We quantitatively analyzed particle tracking data on insulin granules expressing fluorescent fusion proteins in MIN6 cells to better understand the motions contributing to intracellular transport and, more generally, the means for characterizing systems far from equilibrium. Care was taken to ensure that the statistics reflected intrinsic features of the individual granules rather than details of the measurement and overall cell state. We find anomalous diffusion. Interpreting such data conventionally requires assuming that a process is either ergodic with particles working against fluctuating obstacles (fractional Brownian motion) or nonergodic with a broad distribution of dwell times for traps (continuous-time random walk). However, we find that statistical tests based on these two models give conflicting results. We resolve this issue by introducing a subordinated scheme in which particles in cages with random dwell times undergo correlated motions owing to interactions with a fluctuating environment. We relate this picture to the underlying microtubule structure by imaging in the presence of vinblastine. Our results provide a simple physical picture for how diverse pools of insulin granules and, in turn, biphasic secretion could arise. Eukaryotic cells package proteins into vesicles for trafficking and spatially localized secretion. These essential functions are highly regulated, and defects in them can lead to disease (1, 2). Although optical microscopy, combined with molecular and cellular biology, can provide important insight into intracellular dynamics, in the past, most measurements detected variations in intensities from many molecular events and thus averaged in some way. These include fluorescence correlation spectroscopy (FCS) (3), fluorescence recovery after photobleaching (FRAP) (4), and image correlation spectroscopy (5). Recent advances in experimental methods now enable tracking single particles in cells (6). Although these measurements still involve a degree of time averaging (7), the resulting individual time trajectories contain more information than the mean values extracted from the aforementioned approaches.Qualitatively, the time trajectories reveal complex behaviors: combinations of random, directed, transiently stalled and constrained motions (e.g., refs. 7, 8). These different types of motion reflect the interplay of various molecular components in crowded environments. Quantifying their relative importance can constrain mechanisms, but extracting this information from the particle tracking data requires new theoretical tools. Operationally, one strategy is to classify segments of trajectories according to their motions (e.g., active and passive) (9, 10), but this requires long trajectories. A less data-demanding approach is to identify different types of anomalous diffusion (11).What features can give rise to the observed anomalous behavior? Simple crowding is insufficient, as it results in standard Brownian motion but with a reduced diffusion coefficient (12). Instead, anomalous b...

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We demonstrate three-dimensional trapping and orientation of individual Au nanorods by using laser light slightly detuned from their longitudinal plasmon mode. Detuning to the long-wavelength side of the resonance allows stable trapping for several minutes, with an exponential dependence of trapping time on laser power (consistent with a Kramer's escape process). Detuning to the short-wavelength side causes repulsion of the rods from the laser focus. Alignment of the long axis of the rods with the trapping laser polarization is observed as a suppression of rotational diffusion about the short axis.

The linear and nonlinear optical properties of nanoparticles are of significant importance for fundamental science and technological applications. 1,2 The second harmonic (SH) response of nanoparticles is particularly interesting due to the potential dependence on particle shape and deviations from high symmetry (e.g., spherical). However, ensemble measurements 3 (e.g. SH from colloidal solutions) obscure deeper insights due to the inevitable inhomogeneity of particle shape and size. Thus, measurements on a single-particle level are necessary to gain a more complete understanding of the optical properties of nanocrystal materials. 4 Undertaking such measurements is, however, impeded by the lack of a methodology allowing precise correlation of optical measurements and highresolution imaging of the particle morphology by transmission electron microscopy (TEM).This communication reports the results of an approach that permits correlation of SH activity with nanoparticle morphology imaged by TEM, which achieves significantly higher resolution than atomic force microscopy and scanning electron microscopy. The essence of our approach is to create position markers on an optical and electron transparent substrate (Si 3 N 4 thin film, ∼100 nm in thickness) that allows both optical measurements and TEM imaging of the identical nanoparticles. 5 The SH activities of single Ag nanoparticles (spheres versus rods) and cluster structures (e.g., dimers and trimers) are compared. The direct determination of the structure and the resultant structure-spectroscopic correlation allows gaining a new insight into the mechanism for the nonlinear (i.e., resonant second harmonic) response from each specific nanoparticle or cluster.The position bar-markers were fabricated by electron-beam lithography on a specially designed Si 3 N 4 window supported on a Si wafer. Briefly, the Si 3 N 4 substrate was first coated with a thin layer (8 nm) of silicon monoxide via thermal evaporation. This changes the hydrophobic Si 3 N 4 surface to hydrophilic, which provides better adhesion and distribution of aqueous Ag nanoparticles. The substrates were then spin-coated with electron resist, followed by electron-beam patterning. The patterned substrate was further deposited with a thin layer (20 nm) of chromium, which acts as the positional marker material, followed by liftoff of electron resist. The substrate with position markers was further coated with a positively charged polymer (polydimethyldidodecylammonia chloride, PDDA), which captures negatively charged Ag nanoparticles synthesized by citrate reduction of AgNO 3 . As shown in Scheme S1 (Supporting Information), SH maps were acquired with a home-built nonlinear optical microscopy system including a tunable Ti:sapphire femtosecond laser (Spectra Physics). The detection optics allowed detection of wavelengths from 400 to 500 nm, while the excitation was tunable from 800 to 1000 nm with ∼100-fs pulse (see Supporting Information).We first investigated the SH activity of single Ag nanoparticles and cl...

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