Elucidating the mechanisms of insulin granule trafficking in pancreatic β-cells is a critical step in understanding Type II
Diabetes and abnormal insulin secretion. In this paper,
rapid-sampling stochastic scanning multiphoton multifocal
microscopy (SS-MMM) was developed to capture fast insulin granule dynamics in vivo. Stochastic scanning of (a
diffractive optic generated) 10×10 hexagonal array of foci with a galvanometer yields a uniformly sampled image with
fewer spatio-temporal artifacts than obtained by conventional or multibeam raster scanning. In addition, segmented
spatio-temporal image correlation spectroscopy (Segmented STICS) was developed to extract dynamics of insulin
granules from the image sequences. Measurements we conducted on MIN6 cells, which exhibit an order of magnitude
lower granule number density, allow comparison of particle tracking with Segmented-STICS. Segmentation of the
images into 8×8 pixel segments (similar to a size of one granule) allows some amount of spatial averaging, which can
reduce the computation time required to calculate the correlation function, yet retains information about the local spatial
heterogeneity of transport. This allows the correlation analysis to quantify the dynamics within each of the segments
producing a "map" of the localized properties of the cell. The results obtained from Segmented STICS are compared
with dynamics determined from particle tracking analysis of the same images. The resulting range of diffusion
coefficients of insulin granules are comparable to previously published values indicating that SS-MMM and segmented-
STICS will be useful to address the imaging challenges presented by β-cells, particularly the extremely large number
density of granules.
We have measured nonlinear scattering from plasmons in individual Au nanorods and have correlated second-harmonic
activity of Ag nanoparticles and clusters to morphology. The measurements reveal novel ultrafast nonlinear phenomena
related to electron confinement. Surprisingly, the coherent plasmon response is suppressed relative to the hot electron
response indicating enhanced plasmon dephasing. In a parallel set of studies we demonstrate nanometer scale
localization of the nonlinear optical response of single nanoparticles and aggregates and correlate this with their
morphology. Position markers are fabricated on an optical and electron-transparent substrate (Si3N4 thin film) that
allows optical measurements and transmission electron microscopy (TEM) imaging of the identical nanoparticles or
aggregates. The second harmonic (SH) activity optical image of individual Ag nanostructures is registered with the
TEM image. Centroid localization of the optical signals allows correlation with better than 25 nm precision. This is
sufficient to determine the origin of optical "hot spots" within multi-particle aggregates.
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