Wide-field time-correlated single photon counting-based fluorescence lifetime imaging microscopy

Wide-field time-correlated single photon counting detection techniques, where the position and the arrival time of the photons are recorded simultaneously using a camera, have made some advances recently. The technology and instrumentation used for this approach is employed in areas such as nuclear science, mass spectroscopy and positron emission tomography, but here, we discuss some of the wide-field TCSPC methods, for applications in fluorescence microscopy. We describe work by us and others as presented in the Ulitima fast imaging and tracking conference at the Argonne National Laboratory in September 2018, from phosphorescence lifetime imaging (PLIM) microscopy on the microsecond time scale to fluorescence lifetime imaging (FLIM) on the nanosecond time scale, and highlight some applications of these techniques

Spectrally and temporally resolved single photon counting in advanced biophotonics applications

Biomedicine requires highly sensitive and efficient light sensors to analyse light-tissue or light-sample interactions. Single-photon avalanche diode (SPAD) sensors implemented with complementary metal-oxide-semiconductor (CMOS) technology have a growing range of applications in this field. Single-photon detection coupled with integrated timing circuits enables us to timestamp each detected photon with high temporal resolution (down to picoseconds). Arrays of SPAD based pixels and CMOS technology offer massively parallel time-resolved single-photon counting for spectrally and temporally resolved analysis of various light phenomena.This thesis examines how time-resolved CMOS SPAD based line sensors with per pixel timing circuits can be utilized to advance biophotonic applications. The study focuses on improving the existing techniques of fluorescence and Raman spectroscopy, and demonstrates for the first time CMOS SPAD based detection in optical coherence tomography (OCT). A novel detection scheme is proposed combining low-coherence interferometry and time-resolved photon counting. In this approach the interferometric information is revealed from spectral intensity measurements, which is supplemented by time-stamping of the photons building up the spectra.Two CMOS SPAD line sensors (Ra-I and its improved version, Ra-II) were characterized and the effect of their parameters on the selected techniques was analysed. The thesis demonstrates the deployment of the Ra-I line sensor in time-resolved fluorescence spectroscopy with indications of the applicability in time-resolved Raman spectroscopy. The work includes integration of the sensor with surrounding electrical and optical systems, and the implementation of firmware and software for controlling the optical setup. As a result, a versatile platform is demonstrated capable of micro- and millisecond sampling of spectral fluorescence lifetime changes in a single transient of fast chemical reactions.OCT operating in the spectral domain traditionally uses CMOS photodiode and charge-coupled device (CCD) based detectors. The applicability of CMOS SPAD sensors is investigated for the first time with focus on the main limitations and related challenges. Finally, a new detection method is proposed relying on both the wave and particle nature of light, recording time-resolved interferometric spectra from a Michelson interferometer. This method offers an alternative approach to analyse luminous effects and improves techniques based on the light’s time of flight. As an example, a proof of concept study is presented for the removal of unwanted reflections from along the sample and the optical path in an OCT setup