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Using our newly developed ultrafast camera described in the companion paper, we reduced the data acquisition periods required for photoactivation/photoconversion localization microscopy (PALM, using mEos3.2) and direct stochastic reconstruction microscopy (dSTORM, using HMSiR) by a factor of ≈30 compared with standard methods, for much greater view-fields, with localization precisions of 29 and 19 nm, respectively, thus opening up previously inaccessible spatiotemporal scales to cell biology research. Simultaneous two-color PALM-dSTORM and PALM-ultrafast (10 kHz) single fluorescent-molecule imaging-tracking has been realized. They revealed the dynamic nanoorganization of the focal adhesion (FA), leading to the compartmentalized archipelago FA model, consisting of FA-protein islands with broad diversities in size (13–100 nm; mean island diameter ≈30 nm), protein copy numbers, compositions, and stoichiometries, which dot the partitioned fluid membrane (74-nm compartments in the FA vs. 109-nm compartments outside the FA). Integrins are recruited to these islands by hop diffusion. The FA-protein islands form loose ≈320 nm clusters and function as units for recruiting FA proteins.

ABSTRACT:An amphiphilic polymer consisting of p-styrenesulfonate (SS), butyl methacrylate (BMA), and a small amount of vinylcarbazole (VCz) was prepared. The fluorescence spectra from the carbazole chromophore covalently attached to the polymer as an emission probe showed a monomer fluorescence and no excimer emission. Whereas the shape and the wavelength of the monomer fluorescence were unchanged with solvents, the intensity varied with the solvents. The fluorescence was most intense in water where the polymers form a latex particle. The intensity was correlated with the solubility parameter of the solvents; it decreased with this parameter in organic solvents. In good solvents for the polymer such as tetrahydrofuran (THF), the expanded form of the polymer chain allows the energy migration between the carbazole units. Then the migration energy is trapped by an energy trap site existing on the polymer chain, resulting in a decrease in intensity. The quenching of the photoexcited carbazole by organic cations was also studied from measurements of the steady-state emission intensity. The quenching behavior in water was quite different from those in THF and dimethylformamide (DMF). In water, the quenching became more effective in more dilute polymer concentration. The incorporation of the quencher due to a hydrophobic and/or electrostatic interactions into the polymer chain promoted the quenching.KEY WORDS Amphiphilic Polymer/ Emission Probe/ Electron Transfer/ Molecular Assembly / The photochemistry in molecular assembly systems has been extensively investigated. Especially photoinduced electron transfer process in microheterogeneous environments with charged interfaces such as micelle, 1 microemulsion,2 vesicle,3 polyelectrolyte, 4 and LB multilayer 5 is now receiving much attention in relation to solar energy conversion. It is necessary to design various molecular assemblies for a desired functionality. To this end, polymer systems are convenient because the monomers with a varied property can be introduced as a comonomer. Amphiphilic polymers consisting of a hydrophobic and a hydrophilic monomer unit can yield a variety of microheterogeneous environment by change in copolymer compositions. 6 Moreover, the third monomer having photoactive properties has been often introduced by terpolymerization to prepare a photoredox reactive site or a probe molecule.