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

Time Correlated Single Photon Counting (TCSPC) is a valuable tool fbr 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 truely 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 Gate Array (FPGA) logic permit a continouos data throughput as high as 3Mcounts/sec. The histogramming time may be set as short as 100 mu s. Every histogram or every single fluorescence photon can be real-time tagged at 200ns 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 60ps and up to 4096 histogramm channels. The overall instrument response function in combination with a low cost picosecond diode laser and an inexpensive photomultiplier tube was found to be 180ps and well sufficient to measure sub-nanosecond fluorescence lifetinmes.