Using Time-Correlated Single Photon Counting (TCSPC) for the purpose of fluorescence lifetime measurements is usually limited in speed due to pile-up. With modern instrumentation this limitation can be lifted significantly but some artefacts due to frequent merging of closely spaced detector pulses (detector pulse pile-up) remains an issue to be addressed. We propose here a data analysis method correcting for this type of artefact and the resulting systematic errors. It physically models the photon losses due to detector pulse pile-up and incorporates the loss in the decay fit model employed to obtain fluorescence lifetimes and relative amplitudes of the decay components. Comparison of results with and without this correction show a significant reduction of systematic errors at count rates approaching the excitation rate. This allows quantitatively accurate fluorescense lifetime imaging (FLIM) at very high frame rates.
Stimulated Emission Depletion (STED) Microscopy has evolved into a well established method offering optical superresolution below 50 nm. Running both excitation and depletion lasers in picosecond pulsed modes allows for highest optical resolution as well as fully exploiting the photon arrival time information using time-resolved single photon counting (TCSPC). Non-superresolved contributions can be easily dismissed through time-gated detection (gated STED) or a more detailed fluorescence decay analysis (FLIM-STED), both leading to an even further improved imaging resolution. Furthermore, these methods allow for accurate separation of different fluorescent species, especially if subtle differences in the excitation and emission spectra as well as the fluorescence decay are taken into account in parallel. STED can also be used to shrink the observation volume while studying the dynamics of diffusing species in Fluorescence Correlation Spectroscopy (FCS) to overcome averaging issues along long transit paths. A further unique advantage of STED-FCS is that the observation spot diameter can be tuned in a gradual manner enabling, for example, determining the type of hindered diffusion in lipid membrane studies. Our completely pulsed illumination scheme allows realizing an improved STED-FCS data acquisition using pulsed interleaved excitation (PIE). PIE-STED-FCS allows for a straightforward online check whether the STED laser has an influence on the investigated diffusion dynamics.
Semisquarylium
dyes use a novel acyloin anchor group to strongly
bind to TiO2 semiconductors. Efficient acyloin anchor mediated
electron injection into nanocrystalline TiO2 is demonstrated,
allowing highly efficient dye-sensitized solar cells with IPCEs >
80%. The acyloin anchor can thus be viewed as a true alternative to
the standard carboxylic acid anchor group. The opto-electronic and
electron injection properties of the most basic semisquarylium dye
SY404 are compared to the modified semisquarylium
dye DD1 and the carboxylic acid anchored indoline dye
D131 using a combination of ultrafast and photoemission
spectroscopy. For SY404, ultrafast injection times of
∼50 fs are found despite a small energetic driving force between
dye excited states and TiO2 conduction band minimum. This
is possible due to the strong electronic coupling of the semisquarylium
dyes to the TiO2 surface mediated by the acyloin anchor.
For a better overlap with the solar spectrum, the semisquarylium dyes
are modified by substitution with a larger donor moiety (DD1). While for DD1 the overall absorption increases, the
injection process slightly slows down; however, it still proves fast
enough for very efficient injection. Compared to the carboxylic acid
anchored indoline dye D131, the SY404 dye
injects more than seven times faster despite a ∼150 meV smaller
driving force.
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