Michael Gösch

First fully integrated 2-D array of single-photon detectors in standard CMOS technology

A two-dimensional (2-D) array (4 by 8) of single-photon avalanche diodes integrated in an industrial complementary metal-oxide-semiconductor (CMOS) process is presented. Each pixel combines a photodiode biased above its breakdown voltage in the so-called Geiger mode, a quenching resistor, and a simple comparator. The pitch between the pixels is 75 /spl mu/m and the diameter of each pixel is 6.4 /spl mu/m. The full integration allows reducing the number of charge carriers in a Geiger pulse. The electroluminescence responsible for optical crosstalks between pixels is then reduced leading to a negligible optical crosstalk probability. Thanks to the cleanness of the fabrication process, no afterpulsing effects are noticed. At room temperature, most of the pixels exhibit a dark-count rate of about 50 Hz. The detection probability is almost identical for all 32 pixels of the array with relative variation in the range of a few percents. This letter demonstrates the feasibility of an array of single-photon detectors sensitive in the visible part of the spectrum. Besides low production costs and compactness, an undeniable benefit lies in the potential to easily modify the design to fit a specific application. Furthermore, the CMOS integration opens the way to on-chip data processing.

Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule

We designed a fluorescence correlation spectroscopy (FCS) system for measurements on surfaces. The system consists of an objective-type total internal reflection fluorescence (TIRF) microscopy setup, adapted to measure FCS. Here, the fluorescence exciting evanescent wave is generated by epi-illumination through the periphery of a high NA oil-immersion objective. The main advantages with respect to conventional FCS systems are an improvement in terms of counts per molecule (cpm) and a high signal to background ratio. This is demonstrated by investigating diffusion as well as binding and release of single molecules on a glass surface. Furthermore, the size and shape of the molecule detection efficiency (MDE) function was calculated, using a wave-vectorial approach and taking into account the influence of the dielectric interface on the emission properties of fluorophores.

Dual-color total internal reflection fluorescence cross- correlation spectroscopy

We present the development and first application of a novel dual-color total internal reflection (TIR) fluorescence system for single-molecule coincidence analysis and fluorescence cross-correlation spectroscopy (FCCS). As a performance analysis, we measured a synthetic DNA-binding assay, demonstrating this dual-color TIR-FCCS approach to be a suitable method for measuring coincidence assays such as biochemical binding, fusion, or signal transduction at solid/liquid interfaces. Due to the very high numerical aperture of the epi-illumination configuration, our setup provides a very high fluorescence collection efficiency resulting in a two- to three-fold increase in molecular brightness compared to conventional confocal FCCS. Further improvements have been achieved through global analysis of the spectroscopic data.

Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements

Dual-color cross-correlation spectroscopy allows the detection and quantification of labeled biomolecules at ultra-low concentrations, whereby the sensitivity of the assay correlates with the measurement time. We now describe a parallel multifocal dual-color spectroscopic configuration employing multiple avalanche photodiodes and hardware correlators. Cross-correlation curves are obtained from several dual-color excitation foci simultaneously. Multifocal dual-color excitation is achieved by splitting each of two laser beams (488 and 633 nm) into four sub-beams with the help of two 2x2 fan-out diffractive optical elements (DOEs), and subsequent superposition of the two sets of four foci. The fluorescence emission from double-labeled biomolecules is detected by two 2x2 fiber arrays.

Prism-based multicolor fluorescence correlation spectrometer

We report the design and application of a prism-based detection system for fluorescence (cross) correlation spectroscopy. The system utilizes a single laser wavelength for the simultaneous excitation of several dyes of different emission spectra. Fluorescence light is spectrally separated with a prismatic setup, and wavelengths are selected by scanning a fiber-coupled avalanche photodiode across the image spots. Multicolor autocorrelations are demonstrated with standard and tandem dyes, and fluorescence cross-correlation measurements of biotinylated nanocontainers and streptavidin are presented. This spectrometer offers high optical stability and no focal volume mismatch for the multicolor detection of molecular dynamics and interactions, with single-molecule sensitivity.

Parallel Confocal Detection of Single Biomolecules using Diffractive Optics and Integrated Detector Units

The past few years we have witnessed a tremendous surge of interest in so-called array-based miniaturised analytical systems due to their value as extremely powerful tools for high-throughput sequence analysis, drug discovery and development, and diagnostic tests in medicine (see articles in Issue 1). Terminologies that have been used to describe these array-based bioscience systems include (but are not limited to): DNA-chip, microarrays, microchip, biochip, DNA-microarrays and genome chip. Potential technological benefits of introducing these miniaturised analytical systems include improved accuracy, multiplexing, lower sample and reagent consumption, disposability, and decreased analysis times, just to mention a few examples. Among the many alternative principles of detection-analysis (e.g.chemiluminescence, electroluminescence and conductivity), fluorescence-based techniques are widely used, examples being fluorescence resonance energy transfer, fluorescence quenching, fluorescence polarisation, time-resolved fluorescence, and fluorescence fluctuation spectroscopy (see articles in Issue 11). Time-dependent fluctuations of fluorescent biomolecules with different molecular properties, like molecular weight, translational and rotational diffusion time, colour and lifetime, potentially provide all the kinetic and thermodynamic information required in analysing complex interactions. In this mini-review article, we present recent extensions aimed to implement parallel laser excitation and parallel fluorescence detection that can lead to even further increase in throughput in miniaturised array-based analytical systems. We also report on developments and characterisations of multiplexing extension that allow multifocal laser excitation together with matched parallel fluorescence detection for parallel confocal dynamical fluorescence fluctuation studies at the single biomolecule level.