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.

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.

The features and the behavior of a modular ultra-fast time correlated single photon counting (TCSPC) system are analyzed. Using a Ti-Sapphire laser as excitation source an instrumental response function (IRF) of less than 17 ps for the total system was achieved. Despite this excellent IRF time there are at present some problems in analyzing fluorescence decay curves with such an ultra-fast IRF because of increased influence of color shift, time-spatial relationship, and electronic noise. Furthermore, we developed a new compact experimental set-up for TCSPC measurements consisting of laserdiodes including generator DL 4000 with pulse width less than 15 ps and the PC plug-in electronic card SPC 300. Thus the financial and spatial expense for TCSPC systems can be extremely reduced. Besides the traditional single channel data acquisition the new electronics allow parallel detection and acquisition of up to 128 channels simultaneously. This offers a lot of new applications for the highly sensitive TCSPC technique.

We will demonstrate the operation of the very compact all solid state fluorescence lifetime measurement system FLUO-TIME BQ 2759A. For this purpose we developed a new type of compact driving generator LD 4000 for a set of ps-laserdiodes with wavelengths between 630 nm and 690 nm, which will produce sub 50 ps pulses with up to 200 mW peak power and 3 MHz repetition rate. Using this miniaturized excitation source we are able to investigate a lot of red and NIR dyes. The fluorescence signal will be detected with single photon counting sensitivity by an ultrafast photomultiplier tube with only the size of the transistor (TO8 housing). Spectral resolution is given by a set of bandpass filters or a compact monochromator. With our recently introduced time correlated single photon counting (TCSPC) electronics SPC 300 (a PC-plug-in-card) we have a powerful instrument for data acquisition with highest data throughput. The instrumental response time (IRF) of the complete measurement system is less than 250 ps, allowing the investigation of fluorescence decay time components down to 25 ps using out deconvolution and analysis software package PHYSFIT. This performance can be improved to less than 90 ps IRF using a microchannel plate photomultiplier tube (MCP-PMT) detector. In this paper we demonstrate also the first practical application of this system to standard fluorescence dyes (oxazine, rhodamin).