Improved Method for Counting DNA Molecules on Biofunctionalized Nanoparticles

In order to accurately determine low numbers (1−100) of immobilized ssDNA molecules at a single, silica 250 nm nanoparticle surface, we hereby propose an integrated approach combining classic single molecule confocal microscopy (SMCM), that is, stepwise photobleaching of labeled ssDNA, with modified total internal reflection fluorescence microscopy (mTIRF). We postulate that SMCM alone is unable to exactly account for all labeled ssDNA because of inherent laser polarization effects; that is, perpendicularly oriented molecules to the sample surface are not (or are only slightly) susceptible to laser excitation and thus are invisible in a classic photobleaching experiment. The SMCM method accounts for at best two-thirds (68%) of the present ssDNA molecules. The principle of the mTIRF technique, which relies on the creation of highly inclined illumination combined with part of the laser remaining in normal Köhler illumination, enables accurate counting of SMCM invisble molecules. The combined approach proposed here circumvents the polarization issue and allows a complete single molecule counting on individual nanoparticles, fully in line with bulk measurements, as will be demonstrated

The Origin of Heterogeneity of Polymer Dynamics near the Glass Temperature As Probed by Defocused Imaging

Single molecule defocused wide-field fluorescence microscopy (SMDWM) has been used to monitor the 3D reorientation of single molecules in a thin polymer film (∼300 nm) of monodisperse poly(n-butyl methacrylate) near the glass temperature (Tg). Stroboscopic illumination allows for estimating reliable correlation times of single molecule rotational diffusion owing to the drastic lengthening of the observable trajectories. We demonstrate that homogeneity is restored ∼19 K above the Tg determined with calorimetry. The rotational correlation times obtained from SMDWM show similar temperature dependence as the ones measured with established bulk measurements, such as dielectric spectroscopy and rheology, on the same polymer sample. Single molecular reorientation is coupled to the segmental rather than terminal relaxation of the surrounding polymer matrix. SMDWM revealed that spatial heterogeneity is more pronounced than temporal heterogeneity within the measurement time scale (hours to days), whereas this information is hidden in the bulk measurement

Unraveling Excited-State Dynamics in a Polyfluorene-Perylenediimide Copolymer

Insight into the exciton dynamics occurring in a polyfluorene-perylenediimide (PF-PDI) copolymer with a reaction mixture ratio of 100 fluorene units to 1 N,N′-bis(phenyl)-1,6,7,12-tetra(p-tert-octylphenoxy)-perylene-3,4,9,10-tetracarboxylic acid diimide (PDI) is presented here. Time-correlated single photon counting and femtosecond transient absorption spectroscopy measurements on the PF-PDI copolymer have been employed to investigate the excited-state properties of the polyfluorene subunit where the exciton is localized (PF) and the incorporated PDI chromophore. The experimental results were compared with those obtained from a polyfluorene polymer (model PF) and a N,N′-bis(2,6-diisopropylphenyl)-1,6,7,12-tetra(p-tert-octylphenoxy)-perylene-3,4,9,10-tetracarboxylic acid diimide (model PDI) which were used as reference compounds. Because of the high polydispersity of the PF-PDI copolymer, there is a polymer fraction present that contains no PDI chromophores (polyfluorene polymer fraction (PF polymer fraction)), and wide-field imaging of single polymers chains of the synthesized PF-PDI copolymer was used to estimate this PF polymer fraction. Following the primary excitation of the PF in the PF-PDI copolymer, energy hopping between PF’s can occur. A fraction of the energy of the absorbed photons will be transferred to a PDI chromophore via energy transfer from a PF. In a polar solvent, a charge transfer state having the S1 of the PDI moiety as a precursor state is found to form with high efficiency on a nanosecond time scale. The data suggest that a fraction of the absorbed energy is directed, transferred, and used in charge separation, providing a clear view of a multistep mechanism of exciton dissociation into charges

Synthesis, Ensemble, and Single Molecule Characterization of a Diphenyl-Acetylene Linked Perylenediimide Trimer

Perylenediimide (PDI) dyes have attracted a great deal of attention as they possess excellent photochemical stability, high extinction coefficients, and fluorescence quantum yields. The use of multiple PDI chromophores in one synthetic architecture increases their versatile use and functionality even more. However, bringing multiple chromophores in close proximity also leads to interactions among the chromophores and opens up new photophysical pathways. Here, the synthesis and photophysical characterization, both at the ensemble and single molecule level, of a diphenyl-acetylene linked perylenediimide trimer (3PDIAc) is presented. Förster type energy transfer processes like energy hopping and singlet−singlet annihilation among the chromophores are investigated. Despite the lower singlet−singlet annihilation rate of the phenoxy substituted perylenediimide chromophores (356 ps) versus for example perylenemonoimide (10 ps), the system still behaves as a single photon emitter. Sequential fitting of the dipole emission pattern recorded with defocused wide field imaging of single 3PDIAc, immobilized in a PMMA polymer film, demonstrated that emission can switch between sequential emission of all of the chromophores or emission from one chromophore that likely is the lowest in energy

Synthesis, Ensemble, and Single Molecule Characterization of a Diphenyl-Acetylene Linked Perylenediimide Trimer

Perylenediimide (PDI) dyes have attracted a great deal of attention as they possess excellent photochemical stability, high extinction coefficients, and fluorescence quantum yields. The use of multiple PDI chromophores in one synthetic architecture increases their versatile use and functionality even more. However, bringing multiple chromophores in close proximity also leads to interactions among the chromophores and opens up new photophysical pathways. Here, the synthesis and photophysical characterization, both at the ensemble and single molecule level, of a diphenyl-acetylene linked perylenediimide trimer (3PDIAc) is presented. Förster type energy transfer processes like energy hopping and singlet−singlet annihilation among the chromophores are investigated. Despite the lower singlet−singlet annihilation rate of the phenoxy substituted perylenediimide chromophores (356 ps) versus for example perylenemonoimide (10 ps), the system still behaves as a single photon emitter. Sequential fitting of the dipole emission pattern recorded with defocused wide field imaging of single 3PDIAc, immobilized in a PMMA polymer film, demonstrated that emission can switch between sequential emission of all of the chromophores or emission from one chromophore that likely is the lowest in energy