Time-Resolved Single Molecule Spectroscopy

The techniques described under Photon Correlation exploit the correlation between the photons emitted by single molecules and by a small number of molecules. Picosecond photon correlation techniques investigate effects driven by the absorption of a single photon of the excitation light. The effects investigated by FCS are driven by Brownian motion, rotation, diffusion effects, intersystem crossing, or conformational changes. Because of these random and essentially sample-internal stimulation mechanisms, correlation techniques do not necessarily depend on a pulsed laser. 

A second way to obtain information about single molecules is time-resolved spectroscopy with pulsed excitation at high repetition rate. The borderline between 

The optieal systems used for both techniques are essentially the same. A small sample volume is obtained by confocal deteetion or two-photon excitation in a microscope. Several detectors are used to deteet the fluorescence in different spectral ranges or under different polarisation angles. Therefore correlation techniques ean be combined with fluorescence lifetime deteetion, and the typical time-resolved single-molecule techniques may use eorrelation of the photon data. The paragraphs below focus on single-molecule experiments that not only use, but are primarily based on pulsed excitation and time-resolved detection. 

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Spectroscopy of single molecules is based on fluorescence correlation, photoncounting histograms, or burst-integrated-lifetime techniques. Each case requires recording not only the times of the photons in the laser period, but also their absolute time. Modem time-resolved single molecule techniques therefore use almost exclusively the FIFO (time-tag) mode of TCSPC. The FIFO mode records all information about each individual photon, i.e. the time in the laser pulse sequence (micro time), the time from the start of the experiment (macro time), and the number of the detector that detected the photon (see Sect. 3.6, page 43). 

Hiiize G, Metivier R, Nolde F, Milllen K, Basche T (2008) Intramolecular electronic excitatirai energy transfer in donor/acceptor dyads studied by time and freqntarcy resolved single molecule spectroscopy. J ChemPhys 128 124516... 

Fries J R, Brand L, Eggeling C, Kdllner M and Seidel CAM 1998 Quantitative identification of different single molecules by selective time-resolved confocal fluorescence spectroscopy J. Phys. Chem. A 102 6602-13... 

Fluorescence microscopy techniques are now available which are capable of studying supramolecular interfacial assemblies with excellent spatial and temporal resolution as well as exceptional sensitivity. These methods were initially developed for use in cellular biology, but are finding increasing application in interfacial supramolecular chemistry. This trend is set to continue as methods in single-molecule spectroscopy and time-resolved microscopy evolve. 

Lakowicz, J. and Fu, Y. (2006) Enhanced Fluorescence of Cy5-Labeled DNA Tethered to Silver Island Films Fluorescence Images and Time-Resolved Studies Using Single-Molecule Spectroscopy Analytical Chemistry 78 6238 - 6245. 

Fluorescence of Cy5-labelled DNA tethered to silver island films fluorescence images and time-resolved studies using single-molecule spectroscopy. Anal. Chem., 78 6238. 

An additional push can be expected from new technical developments in TCSPC itself. The largest potential is probably in the development of new detectors. The introduction of direct (wide-field) imaging techniques is clearly hampered by the limited availability of position-sensitive detectors. In addition the selection of multianode PMTs is still very limited, especially for NIR-sensitive versions. Large-area detectors with 64 or more channels may result in considerable improvements in DOT techniques. Single photon APDs with improved timing stability are urgently required for single-molecule spectroscopy and time-resolved microscopy. 

Keywords Nonradiative excitation energy transfer Time-resolved fluorescence anisotropy Dynamics of polymer chains ir-Conjugated polymers Single-molecule spectroscopy... 

Because this problem is complex several avenues of attack have been devised in the last fifteen years. A combination of experimental developments (protein engineering, advances in x-ray and nuclear magnetic resonance (NMR), various time-resolved spectroscopies, single molecule manipulation methods) and theoretical approaches (use of statistical mechanics, different computational strategies, use of simple models) [5, 6 and 7] has led to a greater understanding of how polypeptide chains reach the native confonnation. 

TNP-ATP complex obtained by the single-molecule time-resolved spectroscopy, together with a fluorescence decay curve of TNP-ATP obtained by a bulk measurement. Both curves were well fitted to biexponential functions. The instrument-response function in 195-ps fwhm is also displayed. (B) Representative fluorescence spectrums of two individual enzyme-TNP-ATP complexes showing different emission peaks. A fluorescence spectrum of TNP-ATP obtained from a bulk measurement is also displayed for comparison. All spectrums were normalized to unity at their maximum. (From Ref. 18.)... 

Fluorescence-based detection methods are the most commonly used readouts for HTS as these readouts are sensitive, usually homogeneous and can be readily miniaturised, even down to the single molecule level.7,8 Fluorescent signals can be detected by methods such as fluorescence intensity (FI), fluorescence polarisation (FP) or anisotropy (FA), fluorescence resonance energy transfer (FRET), time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence intensity life time (FLIM). Confocal single molecule techniques such as fluorescence correlation spectroscopy (FCS) and one- or two-dimensional fluorescence intensity distribution analysis (ID FID A, 2D FIDA) have been reported but are not commonly used. 

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