Successive Molecular Decay Measurement by Time-Correlated Multiphoton Counting (TCMPC)

Fluorescence spectrum, intensity and decay measurements are powerful outputs to analyze molecular structure and energy transition in cellular biology and cytometry. Frequently, fluorescence measurements are made by photocurrent detection using Photomultiplier Tubes (PMTs) and recently using Avalanche Photo Diodes (APDs). To address the challenge of measuring nanosecond order fluorescence decay times on single cells, single photon detection technology has been developed using pixel-coupled Silicon Photo Multiplier (SiPM), GHz electronics and waveform analysis. As a result, photoelectron (PE) pulse widths of 500ps and saturation count of 1Gcps with 7 LOG dynamic range have been achieved. This capability enables detection of multiple fluorescence PE pulses during one START-STOP time interval measurement. Combined with high-speed pulsed excitation, it is possible to measure fluorescence intensity during the pulse excitation 10MHz repetition, 80ns ON time, and fluorescence decay during the 20ns OFF time. Statistically, measured decay time is different from currently defined decay time by impulse excitation. We named these techniques "time-correlated multi-photon counting (TCMPC)" and "successive molecular decay (SMD)" to distinguish from conventional definitions and methods. To confirm the techniques, the decay time of Rhodamine in polymer and other fluorescence materials was measured. To apply single photon detection to quantum flow cytometry, which involves several microseconds per event, full spectral detection with 42 channels from UV to IR wavelength and 10Gsps(100ps) data acquisition electronics is under development. SMD and TCMPC are innovative techniques to analyze single molecule behavior and structure and will be powerful tools to understand the quantum nature of biology.