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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.