Fluorescence correlation spectroscopy (FCS) and related fluctuation spectroscopy and microscopy methods have
become important research tools that enable detailed investigations of the chemical and physical properties of
molecules and molecular systems in a variety of complex environments. When analyzed successfully fluctuation
measurements often provide unique information that is otherwise difficult to measure, such as molecular
concentrations and interaction stoichiometry. However, information recovery via curve fitting of fluctuation data
can present challenges due to limited resolution and/or problems with fitting model verification. We discuss a new
approach to fluctuation data analysis coupling multi-modal fluorescence measurements and global analysis, and
demonstrate how this approach can provide enhanced sensitivity and resolution in fluctuation measurements. We
illustrate the approach using a combination of FCS and fluorescence lifetime measurements, here called τFCS, and
demonstrate the capability to recover the concentration of two independent molecular species in a two component
mixture even when the species have identical diffusion coefficients and molecular brightness values. This work was
partially supported by NSF grants MCB0817966 and DMR0907435.
We use Fluorescence Lifetime Imaging Microscopy (FLIM) and Second Harmonic Imaging Microscopy (SHIM) to
investigate the fundamental molecular mechanisms responsible for nucleation and growth of amyloidogenic-derived
nanomaterials. The nanomaterials are assembled from of Amyloid-β(16-22), specifically Ac-KLVFFAE-NH2, the
nucleating core of the Alzheimer's Amyloid-β protein. We describe how FLIM and SHIM can be used to follow
different nucleation pathways and to quantify structural heterogeneities within these complex nanomaterials. New
evidence suggests that different structures emerge from distinct nucleation pathways and these insights inform our
understanding of the peptide self-assembly mechanisms. We discuss these insights in the context of a top down
understanding of amyloidogenic diseases, the bottom up control of functional nanomaterials and the discovery of realtime
structural indicators for nanofabrication strategies.
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