No abstract

No abstract

Time-Correlated Single Photon Counting (TCSPC) is a very powerful method for sensitive time-resolved optical measurements. Its main application was historically the measurement of fluorescence lifetime. This application is still important, however, improvements over the early designs allow the recovery of much more information from the collected photons and enable entirely new applications. In conventional TCSPC instruments the timing signals are processed by a Time to Amplitude Converter (TAC) and subsequent Analog to Digital Converter (ADC) which provides digital values to address a histogrammer. TAC and ADC must guarantee a very good linearity and short dead time. These criteria are difficult to meet simultaneously, particularly at high resolution. Even with highest technically feasible ADC resolution, the time span such a system can measure at high temporal resolution is very limited. Here we present a new TCSPC system overcoming these limitations, based on Time to Digital Converters (TDC). This allows not only picosecond timing, but can also extend the measurable time span to virtually any length by means of digital counters. Our new design uses two such circuits that work independently on each input channel but with a common crystal clock. The timing circuits are therefore precisely synchronized and provide picosecond arrival times that can be processed further in any conceivable manner. Due to the symmetrical design without any ab initio assignment of dedicated start and stop inputs, the processing provides significantly more options than in conventional TCSPC systems, while still embracing the classical case. We present measurement data and results from applications in general quantum physics as well as analytical applications including confocal time resolved fluorescence microscopy at the single molecule level.

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.

No abstract