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 <i>a</i>-subunit of the F<sub>o</sub>F<sub>1</sub>-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 <i>a</i>- and <i>c</i>-subunit of the F<sub>o</sub>F<sub>1</sub>-ATP synthase respectively.
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 electro-optical 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 more than 1000-fold. Conformational changes of single FRET-labeled membrane enzymes F<sub>o</sub>F<sub>1</sub>-ATP synthase can be detected in the ABELtrap.
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.
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.
The principle of the hybrid PMT is known for about 15 years: Photoelectrons emitted by a photocathode are accelerated
by a strong electrical field, and directly injected into an avalanche diode chip. Until recently, the gain of hybrid PMTs
was too low for picosecond-resolution photon counting. Now devices are available that reach a total gain of a few
100,000, enough to detect single photons at ps resolution. Compared with conventional PMTs, multi-channel PMTs, and
SPADs (single-photon avalanche photodiodes) hybrid PMTs have a number of advantages: With a modern GaAsP
cathode the detection quantum efficiency reaches the efficiency of a SPAD. However, the active area is on the order of
5 mm<sup>2</sup>, compared to 2.5 10<sup>-3</sup> mm<sup>2</sup> for a SPAD. A hybrid PMT can therefore be used for non-descanned detection in a
multiphoton microscope. The TCSPC response is clean, without the bumps typical for PMTs, and without the diffusion
tail typical for SPADs. Most important, the hybrid PMT is free of afterpulsing. So far, afterpulsing has been present in
all photon counting detectors. It causes a signal-dependent background in FLIM measurements, and a typical
afterpulsing peak in FCS. With a hybrid PMT, FLIM measurements reach a much higher dynamic range. Clean FCS
data are obtained from a single detector. Compared to cross-correlation of the signals of two detectors an increase in
FCS efficiency by a factor of four is obtained. We demonstrate the performance of the new detector for a number of
Currently used TCSPC FLIM systems are characterised by high counting efficiency, high time resolution, and multiwavelength
capability. The systems are, however, restricted to count rates on the order of a few MHz. In the majority of
applications, such as FRET or tissue autofluorescence, the photostability of the samples limits the count rate to much
lower values. The limited counting capability of the hardware is therefore no problem. However, if FLIM is used for
samples containing highly photostable fluorophores at high concentrations the available count rates can exceed the
counting capability of a single TCSPC channel. In this paper we describe a TCSPC FLIM system that uses 8 parallel
TCSPC channels to record FLIM data at a peak count rate on the order of 50•10<sup>6</sup> s<sup>-1</sup>. By using a polychromator for
spectral dispersion and a multi-channel PMT for detection we obtain multi-spectral FLIM data at acquisition times on
the order of one second. We demonstrate the system for recording transient lifetime effects in the chloroplasts in live