Jörg Enderlein

Fast fitting of multi-exponential decay curves

Abstract In the analysis of time-resolved measurements of fluorescence decays, one is usually confronted with the essentially non-linear fitting problems. There are several standard methods for non-linear minimisation, but they are all very sensitive to initial guess parameters and are time-consuming. Recently, Sasaki and Masuhara and independently Apanasovich and Novikov proposed and elegant method of quasi-linearising the problem of multi-exponential fitting. In the present paper it will be shown, that their method can be improved by better taking into account the statistical character of the measured data.

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.

Time-Resolved Luminescence Imaging of Hydrogen Peroxide Using Sensor Membranes in a Microwell Format:

We demonstrate an optical imaging scheme for hydrogen peroxide in a microwell-based format using the europium(III) tetracycline complex as the fluorescent probe, which is incorporated into a polyacrylonitrile-co-polyacrylamide polymer matrix. The resulting sensor membranes are integrated into a 96-microwell plate. Hydrogen peroxide can be visualized by means of time-resolved luminescence lifetime imaging. The imaging system consists of a fast, gated charge-coupled device (CCD) camera and a pulsed array of 96 light emitting diodes (LEDs). Fluorescence lifetime images are acquired in different modes (rapid lifetime determination, RLD, and phase delay rationing, PDR) and compared with conventional intensity-based methods with respect to sensitivity and the dynamic range of the sensor. The lowest limits of detection can be achieved by the RLD method. The response time of the sensor is comparatively high, typically in the range of 10 to 20 minutes, but the response is reversible. The largest signal changes are observed at pH values between 6.5 and 7.5.

Efficient detection of single molecules eluting off an optically trapped microsphere

We demonstrate efficient detection of single fluorescent molecules eluting off a polystyrene microsphere optically trapped in a flowing sheath stream. A 1μm diameter analyte doped microsphere was positioned ∼20 μm upstream of a 16 μm diameter probe laser without significant degradation of the detection signal-to-noise ratio due to scattered laser light and fluorescence from the microsphere. In comparison to more standard capillary sample introduction, the microsphere causes only small perturbations to the sheath fluid flow. The small diameter of the analyte stream eluting from the microsphere results in a greater than 90% detection efficiency for single rhodamine-6G molecules, limited primarily by the photostability of the dye.

Comparison between time-correlated single photon counting and fluorescence correlation spectroscopy in single molecule identification

Currently, two methods of detection and identification of single molecules are widely used: fluorescence correlation spectroscopy (FCS) and time-correlated single photon counting (TCSPC). We present a thorough theoretical analysis of the error rates for identifying single molecules according to their diffusion coefficients (using FCS), and to their fluorescence lifetimes (using TCSPC). In most cases, the error rate using TCSPC is much lower. TCSPC is thus proven to be more versatile for analyzing single molecule events. The study is significant for a broad range of ultra-sensitive fluorescence detection applications.

Front Matter: Volume 7185

This PDF file contains the front matter associated with SPIE Proceedings Volume 7185, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing

Fluorescence detection of single molecules applicable to small volume assays

In this chapter we give an overview of optical detection of single molecules with respect to possible application to high throughput screening of substance libraries. We restrict ourselves to single molecule detection (SMD) because the field of ultrasensitive detection is too broad to be covered by a single chapter; thus we will concentrate on the ultimate detection limit In the future SMD will be the method of choice because of the ultimate small amount of analyte needed in connection with extremely high speed screening of vast libraries. Besides the topic of high-speed DNA sequencing (see below), the high sensitivity received attention also in the context of combinatorial chemistry (Eigen, 1984; Pluckthun,1991; Eigen, 1994; Xiang, 1995) and molecular computation (Adleman, 1994; Lipton, 1995). The basic idea of combinatorial chemistry is to synthesize large arrays of slightly different molecules, followed by screening and selection of molecules with desired properties. In the case of molecular computation, a random chemical synthesis is used to find solutions of numerically difficult problems; again, a fast and ultrasensitive screening method is needed to extract the result of the “computation”.

Comparison of One-Focus and Two-Foci Setup in Single-Molecule Detection Experiments

Single molecule detection in fluid flow has become a widely used technique in ultrasensitive fluorescence detection. The detection of a single molecule in a fluid flow at room temperature is limited by effects such as background signals from the surrounding liquid and possible contamination in it, photobleaching effects which restrict the overall number of detectable photons from one molecule, and diffusion can cause the molecule to miss the exciting laser beam when flowing along the capillary. A method for enhancing the single molecule detection efficiency is the use of multiple excitation-detection regions along the capillary and the application of an analysis for correlating the photon detection data from the different regions. In this note we demonstrate that, at a constant laser power, no improvement results using time-correlated detection from multiple-foci regions. (AIP) {copyright} {ital 1997} {ital Society for Applied Spectroscopy}