Photon counting detectors currently used in fluorescence lifetime microscopy have a number of deficiencies that result in less-than-ideal signal-to-noise ratio of the lifetimes obtained: either the quantum efficiency is unsatisfactory or the active area is too small, and afterpulsing or tails in the temporal response contribute to overall timing inaccuracy. We have therefore developed a new FLIM detector based on a GaAsP hybrid photomultiplier. Compared with conventional PMTs and SPADs, GaAsP hybrid detectors have a number of advantages: The detection quantum efficiency reaches or surpasses the efficiency of fast SPADs, and the active area is on the order of 5 mm², compared with 2.5 10⁻³ mm² for a SPAD. The TCSPC response is clean, without the bumps and the diffusion tails typical for PMTs and SPADs. Most important, the hybrid detector is intrinsically free of afterpulsing. FLIM results are therefore free of signal-dependent background, and FCS curves are free of the known afterpulsing peak. We demonstrate the performance of the new detector for multiphoton NDD FLIM and for FCS.

To monitor conformational changes of individual membrane transporters in liposomes in real time, we attach two fluorophores to selected domains of a protein. Sequential distance changes between the dyes are recorded and analyzed by Förster resonance energy transfer (FRET). Using freely diffusing membrane proteins reconstituted in liposomes, observation times are limited by Brownian motion through the confocal detection volume. A. E. Cohen and W. E. Moerner have invented and built microfluidic devices to actively counteract Brownian motion of single nanoparticles in electrokinetic traps (ABELtrap). Here we present a version of an ABELtrap with a laser focus pattern generated by electrooptical beam deflectors and controlled by a programmable FPGA. This ABELtrap could hold single fluorescent nanobeads for more than 100 seconds, increasing the observation times of a single particle by more than a factor of 1000. Conformational changes of single FRET-labeled membrane enzymes FoF1-ATP synthase can be detected in the ABELtrap.

We present a lifetime imaging technique that simultaneously records fluorescence and phosphorescence lifetime images in laser scanning systems. It is based on modulating a high-frequency pulsed laser by a signal synchronous with the pixel clock of the scanner, and recording the fluorescence and phosphorescence signals by multi-dimensional TCSPC. Fluorescence is recorded during the on-phase of the laser, phosphorescence during the off-phase. The technique does not require a reduction of the laser pulse repetition rate by a pulse picker, and eliminates the need of using excessively high pulse power for phosphorescence excitation. Laser modulation is achieved either by electrically modulating picosecond diode lasers, or be controlling the lasers via the AOM of a standard confocal or multiphoton laser scanning microscope. Downloaded From: http://proceedings.spiedigitallibrary.org/ on 05/19/2015 Terms of Use: http://spiedl.org/terms Proc. of SPIE Vol. 7903 790320-5 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 05/19/2015 Terms of Use: http://spiedl.org/terms Proc. of SPIE Vol. 7903 790320-6 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 05/19/2015 Terms of Use: http://spiedl.org/terms

Observation times of freely diffusing single molecules in solution are limited by the photophysics of the attached fluorescence markers and by a small observation volume in the femtolitre range that is required for a sufficient signal-to-background ratio. To extend diffusion-limited observation times through a confocal detection volume, A. E. Cohen and W. E. Moerner have invented and built the ABELtrap -a microfluidic device to actively counteract Brownian motion of single nanoparticles with an electrokinetic trap. Here we present a version of an ABELtrap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA chip. This ABELtrap holds single fluorescent nanoparticles for more than 100 seconds, increasing the observation time of fluorescent nanoparticles compared to free diffusion by a factor of 10000. To monitor conformational changes of individual membrane proteins in real time, we record sequential distance changes between two specifically attached dyes using Förster resonance energy transfer (smFRET). Fusing the asubunit of the F o F 1 -ATP synthase with mNeonGreen results in an improved signal-to-background ratio at lower laser excitation powers. This increases our measured trap duration of proteoliposomes beyond 2 s. Additionally, we observe different smFRET levels attributed to varying distances between the FRET donor (mNeonGreen) and acceptor (Alexa568) fluorophore attached at the a-and c-subunit of the F o F 1 -ATP synthase respectively.

We present a technique that records transient effects in the fluorescence lifetime of a sample with spatial resolution along a one-dimensional scan. The technique is based on scanning a sample with a high-frequency pulsed laser beam, and building up a photon distribution over the distance along the scan, the arrival times of the photons after the excitation pulses, and the time after a stimulation of the sample. The maximum resolution at which lifetime changes can be recorded is given by the line scan time. With repetitive stimulation and triggered accumulation transient lifetime effects can be resolved at a resolution of about one millisecond.