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.
The application of fluorescent proteins in live cells has greatly improved our ability to study molecular mobility, which both reflects molecular function in live cells and reveals the properties of the local environment. Although measuring molecular mobility with fluorescent fusion proteins is powerful and convenient, certain experiments still require exogenous macromolecules to be loaded into cells. Cell viability provides a rough gauge of cellular damage following membrane permeabilization, but it is unknown how permeabilization will affect intracellular mobility. We have used fluorescence correlation spectroscopy to measure the intracellular dynamics of the enhanced green fluorescent protein (EGFP) in living human embryonic kidney (HEK) cells under conditions where the EGFP is either expressed or loaded using streptolysin O (SLO) permeabilization to determine how permeabilization effects mobility. We found that purified EGFP loaded with SLO has the same mobility as the expressed EGFP, while the mobility of the expressed EGFP after SLO permeabilization treatment becomes slightly slower. Our results indicate that SLO permeabilization is often accompanied by the loss of cellular soluble proteins to the surrounding medium, which explains the apparent decrease in diffusion rates following treatment. These measurements are also relevant to the role of molecular crowding in the intracellular mobility of proteins.
While the growth and structure of amyloid fibers with ß-sheet secondary structure has been
widely investigated in recent years, the mechanism of self-assembly remains poorly understood.
Multiple intermediate species have been proposed to play important roles in the self assembly
process, yet many of these remain poorly defined or have not been clearly observed.
Fluorescence microscopy and spectroscopy should provide powerful tools to amyloid formation
mechanisms, although given the tight packing of molecules within amyloid structures one must
be concerned about the extent to which the coupling of fluorescent probes will interfere with the
amyloid formation process. We have performed systematic characterization of the self assembly
and interactions between a model amyloid forming peptide, residues 16-22 from the amyloid beta peptide, together with two different rhodamine conjugated forms of this same peptide sequence. We observe that in some cases, the fluorescent dye does appear to alter the morphology of assembled amyloid structures. We also report on amyloid formation using mixtures of labeled and unlabeled peptides which does not perturb the morphology of the amyloid fibers and tubes, and appears to provide an excellent system for further investigation of amyloid formation.
KEYWORDS: Fluorescence correlation spectroscopy, Luminescence, Fluorescence spectroscopy, Spectroscopy, Point spread functions, 3D modeling, Objectives, Data modeling, Statistical modeling, 3D metrology
Information recovery in fluorescence fluctuation spectroscopy requires accurate models both for the physical dynamics observed and for the effective size and shape of the sample region from which fluorescence signals are measured. In both one- and two-photon fluctuation spectroscopy, the so called observation volume is assumed to be well approximated by a three dimensional Gaussian (3DG) function. Here, we present wave optics computations that provide an accurate representation of the true profile for the fluorescence measurement with two-photon excitation. Fluorescence correlation spectroscopy (FCS) curves are computed for these true profiles for a variety of optical configurations, and we demonstrate that under most illumination conditions the 3DG based FCS models do provide reasonable approximations to the measured FCS curves.
Fluorescence correlation spectroscopy (FCS) and related distribution analysis techniques have become extremely important and widely used research tools for analyzing the dynamics, kinetics, interactions, and mobility of biomolecules. However, it is not widely recognized that photophysical dynamics can dramatically influence the calibration of fluctuation spectroscopy instrumentation. While the basic theories for fluctuation spectroscopy methods are well established, there have not been quantitative models to characterize the photophysical-induced variations observed in measured fluctuation spectroscopy data under varied excitation conditions. We introduce quantitative models to characterize how the fluorescence observation volumes in one-photon confocal microscopy are modified by excitation saturation as well as corresponding models for the effect of the volume changes in FCS. We introduce a simple curve fitting procedure to model the role of saturation in FCS measurements and demonstrate its accuracy in fitting measured correlation curves over a wide range of excitation conditions.
Excitation saturation and other photophysical dynamics can have a dramatic influence on the effective imaging point spread function (psf) in fluorescence microscopy. Specifically, saturation leads to increased fluorescence observation volumes and altered spatial profiles for the psf. These changes have important implications for both fluorescence correlation spectroscopy (FCS) and imaging applications. A detailed characterization of these changes is required for accurate interpretation of FCS measurements. We here introduce a method to calculate molecular excitation profiles that represent the true fluorescence observation volume under the influence of excitation saturation in two-photon microscopy. An analytical model that accounts for pulsed excitation is developed to calculate the influence of saturation at any location within the excitation laser profile, and the overall saturation influenced molecular excitation profiles are evaluated numerically. Fluorescence signals measured with a solution of Rhodamine 6G are presented, showing good agreement with these calculations.
Fluorescence correlation spectroscopy (FCS) is a sensitive research tool for studying molecular dynamics at the single molecule level. Photophysical dynamics often dramatically influence FCS measurements, as we show here in characterizing the role of excitation saturation in two-photon fluorescence correlation measurements. We introduce a physical model that characterizes the influence of excitation saturation on the two-photon fluorescence observation volume, and derive an analytical expression for the correlation function that includes the influence of saturation. With this model, we can accurately describe both the temporal decay and the amplitude of measured fluorescence correlation functions over a wide range of illumination powers.
Deep tissue imaging may have important biomedical applications in the areas of skin disease diagnosis, wound healing, and tissue engineering. For the study oftissue physiology with microscopic resolution, we used two-photon microscopic imaging based on the excitation of endogenous fluorophores. While autofluorescence is observed ubiquitously in many tissue types, the identities and distributions of these fluorophores have not been completely characterized. The different fluorescent species are expected to have different fluorescence excitation and emission spectra. Self-modeling curve resolution (SMCR) can be applied to extract spectroscopic components from two-photon images. In ex vivo human skin, we were able to acquire a four-dimensional data set (3D space + excitation spectra). We extracted the major spectral components from this data set using multivariate curve resolution and correlated these species with known tissue structures. From the SMCR analysis, it was determined that there are approximately seven factors that contribute to most of the autofluorescence from human skin. This analysis provides us with the concentration ofthe species at different depths within the skin and also with a reconstructed image of the skin due to each single factor alone. Several ofthese chemical components have been identified, such as collagen, elastin, and NAD(P)H. In addition to providing insight into tissue physiology, we are able to optimize the excitation wavelength for each biochemical species for skin imaging applications.
Fluorescence correlation spectroscopy (FCS) is rapidly growing in popularity as a research tool in biological and biophysical research. Under favorable conditions, FCS measurements can produce an accurate characterization of the chemical, physical, and kinetic properties of a biological system. However, interpretation of FCS data quickly becomes complicated as the heterogeneity of a molecular system increases, as well as when there is significant non-stationery fluorescence background (e.g. intracellular autofluorescence). Use of multi-parameter correlation measurements is one promising approach that can improve the fidelity of FCS measurements in complex systems. In particular, the use of dual-color fluorescence assays, in which different interacting molecular species are labeled with unique fluorescent indicators, can "tune" the sensitivity of FCS measurements in favor of particular molecular species of interest, while simultaneously minimizing the contribution of other molecular species to the overall fluorescence correlation signal. Here we introduce the combined application of two-photon fluorescence excitation and dual-color cross-correlation analysis for detecting molecular interactions in solution. The use of two-photon excitation is particularly advantageous for dual-color FCS applications due to the uncomplicated optical alignment and the superior capabilities for intracellular applications. The theory of two-photon dual-color FCS is introduced, and initial results quantifying hybridization reactions between three independent single stranded DNA molecules are presented.
The attainable frequency range of ordinary heterodyning and super-heterodyning frequency- domain fluorescence instrumentation is limited by the response of the optical detector rather than by the harmonic content of the light source. The replacement of the photomultiplier detector by a 6 (mu) microchannel plate detector has improved the frequency response from 500 MHz to 10 GHz. A new method is developed to detect fast, excited state processes by extending to the frequency-domain the well known pump/probe (absorption) technique used in the time-domain. The upper frequency limit attainable with this method is limited only by the pulse width of the light sources. For picosecond pulse lasers this limit extends to hundreds of gigahertz. A theoretical determination of the basic equations is given, and data are shown for the excited state decay of a rhodamine 6G sample in ethylene glycol.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.