Jay R. Knutson

Mechanism of fluorescence concentration quenching of carboxyfluorescein in liposomes: energy transfer to nonfluorescent dimers.

When 5(6)-carboxyfluorescein (6CF) is encapsulated in liposomes at 0.2 M, 97-98% of the fluorescence is quenched. We have studied the mechanism of this effect. The dye-liposome system is a special case of concentration quenching of dyes, a phenomenon recognized for 100 years. Absorption spectra of encapsulated dye show that 6CF dimerizes, and the dimer is nonfluorescent. The dimerization constant was estimated, and it was concluded that dimerization can account for only part of the quenching. In 6CF solutions, the fluorescence lifetime decreased drastically as concentration was changed over the narrow range 0.02-0.05 M, a finding which was attributed to energy transfer to dimers. Inhibition of dimerization by propylene glycol also inhibited the shortening of lifetime. Förster critical transfer distances were calculated to be 51 and 57 A for monomer-monomer and monomer-dimer transfer, respectively. Monomer-monomer transfer was demonstrated directly by steady-state or time-resolved anisotropy experiments, while transfer to dimer was modeled by using sulforhodamine B, which has a critical transfer distance like that for the dimer and also quenches 6CF emission. No direct evidence for collisional self-quenching of 6CF could be found, although a model compound, salicylate, did quench weakly. For xanthene dyes, the rate of energy transfer is much faster than that for quenching collisions, implying that collisional quenching in the usual 6CF-liposome system is insignificant. The reason why 6CF is not 100% quenched in liposomes is attributed to dye interaction with lipid as evidenced by (i) multiexponential decay of 6CF in liposomes with a long component of 3-4 ns, (ii) inhibition of dimerization in liposomes, (iii) partial protection of dye from quenching by KI, (iv) differing amounts of dimerization in liposomes made from different kinds of phospholipid, and (v) enhancement of fluorescence lifetime in the presence of Triton X-100.

LOW-COHERENCE REFLECTOMETRY FOR STATIONARY LATERAL AND DEPTH PROFILING WITH ACOUSTO-OPTIC DEFLECTORS AND A CCD CAMERA

We describe a new optical low-coherence reflectometer for depth and lateral scanning without moving parts. The reflectometer covers a range of 0.4 and 1 mm in the depth and lateral dimensions, respectively. This level was accomplished by an acousto-optic deflector for lateral scanning and temporal-coherence gating for depth resolution. The ac component of the reflected light was captured by a cooled 16-bit CCD camera with a special readout scheme. As a proof of principle, optical depths of a staggered stack of glass plates were measured.

Direct measurement of association and dissociation rates of DNA binding in live cells by fluorescence correlation spectroscopy

Measurement of live-cell binding interactions is vital for understanding the biochemical reactions that drive cellular processes. Here, we develop, characterize, and apply a new procedure to extract information about binding to an immobile substrate from fluorescence correlation spectroscopy (FCS) autocorrelation data. We show that existing methods for analyzing such data by two-component diffusion fits can produce inaccurate estimates of diffusion constants and bound fractions, or even fail altogether to fit FCS binding data. By analyzing live-cell FCS measurements, we show that our new model can satisfactorily account for the binding interactions introduced by attaching a DNA binding domain to the dimerization domain derived from a site-specific transcription factor (the vitellogenin binding protein (VBP)). We find that our FCS estimates are quantitatively consistent with our fluorescence recovery after photobleaching (FRAP) measurements on the same VBP domains. However, due to the fast binding interactions introduced by the DNA binding domain, FCS generates independent estimates for the diffusion constant (6.7 +/- 2.4 microm2/s) and the association (2 +/- 1.2 s(-1)) and dissociation (19 +/- 7 s(-1)) rates, whereas FRAP produces only a single, but a consistent, estimate, the effective-diffusion constant (4.4 +/- 1.4 microm2/s), which depends on all three parameters. We apply this new FCS method to evaluate the efficacy of a potential anticancer drug that inhibits DNA binding of VBP in vitro and find that in vivo the drug inhibits DNA binding in only a subset of cells. In sum, we provide a straightforward approach to directly measure binding rates from FCS data.

Spatial localization of absorbing bodies by interfering diffusive photon-density waves

The use of the destructive interference of diffusive photon-density waves for the localization of an absorbing (and scattering) body in a scattering medium was studied. The objectives of this approach in the reflectance mode were as follows: first, to reduce sensitivity to absorption features occurring in superficial layers while sensitivity to bodies lying deeper is maintained; second, to establish a confined depth region of maximum sensitivity in which the distance of an absorbing body could be determined through phase measurement. Intensity and phase data were acquired with a modified frequency-domain spectrometer at modulation frequencies up to 600 MHz as an absorbing body was moved in three dimensions. The experimental results are compared with simulations based on a numerical solution of a time-dependent photon-diffusion equation.

Cross-Validating FRAP and FCS to Quantify the Impact of Photobleaching on In Vivo Binding Estimates

Binding can now be quantified in live cells, but the accuracy of such measurements remains uncertain. To address this uncertainty, we compare fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) measurements of the binding kinetics of a transcription factor, the glucocorticoid receptor, in the nuclei of live cells. We find that the binding residence time measured by FRAP is 15 times longer than that obtained by FCS. We show that this discrepancy is not likely due to the significant differences in concentrations typically used for FRAP and FCS, nor is it likely due to spatial heterogeneity of the nucleus, improper calibration of the FCS focal volume, or the intentional FRAP photobleach. Instead, our data indicate that photobleaching of bound molecules in FCS is mainly responsible. When this effect is minimized, FRAP and FCS measurements nearly agree, although cross-validation by other approaches is now required to rule out mutual errors. Our results demonstrate the necessity of a photobleach correction for FCS measurements of GFP-tagged molecules that are bound for >0.25 s, and represent an important step forward in establishing a gold standard for in vivo binding measurements.

Flow cytometric measurement of fluorescence (Förster) resonance energy transfer from cyan fluorescent protein to yellow fluorescent protein using single-laser excitation at 458 nm.

Use of distinct green fluorescent protein (GFP) variants permits the study of protein–protein interactions and colocalization in viable transfected cells by fluorescence (Förster) resonance energy transfer (FRET). Flow cytometry is a sensitive method to detect FRET. However, the typical dual‐laser methods used in flow cytometric FRET assays are not generally applicable because they require a specialized krypton ultraviolet (UV) laser. The purpose of this work was to develop a flow cytometric method to detect FRET between cyan fluorescent protein (CFP; donor) and yellow fluorescent protein (YFP; acceptor) by using the 458‐nm excitation from a single tunable argon‐ion laser.

Acousto‐optic scanning and interfering photon density waves for precise localization of an absorbing (or fluorescent) body in a turbid medium

In most optical methods proposed for imaging an absorbing object embedded in a turbid medium, data are collected using a single source and detector scanned mechanically across the surface of the medium. In our setup, we exploited destructive interference of diffusive photon density waves originating from two sources to localize an absorbing (or fluorescent) body in a scattering medium. A frequency‐domain instrumentation is described that scans several laser‐beam spots across the surface of a turbid medium using 1D (or 2D) acousto‐optical deflectors. An additional acousto‐optic deflector is used to establish arbitrary phase shifts for the interfering photon‐density waves. A destructive interference pattern was created to laterally localize an absorbing (or fluorescent) body in the reflection and transmission modes. In some experiments the destructive interference pattern was altered by modulating the individual beam intensities to improve sensitivity and ameliorate surface texture problems. The experimental results were retrieved from a gated intensified CCD camera at 246 MHz modulation frequency. Results indicate that less than a 1 mm displacement of a small object embedded 10 mm in a medium with optical characteristics similar to bloodless skin tissue can be detected.

Activation of moesin, a protein that links actin cytoskeleton to the plasma membrane, occurs by phosphatidylinositol 4,5-bisphosphate (PIP2) binding sequentially to two sites and releasing an autoinhibitory linker.

Background: Phosphatidylinositol 4,5-bisphosphate (PIP2) activates moesin via two binding sites whose roles are poorly understood. Results: Critical residues are identified in both sites and an inhibitory linker (FLAP) is characterized. Conclusion: Activation of moesin requires PIP2 binding to each site and release of the FLAP. Significance: The results fit a sequential activation model involving conformational change and interfacial release of FLAP. Many cellular processes depend on ERM (ezrin, moesin, and radixin) proteins mediating regulated linkage between plasma membrane and actin cytoskeleton. Although conformational activation of the ERM protein is mediated by the membrane PIP2, the known properties of the two described PIP2-binding sites do not explain activation. To elucidate the structural basis of possible mechanisms, we generated informative moesin mutations and tested three attributes: membrane localization of the expressed moesin, moesin binding to PIP2, and PIP2-induced release of moesin autoinhibition. The results demonstrate for the first time that the POCKET containing inositol 1,4,5-trisphosphate on crystal structure (the “POCKET” Lys-63, Lys-278 residues) mediates all three functions. Furthermore the second described PIP2-binding site (the “PATCH,” Lys-253/Lys-254, Lys-262/Lys-263) is also essential for all three functions. In native autoinhibited ERM proteins, the POCKET is a cavity masked by an acidic linker, which we designate the “FLAP.” Analysis of three mutant moesin constructs predicted to influence FLAP function demonstrated that the FLAP is a functional autoinhibitory region. Moreover, analysis of the cooperativity and stoichiometry demonstrate that the PATCH and POCKET do not bind PIP2 simultaneously. Based on our data and supporting published data, we propose a model of progressive activation of autoinhibited moesin by a single PIP2 molecule in the membrane. Initial transient binding of PIP2 to the PATCH initiates release of the FLAP, which enables transition of the same PIP2 molecule into the newly exposed POCKET where it binds stably and completes the conformational activation.

Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector

We have constructed a device that maximizes the probability of collecting all of the scattered and ballistic light isotropically generated at the focal spot of multiphoton excited emissions (MPE) to optimize the signal‐to‐noise ratio (SNR) for micro‐imaging. This was accomplished by optically coupling a parabolic reflector (that surrounds the sample and top of the objective) to a pair of collimating lenses (above the sample) that redirects emitted light to a separate detector. These additional optics, combined with the objective, allow the total emission detection (TED) condition to be approached. Numerical simulations suggest an approximately 10‐fold improvement in SNR with TED. Comparisons between the objective detection and TED reveal an enhancement of 8.9 in SNR (77% of predicted) for GFP‐labelled brain slices and similar results for fluorescent beads. This increase in SNR can be used to improve time resolution, reduce laser power requirements/photodynamic damage, and, in certain cases, detection depth, for MPE imaging techniques.

In Vivo Fluorescence Lifetime Imaging Monitors Binding of Specific Probes to Cancer Biomarkers

One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the “image and treat” concept, especially for early evaluation of the efficacy of the therapy.