Rainer Erdmann

Hardware solution for continuous time resolved burst detection of single molecules in flow

Time Correlated Single Photon Counting (TCSPC) is a valuable tool for Single Molecule Detection (SMD). However, existing TCSPC systems did not support continuous data collection and processing as is desirable for applications such as SMD for e.g. DNA-sequencing in a liquid flow. First attempts at using existing instrumentation in this kind of operation mode required additional routing hardware to switch between several memory banks and were not truly continuous. We have designed a hard- and software system to perform continuous real-time TCSPC based upon a modern solid state Time to Digital Converter (TDC). Short dead times of the fully digital TDC design combined with fast Field Programmable Gay Array logic permit a continuous data throughput as high as 3 Mcounts/sec. The histogramming time may be set as short as 100 microsecond(s) . Every histogram or every single fluorescence photon can be real-time tagged at 200 ns resolution in addition to recording its arrival time relative to the excitation pulse. Continuous switching between memory banks permits concurrent histogramming and data read-out. The instrument provides a time resolution of 60 ps and up to 4096 histogram channels. The overall instrument response function in combination with a low cost picosecond diode laser and an inexpensive photomultiplier tube was found to be 180 ps and well sufficient to measure sub-nanosecond fluorescence lifetimes.

Dead-time effects in TCSPC data analysis

In Time Correlated Single Photon Counting (TCSPC) the maximum signal throughput is limited by the occurrence of classical pile-up and dead-time effects: At a given photon rate characteristic distortions become visible in the TCSPC histogram. How to describe these distortions in mathematical terms is well known1,2. While the approach of correcting these distortions directly by operations on the raw data has drawbacks, e.g. with respect to calculation effort as well as numerical stability, it is comparably straightforward to include corrections in the models describing the data. We demonstrate the applicability of a model based approach on decay data which are heavily distorted by dead-time and pile-up effects.

Two-channel fluorescence lifetime microscope with two colour laser excitation, single-molecule sensitivity, and submicrometer resolution

We present results from a two channel confocal microscope set-up allowing one to efficiently record two-colour as well as polarization resolved time-correlated single molecule fluorescence data. In addition to their spectral characteristics, single molecules can be distinguished by their fluorescence lifetime and polarization. This provides independent distinctive information and results in enhanced detection sensitivity. The set-up we present uses two picosecond diode lasers (440nm and 635 nm) for fluorescence excitation and a piezo scanner for sample movement. A learning scan algorithm permits very fast piezo scanner movement and offers a superior positioning accuracy on single molecules. The time-correlated photon counting system uses Time-Tagged Time-Resolved (TTTR) data aquisition, in which each photon is recorded individually. This method allows for the reconstruction not only fluorescence decay constants of each pixel for the purpose of Fluorescence Lifetime Imaging (FLIM) but also to analyze the fluorescence fluctuation correlation function on a single spot of interest. Cross-correlation between two channels can be used to eliminate detector artifacts. Finally, fluorescence antibunching can also be analyzed. We show results obtained with immobilized and diffusing red and blue excited fluorescently labelled latex microspheres, as well as from single fluorophore molecules.

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

Application of sub-ns pulsed LEDs in fluorescence lifetime spectroscopy

Lifetime analysis of laser induced fluorescence by means of Time-Correlated Single Photon Counting (TCSPC) provides a powerful discrimination method to distinguish molecules of interest from background and other species. This has made the technique extremely valuable for sensitive analysis down to the single molecule level. We have developed the first complete range of compact picosecond to nanosecond excitation sources for fluorescence lifetime measurements based on laser diodes and LEDs. Using a common driver with interchangeable LED and laser heads the system is adaptable to almost all of the needs for sensitive chemical and biochemical analysis. The sources provide pulse durations under one nanosecond and repetition rates up to 80 MHz. These features qualify them for use in fast TCSPC applications, in particular where short data acquisition time is crucial. The sources can be used in combination with common inexpensive single photon detectors such as Photomultiplier Tubes and Single Photon Avalanche Photodiodes. Compact, low cost and easy to use fluorescence lifetime spectrometers can be built from these sources together with integrated TCSPC electronics. We will demonstrate the performance of the sources and complete systems in terms of power, repetition rate, stability, IRF and fluorescence decay fit quality in various setups and with different fluorescent materials.

Time-resolved confocal fluorescence microscopy: novel technical features and applications for FLIM, FRET and FCS using a sophisticated data acquisition concept in TCSPC

In recent years time-resolved fluorescence measurement and analysis techniques became a standard in single molecule microscopy. However, considering the equipment and experimental implementation they are typically still an add-on and offer only limited possibilities to study the mutual dependencies with common intensity and spectral information. In contrast, we are using a specially designed instrument with an unrestricted photon data acquisition approach which allows to store spatial, temporal, spectral and intensity information in a generalized format preserving the full experimental information. This format allows us not only to easily study dependencies between various fluorescence parameters but also to use, for example, the photon arrival time for sorting and weighting the detected photons to improve the significance in common FCS and FRET analysis schemes. The power of this approach will be demonstrated for different techniques: In FCS experiments the concentration determination accuracy can be easily improved by a simple time-gated photon analysis to suppress the fast decaying background signal. A more detailed analysis of the arrival times allows even to separate FCS curves for species which differ in their fluorescence lifetime but, for example, cannot be distinguished spectrally. In multichromophoric systems like a photonic wire which undergoes unidirectional multistep FRET the lifetime information complements significantly the intensity based analysis and helps to assign the respective FRET partners. Moreover, together with pulsed excitation the time-correlated analysis enables directly to take advantage of alternating multi-colour laser excitation. This pulsed interleaved excitation (PIE) can be used to identify and rule out inactive FRET molecules which cause interfering artefacts in standard FRET efficiency analysis. We used a piezo scanner based confocal microscope with compact picosecond diode lasers as excitation sources. The timing performance can be significantly increased by using new SPAD detectors which enable, in conjunction with new TCSPC electronics, an overall IRF width of less than 120 ps maintaining single molecule sensitivity.

High-speed electronics for the detection of time-resolved fluorescence in a continuous flow system

In single molecule detection by laser induced fluorescence, a main problem is the low signal to noise ratio due to scattering of the exciting laser light. One common approach to solve this problem is the application of time resolved techniques. Here we present a high speed electronic (based on a pair of PC cards) specially suited for detection of TCSPC curves in a continuous flow system. The whole system works on two different time scales: a millisecond time scale (every millisecond a complete TCSPC curve is measured and stored) and a picosecond time scale (showing the fluorescence decay). The technique present here is of particular interest for applications such as fast DNA sequencing, where a distinction between the different bases solely by the decay times of the attached fluorescence labels is conceivable.

Comparison of a Yb-doped fiber and a semiconductor taper for amplification of picosecond laser pulses

In this paper, we compare two amplifier technologies, an ytterbium-doped fiber, and a semiconductor taper, regarding their suitability to amplify sub-100ps pulses. With both setups, we obtain amplification of the pulse energy by more than 10dB. However, suppression of self-lasing in the pulse intervals is critical, particularly in the case of the semiconductor amplifier which has a very short upper state lifetime. The seed pulses were generated by a gain-switched distributed feedback (DFB) laser at arbitrary repetition rates. They have widths of below 100ps FWHM, pulse energies of over 100pJ and a peak power of over 700mW. This makes them very suitable for optical amplification. With the fiber amplifier, the pulse form remained unchanged, while with the semiconductor amplifier, it broadened slightly. With both amplifiers, pulse peak powers of over 8W were obtained, which represents an amplification by more than 10dB.

High-repetion-rate picosecond diode lasers for potential use in single molecule detection

Recently, major advances have been reached in the fluorescence detection of small amounts of molecules in liquids, making possible even the detection of single molecules in liquid flows. Significant improvements of fluorescence detection techniques make single molecule detection feasible for many applications, especially in the field of molecular biology and genetics. For such techniques new compact and inexpensive lasers are desirable. laser diode systems are the most favorable candidates for such light sources. With the expanding number of available NIR-fluorescent dyes, the importance of cheap and reliable laser light sources above 630 nm will increase. But not only cw-laser systems are of growing interest. In a number of recent papers, the application of time-resolved fluorescence detection down to a single molecule level was shown to be of great use for further improving detection efficiency. Thus, one needs high- repetition rate pulsed laser diode systems with good time and optical performance, and detection electronics with high-speed and large data throughput. Here we present such a system, combining a pulsed diode laser system with excellent electrical and optical parameters, and a high speed electronic for time correlated single photon counting. This system is suitable for a broad range of applications in ultra sensitive fluorescence detection.

Simultaneous detection of time-resolved emission spectra using a multianode PMT and new time-correlated single-photon counting (TCSPC) electronics with a 5-MHz count rate

We present data of simultaneously time and spectrally resolved measurements with the time correlated single photon counting technique using our new SPC-300 PC plug-in module combined with a 64 multianode PMT from Philips and a specially designed router. The SPC- 300 card records in up to 128 time channels simultaneously with a count rate of up to 5 MHz. In this paper we show the performance of the electronics for single decay curves using a MCP-PMT detector from Hamamatsu (R 3809U) and for wavelength resolved detection using an 8*8 multi-anode PMT from Philips (XP1702). An ultimate instrumental response function (IRF) of 34 ps and a maximum count rate of 300 000 cps was achieved by using the ultrafast MCP-PMT and a subtractive double monochromator. By using the multimode PMT coupled to a polychromator we got an IRF of 800 ps at 2.3*106 cps. The fluorescence signal was recorded at 8 different wavelengths simultaneously. For the test measurements we used pure Fluorescein, Rhodamin 6G and DODCI solution as well as a mixture of Fluorescein and DODCI. We got an excellent distinction between the two species. The decay times (3.9 ns, 1.1 ns) are in good agreement with the single curve measurement at a fixed wavelength.