Depending on the semiconductor material, the luminescence lifetime of semiconductor wafers can vary over a broad
range from microseconds for Si-wafers down to sub-nanoseconds for III/V and II/VI based thin film or organic
materials. The lifetime of a given wafer sample depends on the free charge carrier dynamics and can therefore be
affected by several parameters. An important example is the influence of bulk or surface defects [1], thus the lifetime is a possible indicator for wafer quality. On dye-sensitized solar cells, lifetime measurements are also useful to characterize the energy transfer process from the sensitizer to the conduction band [2]. We have developed a setup for time-resolved photoluminescence measurements (TRPL) based on pulsed diode lasers and time-correlated single photon counting (TCSPC) with highly sensitive single photon detectors. Depending on the detector type, the instrument response function (IRF) can be as short as 100 ps and the laser pulse rate can be adapted to the luminescence lifetime of the material. The resolvable lifetimes extend from approx. 50 ps up to several hundred
microseconds. The electronics can also be integrated into a microscope based setup for imaging with a lateral resolution down to the sub-μm range [3] as well as testing the lifetime behaviour at different injection levels. We will show measurement results of the system on an GaAsP-based Quantum Well.
KEYWORDS: Atomic force microscopy, Luminescence, Confocal microscopy, Molecules, Microscopes, Fluorescence lifetime imaging, Microscopy, Data acquisition, Imaging systems, Green fluorescent protein
Time-resolved confocal microscopy is well established to image spectral and spatial properties of samples in biology and
material science. Atomic Force Microscopy (AFM) in addition enables to investigate properties which are not optically
addressable or are hidden by the diffraction limited optical resolution.
We present a straight forward combination of single molecule sensitive time-resolved confocal microscopy with different
commercially available AFMs. Besides an extra of information about for example a cell surface, the AFM tip can also be
used to manipulate the sample on a nanometer scale down to the single molecule level.
The interest in super-resolution microscopy techniques has dramatically increased in the last years due to the
unprecedented insight into cellular structure which has become possible [1]. In all widefield-based techniques, such as
Stochastical Optical Reconstruction Microscopy (STORM) or
Photo-activation localization microscopy (PALM), the
dye-sensor-molecules are switched between a bright and a dark state. Many organic fluorophores exhibit intrinsic dark
states with a lifetime that can be tuned by adjusting the level of oxidants and reductants in the buffer, thereby allowing to
reversibly switch individual fluorophores between an on- and
off-state [2]. This behavior is used in the dSTORM
method.
We exploited this redox-level adjusted photoswitching behaviour based on addition of millimolar amounts of reducing
thiols for high-resolution imaging on a setup based on an inverse microscope coupled with ultrasensitive CCD camera
detection. In order to quickly control the quality of the measurement, we used real-time computation of the
subdiffraction-resolution image [3]. This greatly increases the applicability of the method, as image analysis times are
greatly reduced.
The combination of simultaneous spectral detection together with Fluorescence Lifetime Imaging (sFLIM)
allows collecting the complete information inherent to the fluorescence signal. Their fingerprint of lifetime and
spectral properties identify the fluorescent labels unambiguously. Multiple labels can be investigated in parallel
and separated from inherent auto-fluorescence of the sample. In addition, spectral FLIM FRET has the prospect
to allow simultaneous detection of multiple FRET signals with quantitative analysis of FRET-efficiency and
degree of binding.
Spectral FLIM measurements generate huge amount of data. Suitable analysis procedures must be found to
condense the inherent information to answer the scientific questions in a straightforward way. Different analysis
techniques have been evaluated for a diversity of applications as multiplex labeling, quantitative determination
of environmental parameters and distance measurements via FLIM FRET.
In order to reach highest sensitivity in single photon detection, different detector types are investigated and
developed. SPAD arrays equipped with micro-lenses promise superior detection efficiency while the integration
of a spectrograph with a PMT array is easier to realize and allows for a higher number of detection channels.
High detection speed can be realized through parallel TCSPC channels. In order to overcome the limits of the
USB 2.0 interface, new interface solutions have been realized for the multichannel TCSPC unit HydraHarp 400.
We evaluate the potential ability of c-shaped apertures milled in aluminum thin films to reduce the effective
measurement volume and to enhance the fluorescence signal for fluorescence correlation spectroscopy of ATTO655 dye
dissolved in a HEPES buffer solution. Previous studies have shown that by morphing a square aperture into a rectangular
aperture while holding the cross-sectional area constant will yield strong polarization dependence in the reduction of the
effective volume and about a factor of 2-3 enhancement in the fluorescence count rate per molecule. By morphing the
rectangular aperture into a c-shaped aperture we gain further reduction in focal volume while maintaining the count rate
enhancements. In particular, we compare c-shaped apertures to squares with the same cross-sectional area and show that
one can achieve one molecule per focal volume at ~3µM (about a 1000 times reduction in effective volume compared to
confocal FCS) while maintaining a fluorescence count rate per molecule of about an order of magnitude higher than for
bulk diffusing dyes. Two orthogonal polarizations for the incident field have been studied to explore the effects on the
focal volume reduction and fluorescence count rate enhancements.
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