Total Emission Detection for Efficient and Affordable Two-Photon Fluctuation Correlation Spectroscopy (2P-Fcs)

Abstract

Two-photon excitation is a convenient means to generate a tiny “privileged” excitation volume in solution or within a cell; within that sub-fL volume, one may glean photophysical properties: triplet quantum yield, diffusion rate, blinking probability, brightness, conformational mobility, etc. We recently developed the Total Emission Detection (TED) method and devices to recover the entirety of emission from cells and tissues being probed with multiphoton excitation (Combs et al., J Microsc. 2007, v.228,pp.330-7). TED is being exploited now for single (few) molecule spectroscopy studies such as FCS, PCHA, FLIM-FCS, etc. These approaches demand not only high light collection and detection efficiency but also negligible contribution from artifacts arising from detector dark noise and after-pulsing. The latter usually necessitate the use of expensive TE-cooled avalanche photodiodes (APDs) or hybrid PMTs.In this study we show that conventional large-area photomultiplier tubes (PMTs), tightly coupled with TED devices, help overcome challenges arising from highly scattering samples, low signal-to-noise ratios and afterpulses coming after true fluorescence photon detection. In some TED versions, a conventional non-descanned detection (NDD) pathway for fluorescence is still available, so a second PMT can be employed for one- or two-color cross-correlation (FCCS).In short, TED increases “effective” quantum efficiency enough for one to use less expensive PMTs as FCS/FCCS detectors, and this also permits one to select among PMTs with better picosecond, nanosecond or microsecond behaviors compared to APDs.