Phasor imaging with a widefield photon-counting detector

Abstract

Fluorescence lifetime can be used as a contrast mechanism to distinguish fluorophores for localization or tracking, for studying molecular interactions, binding, assembly, and aggregation, or for observing conformational changes via Förster resonance energy transfer (FRET) between donor and acceptor molecules. Fluorescence lifetime imaging microscopy (FLIM) is thus a powerful technique but its widespread use has been hampered by demanding hardware and software requirements. FLIM data is often analyzed in terms of multicomponent fluorescence lifetime decays, which requires large signals for a good signal-to-noise ratio. This confines the approach to very low frame rates and limits the number of frames which can be acquired before bleaching the sample. Recently, a computationally efficient and intuitive graphical representation, the phasor approach, has been proposed as an alternative method for FLIM data analysis at the ensemble and single-molecule level. In this article, we illustrate the advantages of combining phasor analysis with a widefield time-resolved single photon-counting detector (the H33D detector) for FLIM applications. In particular we show that phasor analysis allows real-time subsecond identification of species by their lifetimes and rapid representation of their spatial distribution, thanks to the parallel acquisition of FLIM information over a wide field of view by the H33D detector. We also discuss possible improvements of the H33D detector's performance made possible by the simplicity of phasor analysis and its relaxed timing accuracy requirements compared to standard time-correlated single-photon counting (TCSPC) methods.

Development of new photon-counting detectors for single-molecule fluorescence microscopy

“…Moreover, the use of time-gating [137] or frequencymodulation [138] with intensifier-equipped cameras used in fluorescence lifetime imaging microscopy (FLIM) significantly reduces the effective sensitivity of these detectors, making those approaches inappropriate for single-molecule detection [139].…”

“…Although we managed to detect single QDs [172] and perform FLIM on both live cells and large fluorescent objects such as beads or QD clusters [139] with this prototype (figure 5), the QE was too low for conventional (organic dye) single-molecule analysis. However, this prototype showcased three advantages of this technique for future single-molecule imaging studies:…”

“…-Single-molecule fluorescence lifetime studies can be performed at frame rates of the order of f max /100 [139].…”

“…Indeed, in the same way it is possible to determine the fluorescence lifetime of a single molecule with N 100 photons, using a maximum-likelihood estimation approach [18,176]; such a small number of photons is sufficient to obtain a phasor value with reasonably small variance [139,177]. Therefore, not only does it appear feasible to track individual molecules with approximately 10 nm resolution and kHz frame rate, but it is simultaneously possible to measure their lifetime at the same rate, thus monitoring their electronic environment as they explore it (e.g.…”

The Gray Institute open microscopes applied to radiobiology and protein interaction studies

“…Using this assumption that the phasor will be very near to the universal circle, we can derive a FRET measure based on the phasor FRET trajectory. In the phasor coordinate system (u,v), the phasor for the complex mixture is given by 37 :…”