No abstract

The ability to record images with extreme temporal resolution enables a diverse range of applications, such as fluorescence lifetime imaging, time-of-flight depth imaging and characterization of ultrafast processes. Recently, ultrafast imaging schemes have emerged, which require either long acquisition times or raster scanning and have a requirement for sufficient signal that can only be achieved when light is reflected off an object or diffused by a strongly scattering medium. Here we present a demonstration of the potential of single-photon detector arrays for visualization and rapid characterization of events evolving on picosecond time scales. The single-photon sensitivity, temporal resolution and full-field imaging capability enables the observation of light-in-flight in air, as well as the measurement of laser-induced plasma formation and dynamics in its natural environment. The extreme sensitivity and short acquisition times pave the way for real-time imaging of ultrafast processes or visualization and tracking of objects hidden from view.

Abstract:We demonstrate diffraction limited multiphoton imaging in a massively parallel, fully addressable time-resolved multi-beam multiphoton microscope capable of producing fluorescence lifetime images with sub50ps temporal resolution. This imaging platform offers a significant improvement in acquisition speed over single-beam laser scanning FLIM by a factor of 64 without compromising in either the temporal or spatial resolutions of the system. We demonstrate FLIM acquisition at 500 ms with live cells expressing green fluorescent protein. The applicability of the technique to imaging protein-protein interactions in live cells is exemplified by observation of time-dependent FRET between the epidermal growth factor receptor (EGFR) and the adapter protein Grb2 following stimulation with the receptor ligand. Furthermore, ligand-dependent association of HER2-HER3 receptor tyrosine kinases was observed on a similar timescale and involved the internalisation and accumulation or receptor heterodimers within endosomes. These data demonstrate the broad applicability of this novel FLIM technique to the spatio-temporal dynamics of protein-protein interaction. 73-76 (1990). 9. F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2(12), 932-940 (2005). 10. J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23(9), 655-657 (1998) ©2015 Optical Society of America

We present a CMOS chip 256 × 2 single photon avalanche diode (SPAD) line sensor, 23.78 µm pitch, 43.7% fill factor, custom designed for time resolved emission spectroscopy (TRES). Integrating time-to-digital converters (TDCs) implement on-chip mono-exponential fluorescence lifetime pre-calculation allowing timing of 65k photons/pixel at 200 Hz line rate at 40 ps resolution using centre-of-mass method (CMM). Per pixel time-correlated single-photon counting (TCSPC) histograms can also be generated with 320 ps bin resolution. We characterize performance in terms of dark count rate, instrument response function and lifetime uniformity for a set of fluorophores with lifetimes ranging from 4 ns to 6 ns. Lastly, we present fluorescence lifetime spectra of multicolor microspheres and skin autofluorescence acquired using a custom built spectrometer. In TCSPC mode, time-resolved spectra are acquired within 5 minutes whilst in CMM mode spectral lifetime signatures are acquired within 2 ms for fluorophore in cuvette and 200 ms for skin autofluorescence. We demonstrate CMOS line sensors to be a versatile tool for time-resolved fluorescence spectroscopy by providing parallelized and flexible spectral detection of fluorescence decay.