Foerster (or fluorescence) resonance energy transfer (FRET) is a powerful tool for investigating protein-protein interactions, in both living cells and in controlled environments. A typical hetero-FRET pair consists of a donor and acceptor tethered together with a linker. The corresponding energy transfer efficiency of a hetero-FRET pair probe depends upon the donor-acceptor distance, relative dipole orientation, and spectral overlap. Because of the sensitivity of the energy transfer efficiency on the donor-acceptor distance, FRET is often referred to as a “molecular ruler”. Time-resolved fluorescence approach for measuring the excited-state lifetime of the donor and acceptor emissions is one of the most reliable approaches for quantitative assessment of the energy transfer efficiency in hetero-FRET pairs. In this contribution, we provide an analytical kinetics model that describes the excited-state depopulation of a FRET probe as a means to predicts the time-resolved fluorescence profile as a function of excitation and detection wavelengths. In addition, we used this developed kinetics model to simulate the time-dependence of the excited-state population of both the donor and acceptor. These results should serve as a guide for our ongoing studies of newly developed hetero-FRET sensors (mCerulean3–linker–mCitrine) that are designed specifically for in vivo studies of macromolecular crowding. The same model is applicable to other FRET pairs with the careful consideration of their steady-state spectroscopy and the experimental design for wavelength- dependence of the fluorescence lifetime measurements.
Beta-site APP cleaving enzyme 1 (BACE1) is a type I transmembrane aspartyl protease. In the amyloidogenic pathway, BACE1 provides β-secretase activity that cleaves the amyloid precursor protein (APP) that leads to amyloid beta (Aβ) peptides. The aggregation of these Aβ will ultimately results in amyloid plaque formation, a hallmark of Alzheimer’s disease (AD). Amyloid aggregation leads to progressive memory impairment and neural loss. Recent detergent protein extraction studies suggest that the untreated BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. Here, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using fluorescence correlation spectroscopy (FCS). Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal and DIC microscopy to monitor labeled BACE1 localization and distribution within the cell. Our one-photon fluorescence fluctuation autocorrelation of BACE1- EGFP on the plasma membrane of HEK cells is modeled using two diffusing species on the plasma membrane with estimated diffusion coefficients of 1.39 x 10<sup>-7</sup> cm<sup>2</sup>/sec and 2.8 x 10<sup>-8</sup> cm<sup>2</sup>/sec under resting conditions and STA-200 inhibition, respectively. Anomalous diffusion model also provided adequate description of the observed autocorrelation function of BACE1- EGFP on the plasma membrane with an estimate exponent (α) of 0.8 and 0.5 for resting and STA-200 treated cells, respectively. The corresponding hydrodynamic radius of this transmembrane fusion protein was estimated using the measured diffusion coefficients assuming both Stokes-Einstein and Saffman-Delbruck models. Our results suggest a complex diffusion pattern of BACE1-EGFP on the plasma membrane of HEK cells with the possibility for dimer formation, especially under STA-200 inhibition.
Living cells are crowded with macromolecules and organelles. Yet, it is not fully understood how macromolecular crowding affects the myriad of biochemical reactions, transport and the structural stability of biomolecules that are essential to cellular function and survival. These molecular processes, with or without electrostatic interactions, in living cells are therefore expected to be distinct from those carried out in test tube in dilute solutions where excluded volumes are absent. Thus there is an urgent need to understand the macromolecular crowding effects on cellular and molecular biophysics towards quantitative cell biology. In this report, we investigated how biomimetic crowding affects both the rotational and translation diffusion of a small probe (rhodamine green, RhG). For biomimetic crowding agents, we used Ficoll-70 (synthetic polymer), bovine serum albumin and ovalbumin (proteins) at various concentrations in a buffer at room temperature. As a control, we carried out similar measurements on glycerolenriched buffer as an environment with homogeneous viscosity as a function of glycerol concentration. The corresponding bulk viscosity was measured independently to test the validity of the Stokes-Einstein model of a diffusing species undergoing a random walk. For rotational diffusion (ps–ns time scale), we used time-resolved anisotropy measurements to examine potential binding of RhG as a function of the crowding agents (surface structure and size). For translational diffusion (μs–s time scale), we used fluorescence correlation spectroscopy for single-molecule fluctuation analysis. Our results allow us to examine the diffusion model of a molecular probe in crowded environments as a function of concentration, length scale, homogeneous <i>versus </i>heterogeneous viscosity, size and surface structures. These biomimetic crowding studies, using non-invasive fluorescence spectroscopy methods, represent an important step towards understanding cellular biophysics and quantitative cell biology.
Native coenzymes such as the reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine
dinucleotide play pivotal roles in energy metabolism and a myriad of biochemical reactions in living cells/tissues. These
coenzymes are naturally fluorescent and, therefore, have the potential to serve as intrinsic biomarkers for mitochondrial
activities, programmed cell death (apoptosis), oxidative stress, aging, and neurodegenerative disease. In this
contribution, we employ two-photon fluorescence lifetime imaging microscopy (FLIM) and time-resolved anisotropy
imaging of intracellular NADH for quantitative, non-invasive biochemistry on living cells in response to hydrogenperoxide-
induced oxidative stress. In contrast with steady-state one-photon, UV-excited autofluorescence, two-photon
FLIM is sensitive to both molecular conformation and stimuli-induced changes in the local environment in living cells
with minimum photodamage and inherently enhanced spatial resolution. On the other hand, time-resolved, two-photon
anisotropy imaging of cellular autofluorescence allows for quantitative assessment of binding state and environmental
restrictions on the tumbling mobility of intrinsic NADH. Our measurements reveal that free and enzyme-bound NADH
exist at equilibrium, with a dominant autofluorescence contribution of the bound fraction in living cells. Parallel studies
on NADH-enzyme binding in controlled environments serve as a point of reference in analyzing autofluorescence in
living cells. These autofluorescence-based approaches complement the conventional analytical biochemistry methods
that require the destruction of cells/tissues, while serving as an important step towards establishing intracellular NADH
as a natural biomarker for monitoring changes in energy metabolism and redox state of living cells in response to
In the crowded cellular milieu, biological processes require coordinated intermolecular interactions, conformational changes, and molecular transport that span a wide range of spatial and temporal scales. This complexity requires an integrated, noninvasive, multiscale experimental approach. Here, we develop a multimodal fluorescence microspectroscopy system, integrated on a single platform, to gain information about molecular interactions and their dynamics with high spatio-temporal resolution. To demonstrate the versatility of our experimental approach, we use rhodamine 123-labeled mitochondria in breast cancer cells (Hs578T), verified using differential interference contrast (DIC) and fluorescence (confocal and two-photon) microscopy, as a model system. We develop an assay to convert fluorescence intensity to actual concentrations in intact, individual living cells, which contrasts with conventional biochemical techniques that require cell lysates. In this assay, we employ two-photon fluorescence lifetime imaging microscopy (FLIM) to quantify the fluorescence quantum yield variations found within individual cells. Functionally driven changes in cell environment, molecular conformation, and rotational diffusion are investigated using fluorescence polarization anisotropy imaging. Moreover, we quantify translational diffusion and chemical kinetics of large molecular assemblies using fluorescence correlation spectroscopy. Our integrated approach can be applied to a wide range of molecular and cellular processes, such as receptor-mediated signaling and metabolic activation.
The inherent advantages of nonlinear excitation make multiphoton fluorescence microscopy (MPFM) awell-suited imaging technique for extracting valuable information from turbid and thick biological samples. These advantages include high three-dimensional spatial resolution, large penetration depth, minimum out-of-focus cellular photodamage, and high signal-to-noise contrast. We have investigated the nonlinear spectroscopy of biologically important molecules such as NADH, flavins, and intrinsically fluorescent proteins. Fundamental understanding of the molecular spectroscopy and dynamics of these biomolecules is essential for advancing their applications in biological research. MPFM has been utilized for monitoring a large spectrum of biological processes including metabolic activity and exocytosis. We will discuss two-photon (2P) redox fluorescence microscopy of NADH, which gives a quantitative measure of the respiratory chain activity, thus allowing functional imaging of energy metabolism in neurons and native brain tissue. Finally, a rational design strategy, based on donor-acceptor-donor configuration, will be elucidated for fluorescent probes with large 2P-excitation cross-section. These dyes are water-soluble, yet possess a high affinity to organic phases with site-specific labeling and Ca<sup>+2</sup> sensitivity (K<sub>d</sub> ~ 350 nM). A brief account on the biological application of nanocrystals and second harmonic imaging will be reviewed.
Photochemical reactions which can be activated by the simultaneous absorption of two photons provide a means for single-step fabrication of complex 3D microstructures. These types of structures are needed for a wide range of applications, including microfluidics, electrooptics, and micro-electromechanical systems. We have shown that chromophores can be engineered to have both large two-photon absorptivities as well as an efficient means for activating chemical processes, such as radical polymerization, subsequent to the photoexcitation. Chromophores designed following this strategy two-photon-activate the radical polymerization of acrylates at lower incident laser powers than conventional UV initiators. Efficient two-photon photopolymer resins based on these chromophores were used in the fabrication of complex microarchitectures, such as photonic bandgap structures and tapered waveguides. We have devised a strategy which allows this approach to be extended to other chemical systems.