Characterization of DNA-protein complexes by capillary electrophoresis-single molecule fluorescence correlation spectroscopy.

A high-speed capillary electrophoresis mobility shift assay (CEMSA) for determining the binding ratios of DNA-protein complexes in solution is demonstrated. Single molecule fluorescence correlation spectroscopy (FCS) was used to resolve the bound and unbound fluorescently labeled DNA molecules as they flowed continuously through a fused silica capillary under the influence of an applied electric field. Resolution of the bound and unbound complexes was based on the difference in their electrophoretic mobilities, and was accomplished without the need to perform a chemical separation. Data sufficient to perform the analysis was acquired in less than 10 s, compared to the minutes that are normally needed to carry out such measurement via CE separation. The binding ratios were determined with 5 to 10% precision and agreed with the results obtained by CE separation within experimental error. The resolution of the CEMSA based FCS analysis (CEMSA-FCS) was significantly higher than for the analysis performed by conventional diffusional FCS, due to the higher mass sensitivity of the electrophoretic mobility compared to the translational diffusion coefficient. Fluorescently labeled 39-mer single stranded DNA (ssDNA) and the single stranded binding protein (SSB) from Escherichia coli was used as the model system. The dissociation constant of the ssDNA-SSB complex was estimated to be approximately 2 nM based on the CEMSA-FCS analysis.

Spatially correlated fluorescence/AFM of individual nanosized particles and biomolecules.

Individual fluorescent polystyrene nanospheres (<10-100-nm diameter) and individual fluorescently labeled DNA molecules were dispersed on mica and analyzed using time-resolved fluorescence spectroscopy and atomic force microscopy (AFM). Spatial correlation of the fluorescence and AFM measurements was accomplished by (1) positioning a single fluorescent particle into the near diffraction-limited confocal excitation region of the optical microscope, (2) recording the time-resolved fluorescence emission, and (3) measuring the intensity of the excitation laser light scattered from the apex of an AFM probe tip and the AFM topography as a function of the lateral position of the tip relative to the sample substrate. The latter measurements resulted in concurrent high-resolution (approximately 10-20 nm laterally) images of the laser excitation profile of the confocal microscope and the topography of the sample. Superposition of these optical and topographical images enabled unambiguous identification of the sample topography residing within the excitation region of the optical microscope, facilitating the identification and structural characterization of the nanoparticle(s) or biomolecule(s) responsible for the fluorescence signal observed in step 2. These measurements also provided the lateral position of the particles relative to the laser excitation profile and the surrounding topography with nanometer-scale precision and the relationship between the spectroscopic and structural properties of the particles. Extension of these methods to the study of other types of nanostructured materials is discussed.

Detection of the linear carbon cluster C10: rotationally resolved diode-laser spectroscopy.

Detected in interstellar space and as intermediates in soot formation, molecules of pure carbon in the form of linear chains or ring structures have interested researchers for several decades, who attempt to elucidate their physical properties and the processes govering their formation. A high-resolution infrared spectrometer housing a tunable diode laser and combined with an effective laser ablation source for the cluster production has been used to study the molecular properties of small carbon clusters; reported herein is the first gas-phase spectrum of linear C10.

High-throughput flow cytometric DNA fragment sizing.

The rate of detection and sizing of individual fluorescently labeled DNA fragments in conventional single-molecule flow cytometry (SMFC) is limited by optical saturation, photon-counting statistics, and fragment overlap to approximately 100 fragments/s. We have increased the detection rate for DNA fragment sizing in SMFC to approximately 2000 fragments/s by parallel imaging of the fluorescence from individual DNA molecules, stained with a fluorescent intercalating dye, as they passed through a planar sheet of excitation laser light, resulting in order of magnitude improvements in the measurement speed and the sample throughput compared to conventional SMFC. Fluorescence bursts were measured from a fM solution of DNA fragments ranging in size from 7 to 154 kilobase pairs. A data acquisition time of only a few seconds was sufficient to determine the DNA fragment size distribution. A linear relationship between the number of detected photons per burst and the DNA fragment size was confirmed. Application of this parallel fluorescence imaging method will lead to improvements in the speed, throughput, and sensitivity of other types of flow-based analyses involving the study of single molecules, chromosomes, cells, etc.

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.

Detection system for reaction-rate analysis in a low-volume proteinase-inhibition assay.

High-throughput screening of large combinatorial chemical libraries in biochemical assays will benefit from reduced reagent volume and increased speed of measurement. Standard assays typically are performed in 96-well microtiter plates having 200-microL well volumes and up to an hour of incubation time. In this paper, we demonstrate a technique for precise and rapid measurement of the progress of an enzymatic reaction and its inhibition with reduced volume and time (for this work, the assay was mixed at the 200-microL level and detected in 2-microL volumes with minutes of total assay time). Directly measuring the enzyme activity in the small volume format yields a precise value for the median inhibitory concentration (IC50) of an inhibitor compound. The model assay is the endoproteolytic cleavage of a small fluorogenic peptide by human neutrophil collagenase (MMP-8). The fluorogenic peptide was labeled at one end with a UV/blue fluorophore (N-methylanthranilyl) and at the other end with a quencher (dinitrophenol). To generate inhibition data, a hydroxamate peptide analog inhibitor of collagenase, actinonin, was included in the reaction. The experiments were performed using ultraviolet laser illumination (325 nm wavelength) and parallel fluorescence detection by a cooled, charge-coupled-device camera system to increase sensitivity and speed. The assay volume was reduced to 2 microL for data collection, and the total time for mixing, incubation, and measurement was less than 6 min. For comparison to a standard format, the same assay was performed in a 96-well microtiter plate in 200 microL using 30 min of incubation and measurement in a microtiter plate fluorimeter. Median inhibitory concentrations (IC50) for actinonin of 73 +/- 16 and 100 +/- 14 nM were obtained in the 2- and 200-microL assays, respectively. One concern with assay miniaturization and increases in throughput is a potential loss of precision and accuracy. Laser excitation and parallel detection of fluorescence is a promising approach for increased speed and reduced cost without loss of precision for proteinase inhibition assays.

Fluorescence correlation spectroscopy for rapid multicomponent analysis in a capillary electrophoresis system.

We describe a new technique for performing multicomponent analysis using a combination of capillary electrophoresis (CE) and fluorescence correlation spectroscopy (FCS), which we refer to as CE/FCS. FCS is a highly sensitive and rapid optical technique that is often used to perform multicomponent analysis in static solutions based on the different diffusion times of the analyte species through the detection region of a tightly focused laser beam. In CE/FCS, transit times are measured for a mixture of analytes continuously flowing through a microcapillary in the presence of an electric field. Application of an electric field between the inlet and outlet of the capillary alters the transit times, depending on the magnitude and polarity of the applied field and the electrophoretic mobilities of the analytes. Multicomponent analysis is accomplished without the need to perform a chemical separation, due to the different electrophoretic mobilities of the analytes. This technique is particularly applicable to ultradilute solutions of analyte. We have used CE/FCS to analyze subnanomolar aqueous solutions containing mixtures of Rhodamine 6G (R6G) and R6G-labeled deoxycytosine triphosphate nucleotides. Under these conditions, fewer than two molecules were typically present in the detection region at a time. The relative concentrations of the analytes were determined with uncertainties of ∼10%. Like diffusional FCS, this technique is highly sensitive and rapid. Concentration detection limits are below 10(-)(11) M, and analysis times are tens of seconds or less. However, CE/FCS does not require the diffusion coefficients of the analytes to be significantly different and can, therefore, be applied to multicomponent analysis of systems that would be difficult or impossible to study by diffusional FCS.

Single-molecule identification in flowing sample streams by fluorescence burst size and intraburst fluorescence decay rate.

We report a multiplex technique for identification of single fluorescent molecules in a flowing sample stream by correlated measurement of single-molecule fluorescence burst size and intraburst fluorescence decay rate. These quantities were measured simultaneously for single fluorescent molecules in a flowing sample stream containing a dilute mixture of fluorescent species: Rhodamine 6G and tetramethylrhodamine isothiocyanate. Using a detailed Monte Carlo simulation of our experiment, we calculate single-molecule detection efficiencies and confidence levels for identification of these species and identify major sources of error for single-molecule identification. The technique reported here is applicable to distinguishing between fluorophores with similar spectroscopic properties and requires only a single excitation wavelength and single fluorescence emission detection channel.

Infrared laser spectroscopy of jet-cooled carbon clusters: the nu 5 band of linear C9.

The nu 5 antisymmetric stretching vibration of 1 sigma+g C9 has been observed using direct infrared diode laser absorption spectroscopy of a pulsed supersonic cluster beam. Twenty-eight rovibrational transitions measured in the region of 2079-2081 cm-1 were assigned to this band. A combined least squares fit of these transitions with previously reported nu 6 transitions yielded the following molecular constants for the nu 5 band: nu 0 = 2 079.673 58(17) cm-1, B"= 0.014 321 4(10) cm-1, and B'=0.014 288 9(10) cm-1. The IR intensity of the nu 5 band relative to nu 6 was found to be 0.108 +/- 0.006. Theoretical predictions for the relative intensities vary widely depending upon the level of theory employed, and the experimental value reported here is in reasonable agreement only with the result obtained from the most sophisticated ab initio calculation considered (CCSD).

Characterization of silicon-carbon clusters by infrared laser spectroscopy. The nu 1 band of SiC4.

The nu 1 fundamental vibration of linear SiC4 has been observed by infrared diode laser spectroscopy of a supersonic cluster beam. Twenty-four rovibrational transitions were measured in the spectral region of 2094.6 to 2097.1 cm-1, the rotational temperature was 10 K. A combined least-squares fit of these transitions with previously reported microwave data yielded the following molecular constants: nu 1 = 2095.45806(37) cm-1, B" = 0.051161131(52) cm-1, and B' = 0.0509157(96) cm-1. These results are compared to vibrational spectroscopy measurements of SiC4 trapped in a solid Ar matrix and to ab initio calculations.

Characterization of silicon-carbon clusters by infrared laser spectroscopy: the nu 3(sigma u) band of linear Si2C3.

The nu 3(sigma u) fundamental vibration of 1 sigma g+ Si2C3 has been observed using a laser vaporization-supersonic cluster beam-diode laser spectrometer. Forty rovibrational transitions were measured in the range of 1965.8 to 1970.9 cm-1 with a rotational temperature of 10-15 K. A least-squares fit of these transitions yielded the following molecular constants: nu 3(sigma u)=1968.188 31(18) cm-1, B"=0.031 575 1(60) cm-1, and B'=0.031 437 4(57) cm-1. These results are in excellent agreement with recent Fourier transform infrared (FTIR) measurements of Si2C3 trapped in a solid Ar matrix [J. Chem. Phys. 100, 181(1994)] and with ab initio calculations [J. Chem. Phys. 100, 175 (1994)] which suggest cumulenic-like bonding for Si2C3, analogous to the isovalent C5 carbon cluster.

Infrared laser spectroscopy of the linear C13 carbon cluster.

The infrared absorption spectrum of a linear, 13-atom carbon cluster (C13) has been observed by using a supersonic cluster beam-diode laser spectrometer. Seventy-six rovibrational transitions were measured near 1809 wave numbers and assigned to an antisymmetric stretching fundamental in the 1 sigma g+ ground state of C13. This definitive structural characterization of a carbon cluster in the intermediate size range between C10 and C20 is in apparent conflict with theoretical calculations, which predict that clusters of this size should exist as planar monocyclic rings.

Infrared laser spectroscopy of jet-cooled carbon clusters: the bending dynamics of linear C9.

We report improved measurements for the nu 6 antisymmetric stretch fundamental and observation of the (nu 6 + nu 15)-nu 15 and (nu 6 + 2 nu 15)-2 nu 15 hot bands of the linear C9 carbon cluster by direct absorption diode laser spectroscopy of a supersonic carbon cluster beam. Analysis of these bands characterizes C9 as a semirigid molecule with a bending potential similar to that of C5 and further evidences the alternation in degree of rigidity of linear carbon clusters with the g-u symmetry of the HOMO.

Infrared laser spectroscopy of jet-cooled carbon clusters: structure of triplet C6.

We report the first structural characterization of the triplet isomer of C6. Forty-one rovibrational/fine structure transitions in the nu 4(sigma u) antisymmetric stretch fundamental of the C6 cluster have been measured by diode laser absorption spectroscopy of a supersonic carbon cluster beam. The observed spectrum is characteristic of a centrosymmetric linear triplet state with cumulene-type bonding. The measured ground state rotational constant B0 = 0.048 479 (10)cm-1 and the effective bond length r(eff) = 1.2868 (1) angstroms are in good agreement with ab initio predictions for the linear triplet (3 sigma g-) state of C6.

Immunofluorescence and Treponema infection: a method using immunofluorescence to study rabbit testicular tissue infected with T pallidum and T pertenue.

We compared immunofluorescent staining of rabbit testicular tissue infected with Treponema pallidum or T pertenue, and fixed in Bouin's fixative, 95% cold ethyl alcohol with 1% glacial acetic acid, or routine 10% buffered formalin solution. The fixative of choice clearly was Bouin's. Although we studied only rabbit tissue, we assume that these fixatives will work well in human biopsy or autopsy material when identification of pathogenic Treponema is needed.

Effect of various histological fixatives on fluorescent antibody detection of Legionnaires disease bacteria.

Human lung tissue containing the Legionnaires disease bacterium was fixed in seven different histological fixatives, processed, and embedded in paraffin. Deparaffined sections from each were stained by fluorescent antibody and by Dieterle silver impregnation. With the fluorescent antibody stain, the Legionnaires disease bacterium could be detected in tissues prepared with any of the fixatures, but the Dieterle silver impregnation was not satisfactory on Zenker-fixed tissues.