

A compact system for remote and non intrusive in situ analysis of fluorescent tracers using a newly developed pulsed microchip laser coupled to fiber optics was used for in situ rhodamine determinations. By using a crystal doubling in front of the microchip Nd-YAG laser, it is possible to obtain 532 nm at 5 kHz with an energy of 0.6 μJ in a 0.5 ns pulse. Using fiber optics and a passive optode, it was possible to analyze remotely the fluorescence of rhodamine with a compact detection system (monochromator and photomultiplier). Limits of detection down to 10^–10–10^–11 mol/L can be reached depending on the rhodamine studied. Such a laser can be directly implanted in the optode avoiding laser losses when exciting in the U.V. Received: 30 July 1997 / Revised: 6 October 1997 / Accepted: 10 October 1997     

Probability distribution analysis of single-molecule fluorescence anisotropy and resonance energy transfer

Analysis of anisotropy in single-molecule fluorescence experiments using the probability distribution analysis (PDA) method is presented. The theory of anisotropy-PDA is an extension of the PDA theory recently developed for the analysis of F?rster resonance energy transfer (FRET) signals [Antonik, M.; et al. J. Phys. Chem. B 2006, 110, 6970]. The PDA method predicts the shape of anisotropy histograms for any given expected ensemble anisotropy, signal intensity distribution, and background. Further improvements of the PDA theory allow one to work with very low photon numbers, i.e., starting from the level of background signal. Analysis of experimental and simulated data shows that PDA has the major advantage to unambiguously distinguish between shot noise broadening and broadening caused by heterogeneities in the sample. Fitting of experimental histograms yields anisotropy values of individual species, which can be directly compared with those measured in ensemble experiments. Excellent agreement between the ensemble data and the results of PDA demonstrates a good absolute accuracy of the PDA method. The precision in determination of mean values depends mainly on the total number of photons, whereas the ability of PDA to detect the presence of heterogeneities strongly depends on the time window length. In its present form PDA can be also applied to computed fluorescence parameters such as FRET efficiency and scatter-corrected fluorescence anisotropy. Extension of the PDA theory to low photon numbers makes it possible to apply PDA to dynamic systems, for which high time resolution is required. In this way PDA is developed as a sensitive tool to detect biomolecular heterogeneities in space and time.     

Gel-silica optics: Theory and application

A new generation of silica optics has resulted from alkoxide-based sol-gel processing of silica including: net shape transmissive optical elements, surface diffractive optics, inorganic doped GRIN optics, organic impregnated optical composites such as dye lasers and scintillators, optics with internal diffraction gratings, laser densified microoptical lenses and arrays, and laser densified waveguides. Processing control of the ultrastructure of the monolith at the time of gelation and during aging is essential to producing the optical devices together with chemical and thermal stabilization of the surface of the pore network prior to densification or impregnation. The process control variables for gel-silica optics are summarized together with spectroscopic analysis and molecular orbital calculations that explain how and why the thermal-chemical processing controls work at a molecular level.     

Sol-Gel Derived Thin Films for Hydrogen Sulphide Gas Sensing

Utilizing the sol-gel fabrication route, we have successfully modified and tailored the optical absorption in the visible spectrum of thionine dye trapped in an MTMS gel-matrix to coincide with the emission of a red diode laser operating at 660 nm. These thionine-doped MTMS thin films coated onto transparent substrates have shown a remarkable change in optical absorption in the presence of gaseous hydrogen sulphide (H₂S) diluted in air and in the absence of any buffer gas. The rapid response, sensitivity, reversibility and durability shown by this material can be exploited in developing absorption-based optical H₂S sensors in either an integrated optics or all-optical fiber approach using a red diode laser source.     

Compact fluorescence detection using in-fiber microchannels-its potential for lab-on-a-chip applications

This paper reports a compact and practical fluorescence sensor using an in-fiber microchannel. A blue LED, a multimode PMMA or silica fiber and a mini-PMT were used as an excitation source, a light guide and a fluorescence detector, respectively. Microfluidic channels of 100 microm width and 210 microm depth were fabricated in the optical fibers using a direct-write CO(2) laser system. The experimental results show that the sensor has high sensitivity, able to detect 0.005 microg L(-1) of fluorescein in the PBS solution, and the results are reproducible. The results also show that the silica fiber sensor has better sensitivity than that of the PMMA fiber sensor. This could be due to the fouling effect of the frosty layer formed at the microchannel made within the PMMA fiber. It is believed that this fiber sensor has the potential to be integrated into microfluidic chips for lab-on-a-chip applications.     

Multicolor single-molecule spectroscopy with alternating laser excitation for the investigation of interactions and dynamics

We have developed confocal multicolor single-molecule spectroscopy with optimized detection sensitivity on three spectrally distinct channels for the study of biomolecular interactions and FRET between more than two molecules. Using programmable acousto-optical devices as beamsplitter and excitation filter, we overcome some of the limitations of conventional multichroic beamsplitters and implement rapid alternation between three laser lines. This enables to visualize the synthesis of DNA three-way junctions on a single-molecule basis and to resolve seven stoichiometric subpopulations as well as to quantify FRET in the presence of competing energy transfer pathways. Furthermore, the ability to study correlated molecular movements by monitoring several distances within a biomolecular complex simultaneously is demonstrated.     

Analytical applications of a carbon nanotubes composite modified with copper microparticles as detector in flow systems

A simple microchip electrophoresis-laser-induced fluorescence device was constructed and used for separation and determination of catecholamines. On the fabricated glass chip, an extra optical fiber insertion channel, which was perpendicular and extremely close to the separation channel, was directly integrated by nothing operations more than design features on the photomask. The utilization of optical fiber to transmit the excitation light and the integration fiber channel make the fluorescence detection system simple and disposable. For electrophoresis, optimization of separation conditions was investigated for reaching high separation efficiency and sensitivity. A separation efficiency as high as 10⁶ theoretical plate numbers could be obtained for the analytes.     

基于硒化镓纳米片填充的空芯光纤超高二次谐波增强

与传统的光学晶体相比,全光纤功能器件由于和光纤系统的天然兼容性,被认为是下一代集成光学的重要研究方向,吸引了人们的广泛关注。然而,由于二氧化硅固有的中心反演对称性质,光纤中的二阶非线性光学过程仍有待探索,这在可调谐超快激光、全光信号处理、成像和光通信等商业全光纤非线性光学应用中具有重要意义。因此,我们提出了一种新的溶液填充方法,可有效地将具有高非线性的硒化镓纳米片直接沉积在长度达半米的空芯光纤(HCF)的内孔壁上。此外,采用制备的硒化镓纳米片-空芯光纤(GaSe-HCF)作为光频率转换器,其二次谐波(SHG)比嵌入MoS₂的HCF和普通HCF分别提高了2个数量级和3个数量级。我们的研究成果将拓展其它非线性材料在全光纤高端非线性光学和光电子学中的应用,并提供新的制备思路。     

Single-molecule triplet-state photon antibunching at room temperature

We have probed single-molecule metal-to-ligand charge transfer (MLCT) dynamics of a ruthenium complex at room temperature. Using photon antibunching measurements under continuous wave (CW) laser excitation, nonclassical photon statistics, and excitation power dependent measurements, we were able to selectively measure the single-molecule MLCT state lifetime. This work demonstrated, as the first single-molecule photon antibunching measurement of the triplet excited state, a new application of single-molecule spectroscopy on excited-state dynamics and ground-state recovering dynamics of an important class of chemical species that have often been used and studied in energy conversion and electron transfer.