These application notes describe a technique or method that can be applied using PicoQuant systems or instrumentation. This includes, for example, methods such as Time-Correlated Single Photon Counting (TCSPC) or "F-techniques" such as FLIM, FRET, FCS or FLCS.
Quantitative In Vivo Imaging of Molecular Distances Using FLIM-FRET
The application note provides an introduction into FLIM-FRET experiments and the underlying principle of TCSPC measurement compared to frequency-domain method. The potential of this technique is demonstrated by means of various measurement examples including acceptor photo-bleaching, two-photon excitation and dual-channel detection monitoring both the donor and acceptor simultaneously. In addition, a scripting based FLIM-FRET analysis is applied to separate quenched from unquenched donor species.
Time-Correlated Single Photon Counting
The technical note gives a theoretical overview about Time-Correlated Single Photon Counting (TCSPC). It provides information about basic principles such as count rate statistics, pile-up limitation or reverse start-stop mode. The overall timing resolution (Instrument Response Function - IRF) as well as how individual components of a TCSPC setup contribute to it is also explained.
Time Tagged Time-resolved Fluorescence Data Collection in Life Sciences
The technical note gives an introduction into Time-Tagged Time-Resolved (TTTR) fluorescence data collection. It explains the underlying concept and the multiple data analysis possibilities based on this unique data acquisition mode that is exclusively offered by PicoQuant.
Quantitative Fluorescence Correlation Spectroscopy
The quantitative analysis of FCS measurements strongly rely on the size of the confocal volume which has to be determined experimentally. This application note describes three methods to determine the confocal parameters for quantitative FCS. The results of the measurements are compared to each other in respect of their applicability for quantitative determination of concentration and diffusion constants. Experimental details along with theoretical and practical results are given.
Fluorescence Lifetime Correlation Spectroscopy (FLCS)
The application note gives an introduction into the principle of Fluorescence Lifetime Correlation Spectroscopy (FLCS) and highlights remarkable features of this method. Measurement examples of quasi-multichannel detection as well as suppression of scattered light and various parasitic contributions are shown. FLCS uses picosecond time-resolved fluorescence detection to separate different FCS contributions without the need of fitting to a multi-exponential model which leads to improved concentration measurements.
Time-gated Fluorescence Correlation Spectoscopy
The application note describes how Time-gated Fluorescence Correlation Spectroscopy can be used for improved concentration determinations. With this method background signal can be eliminated using pulsed laser excitation and TCSPC for data acquisition. The document introduces the principles of Time-gated FCS and the realization by the PicoQuant setup. An example demonstrates that accurate concentration measurements free of scattered light artifacts are possible using this method.