Simple near-infrared time-correlated single photon counting instrument with a pulsed diode laser and avalanche photodiode for time-resolved measurements in scanning applications

A simple apparatus for time-correlated single photon counting (TCSPC) measurements in the near-infrared (near-IR) region for scanning-type applications has been constructed and examined. The apparatus consisted of five major components including a pulsed diode laser source (lasing wavelength = 780 nm; repetition rate = 80 MHz; power = 5 mW; pulse width = 150 ps), an integrated microscope, a large-photoactive-area avalanche photodiode (APD), a TCSPC PC-board including the electronics, and a Windows-based software package for accumulating the fluorescence decay profiles. The instrument response function (IRF) of this assembly was found to be 460 ps, which is adequate for measuring lifetimes with tau(f) greater than or equal to 500 ps, Due to the small size of the device, it also allowed implementation into scanning experiments where lifetimes were measured. To demonstrate this capability, we scanned a three-well microscope slide containing a near-IR dye. The decay profile of the near-IR dye, aluminum 2,3-naphthalocyanine, was collected and analyzed to obtain its lifetime, which was found to be 2.73 ns, in close agreement with the literature value for this particular dye. In addition, a three-dimensional plot showing the decay profiles (time vs. photocounts) and scan position of aluminum 2,3-naphthalocyanine fluorescence was acquired by scanning the microscope head over this three-well glass slide. In the scanning mode, the IRFs as well as the decays of the dyes were found to be very stable. The device demonstrated a concentration detection sensitivity of 0.44 nM; however, the dynamic range was limited due to the slow time constant (passive quenching) associated with the APD.