Single Photon Avalanche Diodes (SPADs) are valuable detectors in numerous photon counting applications in the fields
of quantum physics, quantum communication, astronomy, metrology and biomedical analytics. They typically feature a
much higher photon detection efficiency than photomultiplier tubes, most importantly in the red to near-infrared range of
the spectrum. Very often SPADs are combined with Time-Correlated Single Photon Counting (TCSPC) electronics for
time-resolved data acquisition and the temporal resolution ("jitter") of a SPAD is therefore one of the key parameters for
selecting a detector. We show technical data and first application results from a new type of red sensitive single photon
counting module ("τ-SPAD"), which is targeted at timing applications, most prominently in the area of Single Molecule
Spectroscopy (SMS). The τ-SPAD photon counting module combines Laser Components' ultra-low noise VLoK silicon
avalanche photodiode with specially developed quenching and readout electronics from PicoQuant. It features an
extremely high photon detection efficiency of 75% at 670 nm and can be used to detect single photons over the 400 nm to
1100 nm wavelength range. The timing jitter of the output of the τ-SPAD can be as low as 350 ps, making it suitable for
time-resolved fluorescence detection applications. First photon coincidence correlation measurements also show that the
typical breakdown flash of SPADs is of comparably low intensity for these new SPADs.
Upgrade kits towards time-resolved measurements for Confocal Microscopes allow new measurement modes like
Fluorescence Lifetime Imaging (FLIM), time-resolved analysis of Fluorescence Correlation Spectroscopy (FCS) and
Fluorescence Resonance Energy Transfer (FRET). Microscope users would typically like to use the same excitation
wavelength for time-resolved measurements as for steady-state measurements, because their fluorophores are
designed for the CW-laser wavelengths usually provided with the system. Pulsed diode lasers, which are ideally
used for these upgrade kits are, however, not available for every spectral region of interest. Especially for "green"
excitation around 530 nm this is still a problem, as there are no direct emitting laser diodes available.
We present a new picosecond pulsed laser system for 530 nm emission with variable repetition rate and pulse
energy, which is ideally suited for time-resolved measurements using Time-Correlated Single Photon Counting
(TCSPC), and demonstrate its integration into a confocal microscope as well as first results of FLIM and FCS
Time-resolved techniques to measure the fluorescence lifetime can reveal important information about the local
environment of a given fluorescent probe, help to distinguish fluorophores with similar spectral properties or reveal
different conformations of a single fluorophore. We have developed a stable and easy to use upgrade for standard
laser scanning confocal microscopes towards a time-resolved system, which is based on picosecond pulsed lasers,
fast detectors and sophisticated single photon counting electronics.
We demonstrate the capabilities of the time-resolved approach by using fluorescence lifetime measurements to
detect fluorescence resonance energy transfer (FRET) in living cells. The results show that different FRET efficiencies
can be spatially resolved within a single cell. Furthermore, the upgrade kit does not only allow to
measure FRET by observing the shortening of the donor lifetime, but also the acceptor decay can be simultaneously
monitored using two spectrally separated detectors and a router.
A very special feature of the upgrade kit is that it uses an unrestricted data acquisition approach. With this approach,
not only Fluorescence Lifetime Imaging Microscopy (FLIM) with single molecule sensitivity is realized, but the
provided information can also be combined with other techniques such as Fluorescence Correlation Spectroscopy
(FCS). This opens the way to complete new analysis and measurement schemes like Fluorescence Lifetime
Correlation Spectroscopy (FLCS) or Pulsed Interleaved Excitation (PIE). FLCS can, for example, be used to remove
the influence of detector afterpulsing, which is classically done by cross correlation between two detectors.
Optical techniques based on photon migration are rapidly emerging as a promising alternative and/or augmentation of existing medical imaging modalities. For example, real time studies of hemodynamic changes in brain tissue are possible as a step towards optical functional brain imaging. Time-resolved implementations of these techniques allow for discrimination between scattering and absorption and for depth resolution. They require sub-nanosecond pulsed light sources with high repetition rate and sufficient power for deep enough tissue penetration. Picosecond diode lasers satisfy the clinical demands of economy, compact size, and reliability almost perfectly. Today multi-channel diode laser devices are commercially available and are widely used in diffuse optical imaging and spectroscopy, in particular in optical tomography and breast cancer detection. However, the output powers of these devices are just about sufficient for moderate tissue penetration depths. An improvement that does not compromise the advantages of the diode laser sources is amplification of the diode laser output by means of solid state tapered amplifiers. We present an amplified light source for use in NIR diffuse optical spectroscopy and imaging, providing pulse widths as short as 100 ps, adjustable repetition rates up to 80 MHz, and peak power levels as high as 7 Watts, corresponding to average power levels exceeding 100 mW. In combination with time-resolved photon counting electronics matching the high throughput demands in conjunction with the new source, state-of-the-art systems for diffuse optical imaging can be built. System design features and possible application examples are presented.
We present the technical integration of state-of-the-art picosecond diode laser sources and data acquisition electronics in conventional laser scanning microscopes. This procedure offers users of laser scanning microscopes an easy upgrade path towards time-resolved measurements. Our setup uses picosecond diode lasers from 375 to 800 nm for excitation which are coupled to the microscope via a single mode fiber. The corresponding emission is guided to a fibre coupled photon counting detector, such as Photomultiplier Tubes (PMT) or Single Photon Avalanche Diodes (SPAD). This combines the outstanding sensitivity of photon counting detectors with the ease of use of diode laser sources, to allow time-resolved measurements of fluorescence decays with resolutions down to picoseconds. The synchronization signals from the laser scanning microscope are fed into the data stream recorded by the TimeHarp 200 TCSPC system, via the unique Time-Tagged Time-Resolved (TTTR) data acquisition mode. In this TTTR data acquisition mode each photon is recorded individually with its specific parameters as detector channel, picosecond timing, global arrival time and, in this special application, up to three additional markers. These markers, in combination with the global arrival time, allow the system software to reconstruct the complete image and subsequently create the full fluorescence lifetime image (FLIM). The multi-parameter data acquisition scheme of the TimeHarp 200 electronics not only records each parameter individually, but offers in addition the opportunity to analyse the parameter dependencies in a multitude of different ways. This method allows for example to calculate the fluorescence fluctuation correlation function (FCS) on any single spot of interest but also to reconstruct the fluorescence decay of each image pixel and detector channel for advanced Forster Resonance Energy Transfer (FRET) analysis. In this paper, we present some selected results acquired with standard laser scanning microscopes upgraded towards temporal resolution.