W. Patrick Ambrose

Single-Molecule Fluorescence Analysis in Solution

Over the past five years, several groups have developed the capability to detect and identify single fluorescent molecules in solution as the molecules flow through a focused laser beam. The history of the approach to single-molecule detection in fluid solution is shown in Fig. 1. Approximately one dozen molecular species have been detected at this level of sensitivity. Fluorescence-based, single-molecule detection techniques are expected to have a significant impact in fields where fluorescence detection and quantification are broadly applied, e.g., analytical chemistry, biology, and medicine. Single-molecule detection is a new way of doing analytical chemistry, and new applications will arise. In this article, we describe our approach to single-molecule detection and explore assays that can be done at the single-species level that would be difficult or impossible with bulk measurements.

Detection and lifetime measurement of single molecules in flowing sample streams by laser-induced fluorescence

The detection and measurement of fluorescence lifetimes of single Rhodamine‐110 molecules in a flowing, aqueous sample stream is described. Time‐correlated single‐photon counting, used in combination with mode‐locked picosecond pulsed excitation, allows the detection of single fluorescent molecules in the presence of significant solvent Raman and Rayleigh backgrounds. The fluorescence lifetime of a detected molecule is estimated from the record of arrival times (relative to the excitation pulse) of photons detected during the molecule’s passage through the ∼1 pL excitation volume.

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.

A maximum likelihood estimator to distinguish single molecules by their fluorescence decays

Abstract We have developed a maximum likelihood estimator to distinguish between similar molecules at the single molecule level based upon fluorescence decay measurements. Time resolved fluorescence measurements for single Rhodamine 6G and tetramethylrhodamine isothiocyanate molecules in fluid flow are derived from time-correlated single photon counting. A maximum likelihood estimator is developed and applied to data from a mixture of molecules. Single molecules are identified and distinguished by their fluorescence time decays. Comparison is made between identification error rates and theoretical predictions. To our knowledge, this is the first reported example of single molecule identification by fluorescence decay in a mixture.

FLUORESCENCE PHOTON ANTIBUNCHING FROM SINGLE MOLECULES ON A SURFACE

Abstract Fluorescence correlation spectroscopy of individual Rhodamine 6G (R6G) molecules immobilized on a silica surface is performed in air at room temperature using confocal laser scanning optical microscopy (CLSM). The high excitation irradiance in CLSM is used to saturate single R6G molecules, and to observe fluorescence photon antibunching. An experimental arrangement is described that improves the overall coincidence counting efficiency at high irradiance. Photophysical parameters obtained from the saturation data are used to model the antibunching behavior. This is the first reported example of photon antibunching from individual quantum systems on a surface.

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.

Ultrasensitive detection of single molecules in flowing sample streams by laser-induced fluorescence

We report here on the detection and fluorescence lifetime measurement of single Rhodamine- 110 molecules in a flowing, aqueous sample stream. Time-correlated photon counting (TCPC) used in combination with pulsed excitation allows for the detection, in the presence of significant prompt Raman and Rayleigh background, of photon bursts due to single fluorescent molecules passing through a small detection volume (approximately 1 pL). The fluorescence lifetime of a detected molecule is estimated from the decay curve complied from photon arrival times in the burst.

Sizing of DNA fragments by flow cytometry

Individual, stained DNA fragments were sized using a modified flow cytometer with high sensitivity fluorescence detection. The fluorescent intercalating dye ethidium homodimer was used to stain stoichiometrically lambda phage DNA and a Kpn I digest of lambda DNA. Stained, individual fragments of DNA were passed through a low average power, focused, mode-locked laser beam, and the fluorescence from each fragment was collected and quantified. Time-gated detection was used to discriminate against Raman scattering from the water solvent. The fluorescence burst from each fragment was related directly to its length, thus providing a means to size small quantities of kilobase lengths of DNA quickly. Improvements of several orders of magnitude in analysis time and sample size over current gel electrophoresis techniques were realized. Fragments of 17.1, 29.9, and 48.5 thousand base pairs were well resolved, and were sized in 164 seconds. Less than one pg of DNA was required for analysis.

Peer Reviewed: Analytical Applications of Single-Molecule Detection

Single-species-level assays that would be difficult or impossible with conventional bulk measurements can now be performed with laser-induced fluorescence

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.