In recent years, single-molecule fluorescence imaging has been reconciling a fundamental mismatch between optical microscopy and subcellular biophysics. However, the next step in nanoscale imaging in living cells can be accessed only by optical excitation confinement geometries. Here, we review three methods of confinement that can enable nanoscale imaging in living cells: excitation confinement by laser illumination with beam shaping; physical confinement by micron-scale geometries in bacterial cells; and nanoscale confinement by nanophotonics.

dynamics of several replisome components in vivo. Photoactivated localization microscopy (PALM) and single-molecule tracking give a resolution of 20-40 nm, far below the diffraction limit of conventional microscopy, enabling us to localize and track every single protein molecule. In our study, we investigated the dynamics of a number of replisome components under different conditions, including the DNA polymerases PolC, and the b-clamp loader DnaX. We can watch the real time behavior of different replisome components during the DNA synthesis process, and study them quantitatively. Surprisingly, our investigations have revealed that all these replisome components are highly dynamic and exchange more rapidly than previously expected, and we characterize the molecular scale distribution of each replisome component within the dynamic replication machinery.

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DNA replication happens in all living organisms, and assures that the genome is accurately copied and maintained. The replisome is the molecular machine in cells that replicates DNA, and it is composed of several different proteins, including DNA polymerases, which directly synthesize DNA by adding nucleotides. Although the bacterial replisome has been studied extensively in vitro, little is known about the dynamics and architecture of replisome components in vivo. Here we use Bacillus subtilis, a Gram-positive bacterium commonly found in soil, as a model organism in which to study the architecture and dynamics of several replisome components in vivo. Photoactivated localization microscopy (PALM) and single-molecule tracking give a resolution of 20-40 nm, far below the diffraction limit of conventional microscopy, enabling us to localize and track every single protein molecule. In our study, we investigated the dynamics of a number of replisome components under different conditions, including the DNA polymerases PolC and DnaE, and the b-clamp loader DnaX. We can watch the real time behavior of different replisome components during the DNA synthesis process, and study them quantitatively. Surprisingly, our investigations have revealed that all these replisome components are highly dynamic and exchange more rapidly than previously expected, and we characterize the molecular scale distribution of each replisome component within the dynamic replication machinery.

glucose, mannose and galactose with water molecules to calculate spectra of water molecules. We demonstrate that the number of water molecules around typical hydroxyl is different between each monosaccharide. The result shows that the equatorial hydroxyl is more hydrated than the axial one because of steric inhabitation. However, hydroxyl next to oxygen doesn't change between equatorial and axial. Spectra of water molecules in the bulk agree with that obtained by experiments. The spectra calculated by correlation function of relative velocity include three peeks which is related to typical motions of water molecule, angular, stretching and tumbling. The difference spectra between sugar solution and bulk shows that there are two larger changes around two peaks, angular and stretching motions, and the largest peak of angular is galactose, followed by mannose and glucose in this order. These results show the difference of hydration around carbohydrates can be identified by theoretical simulation and we can also identify the spectra change of water molecules.