Single-mode fiber, velocity interferometry.

In this paper, we describe a velocity interferometer system based entirely on single-mode fiber optics. This paper includes a description of principles used in developing the single-mode velocity interferometry system (SMV). The SMV design is based on polarization-insensitive components. Polarization adjusters are included to eliminate the effects of residual birefringence and polarization dependent losses in the interferometers. Characterization measurements and calibration methods needed for data analysis and a method of data analysis are described. Calibration is performed directly using tunable lasers. During development, we demonstrated its operation using exploding-foil bridge-wire fliers up to 200 m/s. In a final test, we demonstrated the SMV in a gas gun experiment up to 1.2 km/sec. As a basis for comparison in the gas gun experiment, we used another velocimetry technique that is also based on single-mode fiber optics: photonic Doppler velocimetry (PDV). For the gas gun experiment, we split the light returned from a single target spot and performed a direct comparison of the homodyne (SMV) and heterodyne (PDV) techniques concurrently. The two techniques had a negligible mean difference and a 1.5% standard deviation in the one-dimensional shock zone. Within one interferometer delay time after a sudden Doppler shift, a SMV unencumbered by multimode-fiber dispersion exhibits two color beats. These beats have the same period as PDV beats-this interference occurs between the "recently" shifted and "formerly unshifted" paths within the interferometer. We believe that recognizing this identity between homodyne and heterodyne beats is novel in the shock-physics field. SMV includes the conveniences of optical fiber, while removing the time resolution limitations associated with the multimode delivery fiber.

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.

Effects of fluorescence excitation geometry on the accuracy of DNA fragment sizing by flow cytometry.

We report on various excitation geometries used in ultrasensitive flow cytometry that yield a linear relation between the fluorescence intensity measured from individual stained DNA fragments and the lengths of the fragments (in base pairs). This linearity holds for DNA samples that exhibit a wide range of conformations. The variety of DNA conformations leads to a distribution of dipole moment orientations for the dye molecules intercalated into the DNA. It is consequently important to use an excitation geometry such that all dye molecules are detected with similar efficiency. To estimate the conformation and the extent of elongation of the stained fragments in the flow, fluorescence polarization anisotropy and autocorrelation measurements were performed. Significant extension was observed for DNA fragments under the flow conditions frequently used for DNA fragment sizing. Classical calculations of the fluorescence emission collected over a finite solid angle are in agreement with the experimental measurements and have confirmed the relative insensitivity to DNA conformation of an orthogonal excitation geometry. Furthermore, the calculations suggested a modified excitation geometry that has increased our sizing resolution.

Single-molecule detection with total internal reflection excitation: comparing signal-to-background and total signals in different geometries.

Excitation of fluorescence with total internal reflection (TIR) excitation yields very low background scattered light and good signal-to-background contrast. The background and its associated noise can be made low enough to detect single fluorescent molecules under ambient conditions. In this paper, different TIR geometries were compared for excitation and detection of single rhodamine 6G (R6G) molecules at air-silica interfaces and single B-phycoerythrin proteins at water-silica interfaces. Through-objective, objective-coverslip, and prism-based TIR geometries were investigated. The signal-to-background ratio (SBR) and the number of photons detected before photobleaching (Nb) were optimum in different geometries. The greatest image contrast was obtained when using prism-TIR (SBR = 11.5), but the largest number of detected signal photoelectrons was obtained by using through-objective TIR for R6G-air-silica ( = 10(4)). The results were discussed in terms of the TIR field enhancements and the modified dipole emission pattern near a dielectric interface. The SBR and total detected photons are important parameters for designing photon-limited experiments.

Micrometer dimension derivatization of biosensor surfaces using confocal dynamic patterning.

Using laser scanning confocal optics in conjunction with avidin/biotin technology, micrometer-sized patterns of biomolecules were fabricated on glassy-carbon and fused-silica surfaces. Photoactive biotin was immobilized using the 325-nm line of a Helium-Cadmium laser, which was focused through a 25x or 100x quartz microscope objective. A three-dimensional piezoelectric micromanipulator was used to position the sample surface in the focal plane of the microscope objective and to create patterns on the focused surface. Biotin patterns with line widths of 5-20 microns were produced by varying the scan speed of the micromanipulator while exposing the surface to the laser. The integrity of the immobilized biotin was confirmed by subsequent derivatization with fluorescently labeled avidin. Fluorescence microscopy with a cooled charge coupled device (CCD) imaging system was used to visualize the distribution of biotin and fluorescent avidin within the patterns created by the laser.

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.

Optical collection efficiency function in single-molecule detection experiments.

The optical collection efficiency function for an optical system such as that used in single-molecule detection experiments is studied. Closed analytical expressions based on a geometrical optics approximation are presented. Comparison is made with exact wave optics calculations.

Reduction of luminescent background in ultrasensitive fluorescence detection by photobleaching.

In luminescence-based ultrasensitive analysis, such as single-molecule detection by flow cytometry, the luminescence background from impurities present in the solvent or reagents can ultimately determine the detection limits. A simple, versatile method for reducing luminescence background is described. The method is based on photobleaching the reagent stream immediately before it enters the detection flow cell. Dramatic reduction (an order of magnitude or more) of both low-level continuous background and single-molecule fluorescence bursts is demonstrated. Application and enhancements of the technique are discussed.

Spatial dependence of the optical collection efficiency in flow cytometry.

The sensitive flow cytometric detection of fluorescent species in liquid sample streams requires efficient collection of light from small [approximately 1 picoliter (pl)] sample volumes. This is often accomplished with high numerical aperture (NA) imaging collection optics used in combination with a spatial filter. A method to measure the spatial variation of the optical collection efficiency within the sample volume, using a submicrometer light source, is described. Measurements of the relative optical collection efficiency are presented for two optical collection systems used in our laboratory for single molecule detection. The measurement are in qualitative agreement with relative optical collection efficiency calculations using a simple geometrical optics model. Absolute measurements of the peak collection efficiencies for the two collection systems are also presented. These absolute collection efficiency measurements are in good quantitative agreement with ideal collection efficiencies calculated using measured transmissions and rated NAs of the collection optics. The utility of this information for the characterization and optimization of sensitive fluorescence detection apparatus is discussed.

Alterations of single molecule fluorescence lifetimes in near-field optical microscopy.

Fluorescence lifetimes of single Rhodamine 6G molecules on silica surfaces were measured with pulsed laser excitation, time-correlated single photon counting, and near-field scanning optical microscopy (NSOM). The fluorescence lifetime varies with the position of a molecule relative to a near-field probe. Qualitative features of lifetime decreases are consistent with molecular excited state quenching effects near metal surfaces. The technique of NSOM provides a means of altering the environment of a single fluorescent molecule and its decay kinetics in a repeatable fashion.

Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry.

Large, fluorescently stained restriction fragments of lambda phage DNA are sized by passing individual fragments through a focused continuous wave laser beam in an ultrasensitive flow cytometer at a rate of 60 fragments per second. The size of the fluorescence burst emitted by each stained DNA fragment, as it passes through the laser beam, is measured in one millisecond. One hundred sixty four seconds of fluorescence burst data allow linear sizing of DNA with an accuracy of better than two percent over a range of 10 to 50 kbp. This corresponds to analyzing less than 1 pg of DNA. Sizing of DNA fragments by this approach is much faster, requires much less DNA, and can potentially analyze large fragments with better resolution and accuracy than with gel-based electrophoresis.

Persistent infrared hole-burning spectroscopy of matrix-isolated CN(-) molecules.

The union of persistent IR hole burning and Fourier-transform IR techniques is used to identify a new type of photophysical vibrational hole-burning center. The persistent signature for matrix-isolated CN(-) complexes in alkali-halide crystals is similar to that found for photochemical hole burning in solids.