Solvent relaxation method (SRM) was used for studying the solvation and behavior of shells of polymeric micelles
formed by linear polystyrene-block-poly(2-vinylpyridine)-block-poly(ethylene oxide) block copolymer (PS-PVP-PEO)
in acidic aqueous media. The shell is formed by partially ionized (protonated) PVP and PEO blocks, while the compact
spherical core is formed by PS. An amphiphilic fluorescent surfactant, PATMAN was used as a probe. It was shown by
independent study that it binds strongly to micelles and that its fluorescent part monitors a thin PEO layer close to the
PVP-PEO interface. The behavior of PATMAN in non-viscous low-molar-mass solvents was studied first and then the
study of PS-PEO, PS-PVP and PS-PVP-PEO micelles was performed. The comparison of SRM data for all studied
systems allows for a plausible explanation of a slightly surprising behavior of PS-PVP-PEO micelles in acidic aqueous
buffers where an unexpected aggregation of micelles is observed at low pH.
Two fluorescence spectroscopy concepts, fluorescence correlation spectroscopy and time correlated single photon
counting (TCSPC) are employed in fluorescence lifetime correlation spectroscopy (FLCS) - a relatively new technique
with several experimental benefits. In FLCS experiments, pulsed excitation is used and data are stored in a special time-tagged
time-resolved mode. Mathematical treatment of TCSPC decay patterns of distinct fluorophores and their mixture
enables to calculate autocorrelation functions of each of the fluorophores and thus their diffusion properties and
concentrations can be determined separately. Moreover, crosscorrelation of the two signals can be performed and
information on interaction of the species can be obtained. This technique is particularly helpful for distinguishing
different states of the same fluorophore in different microenvironments. The first application of that concept represents
the simultaneous determination of two-dimensional diffusion in planar lipid layers and three-dimensional vesicle
diffusion in bulk above the lipid layers. The lifetime in both investigated systems differed because the lifetime of the dye
is considerably quenched in the layer near the light-absorbing surface. This concept was also used in other applications:
a) investigation of a conformational change of a labeled protein, b) detection of small amounts of labeled
oligonucleotides bound to metal particles or c) elucidation of the compaction mechanism of different sized labeled DNA
molecules. Moreover, it was demonstrated that FLCS can help to overcome some FCS experimental drawbacks.
Fluorescence lifetime correlation spectroscopy (FLCS) is a recently developed method which combines the conventional fluorescence correlation spectroscopy (FCS) and time correlated single photon counting (TCSPC). It enables to perform a signal separation of the species which possess different lifetime. Particular diffusion components of a mixture of more fluorescent species can be thus separated. Moreover, the detector afterpulsing can be suppressed by FLCS.
A new multi-focus multi-confocal set-up for performing fluorescence spectroscopy of single molecules in solution is presented. The ultimate goal of the set-up is to track individual molecules during diffusion in solution, when all standard methods of trapping such as optical tweezers or dielectrophoretic traps fail. We present here a detailed description of the experimental setup and show first experimental results.