Multi-Parameter TSCSPC

Recent development of novel single photon counting detectors made it possible to acquire and store the full set of system parameters, completely describing the physical properties of each individual photon:

hv = f [x, y, Dt(TAC), t(abs), l(em), pol],

where xy are the space coordinates of the impingent photon at the photocathode, Dt(TAC) the time difference correlating laser excitation pulse and fluorescence photon (yielding ps/ns fluorescence dynamics), t(abs) the absolute arrival time of each fluorescence photon at the detector (resulting in tracking capabilities and diffusional rates), l(em) its emission wavelength, and pol the direction of polarisation (providing anisotropy dynamics).

Novel microscopes are under construction, combining delay-line (DL) and quadrant-anode (QA) detectors for picosecond-resolved spectroscopy and imaging with a Nipkov disk for confocality. These microscopes are employed in a novel spectroscopic technique, Vehicle-Based Microspectroscopy (VBMS), utilising the tracking capabilities of the system to follow the path of emitters on the sub-second timescale, while acquiring ist ps/ns fluorescence dynamics at each point of the trajectory. The resulting movie captures macromolecular diffusional dynamics and interactions in living cells, fluorescence anisotropy dynamics, and microenvironmental properties along the trajectory of the emitting vehicle. The vehicle is a suitably labelled carrier of probe molecules and can be a virus, a macromolecule, or a latex bead. The multi-dimensional set of photon coordinates is stored in list-mode, providing the option to correlate parameter sub-sets with each other.?