Edward H. Hellen

Fluorescence emission at dielectric and metal-film interfaces

It is well known that the classical optical properties of a bare or metal-film-coated dielectric surface significantly the emission pattern of a fluorophore in close proximity to it. Most previous classical calculations of this perturb model the fluorophore as a continuous fixed-amplitude dipole acting as a simple radiator. However, for effect modeling steady-state excitation, a fixed-power dipole is more appropriate. This modification corresponds to normalizing fixed-amplitude dipole intensities by the total dissipated power, which is itself dependent on fluorophore orientation and proximity to the surface. The results for the fixed-power model differ nontrivially from the fixed-amplitude model. Using the fixed-power dipole model, we calculate the observation-angle-dependent intensity as a function of the fluorophore’s orientation and distance from the surface. The surface can have an intermediate layer of arbitrary thickness on it, which is used to model a metal-film-coated dielectric. In addition, general expressions are derived for the emission power as observed through a circular-aperture collection system (such as a microscope objective) located on either side of the interface. These expressions are applied to several common cases of fluorophore spatial and orientational distributions at bare glass–water and metal-film-coated glass-water interfaces. The results suggest practical experimental approaches for measuring the spatial and orientational distribution of fluorophores adsorbed at a surface, utilizing the distance-dependent fluorescence near a metalized surface and optimizing the collection efficiency from a well-defined volume near a quenching surface.

Kinetics of epidermal growth factor/receptor binding on cells measured by total internal reflection/fluorescence recovery after photobleaching

Total internal reflection fluorescence (TIRF) microscopy is used to measure the dissociation kinetic rate of fluorescein-labeled epidermal growth factor from its specific receptors on the surface of intact but mildly fixed A431 human epidermoid cells in culture. Prior applications of TIRF microscopy have been limited to nonreceptor binding or to model membrane systems. The evanescent field excites fluorescence selectively at the surface of the cell proximal to the coverslip. “Prismless” epiillumination TIR is employed to avoid space limitations and is achieved by passing the excitation laser beam through a high (1.4)-aperture objective so that the light is incident at the glass/water interface beyond the critical angle. Long-term focus is maintained by a special feedback system. Of the possible effects that can influence the time course of the postbleach fluorescence recoveries—the EGF/receptor dissociation ratek2, the bulk solution diffusion rate of EGF, and the cell surface motion of the receptors—we infer that the dissociation ratek2 dominates. Several fitting schemes are compared and indicate the presence of a multiplicity of values fork2, ranging from about 0.05 to 0.004 s−1, with an average value of about 0.012 s−1. These results compare well with values previously obtained by radiolabel/washing techniques. The significance of the results in terms of kinetic models and the advantages of the TIRF technique for these sorts of measurements are discussed.

Noise-Aided Logic in an Electronic Analog of Synthetic Genetic Networks.

We report the experimental verification of noise-enhanced logic behaviour in an electronic analog of a synthetic genetic network, composed of two repressors and two constitutive promoters. We observe good agreement between circuit measurements and numerical prediction, with the circuit allowing for robust logic operations in an optimal window of noise. Namely, the input-output characteristics of a logic gate is reproduced faithfully under moderate noise, which is a manifestation of the phenomenon known as Logical Stochastic Resonance. The two dynamical variables in the system yield complementary logic behaviour simultaneously. The system is easily morphed from AND/NAND to OR/NOR logic.

Electronic Implementation of a Repressilator with Quorum Sensing Feedback.

We investigate the dynamics of a synthetic genetic repressilator with quorum sensing feedback. In a basic genetic ring oscillator network in which three genes inhibit each other in unidirectional manner, an additional quorum sensing feedback loop stimulates the activity of a chosen gene providing competition between inhibitory and stimulatory activities localized in that gene. Numerical simulations show several interesting dynamics, multi-stability of limit cycle with stable steady-state, multi-stability of different stable steady-states, limit cycle with period-doubling and reverse period-doubling, and infinite period bifurcation transitions for both increasing and decreasing strength of quorum sensing feedback. We design an electronic analog of the repressilator with quorum sensing feedback and reproduce, in experiment, the numerically predicted dynamical features of the system. Noise amplification near infinite period bifurcation is also observed. An important feature of the electronic design is the accessibility and control of the important system parameters.

Verifying the diode–capacitor circuit voltage decay

The voltage on a capacitor discharging through a forward biased diode is calculated from basic equations and is found to be in good agreement with experimental measurements. In contrast to the exponential time decay for a RC circuit, the nonlinear characteristics of the diode result in a nonexponential decay for the diode–capacitor circuit. For a silicon diode the decay is predominantly a logarithmic function of time.

Saturation effects in polarized fluorescence photobleaching recovery and steady state fluorescence polarization.

The time-resolved anisotropy produced in polarized fluorescence photobleaching recovery experiments has been successfully used to measure rotational correlation times in a variety of biological systems, however the magnitudes of the reported initial anisotropies have been much lower than the theoretically predicted maximum values. This small time-zero anisotropy has been attributed to fluorophore motion, wobble and rotation, during the photobleaching pulse. We demonstrate that inclusion of the possibility of saturation of the fluorophores transition from its ground state to its excited state during the photobleaching pulse leads to the prediction of reduced time-zero anisotropy. This eliminates the need to rely solely on the assumption of fluorophore motion during the photobleaching pulse as the cause of the reduced initial anisotropy. We present theoretical and experimental results which show that the initial anisotropy decreases as both the bleach pulse intensity is increased and bleach pulse duration is decreased so as to keep the total integrated bleach pulse constant. We also show theoretical and experimental results demonstrating that at high excitation intensity the effects of saturation cause the steady state fluorescence polarization to decrease. We estimate that saturation may occur using common photobleaching conditions.

An automatic focus/hold system for optical microscopes

A system for maintaining long‐term focus of samples under high‐magnification quantitative observation in an epi‐illumination optical microscope is described. A negative feedback signal is generated from focus‐dependent changes in the backreflection of an off‐axis HeNe laser. This reflection is intercepted by a small prism downbeam from the standard trinocular head, and detected by a small two‐photodiode array. Spontaneous drifts in sample focus (presumably due to thermal and mechanical relaxations) are detected as a nonzero difference signal, which is used to drive a dc motor mechanically coupled to the fine‐focus knob of the microscope. This system has several advantages: (1) it is completely compatible and nonobstrusive with concurrent data acquisition of sample intensities; (2) it requires no alteration of the sample, sample stage, or objective; (3) it monitors the focal position of sample areas very near to those under observation; (4) it is inexpensive. In an experimental test, the system can hold a thin glass coverslip sample (a common substrate for biological cell cultures) to within 0.5 μm of its preset focus position, well within the depth of focus of the microscope. Without the system, such samples typically drift several micrometers over periods of 10 min. In response to a disturbance of the focus knob, the system can restore the focus to within 0.5 μm of the preset position.

An Electronic Analog of Synthetic Genetic Networks

An electronic analog of a synthetic genetic network known as the repressilator is proposed. The repressilator is a synthetic biological clock consisting of a cyclic inhibitory network of three negative regulatory genes which produces oscillations in the expressed protein concentrations. Compared to previous circuit analogs of the repressilator, the circuit here takes into account more accurately the kinetics of gene expression, inhibition, and protein degradation. A good agreement between circuit measurements and numerical prediction is observed. The circuit allows for easy control of the kinetic parameters thereby aiding investigations of large varieties of potential dynamics.

Myosin head rotation in muscle fibers measured using polarized fluorescence photobleaching recovery.

The technique of polarized fluorescence photobleaching recovery (PFPR) has been applied for the first time to investigation of the rotational correlation time of the myosin head in muscle fibers. This is a novel application of PFPR because it is the first time PFPR has been applied to a sample which is not cylindrically symmetric about the optical axis. Therefore we present a method for analysis of PFPR results from an oriented sample such as the muscle fibers aligned perpendicularly to the optical axis used here. Control experiments performed on fluorescently labeled myosin heads in solution demonstrate that, under some conditions, our PFPR apparatus can easily measure a rotational correlation time of less than 200 μs. Validity of this application of PFPR to muscle fibers is provided by the agreement of our results with published results from a variety of other spectroscopic techniques. In particular, using glycerinated rabbit psoas muscle fibers, we find that for relaxed fibers and isometrically contracting fibers, the myosin heads undergo high-amplitude rotations on the submillisecond time domain. For fibers in rigor the myosin heads are highly oriented and nearly immobile. For fibers in ADP the myosin heads are highly ordered in a distribution quite different from that in rigor, and they are slightly more mobile than in rigor.

Emission of fluorescence at an interface

Publisher Summary This chapter discusses the classical-optical effects and the emission properties as they might be observed through a microscope, with particular attention to the bare-glass/water and metal film-coated glass/water interfaces. The results suggest some practical experiments that take advantage of the special optical effects at surfaces. These experiments include deducing the relative concentration of fluorophore as a function of distance from the surface, quenching unwanted background fluorescence from fluorophores nonspecifically adsorbed to a substrate, and optimizing the collection of fluorescence by a microscope objective. The presence of the surface affects several aspects of the fluorescence, including its intensity, angular distribution, quantum yield, and lifetime. The effects referred are not quantum chemical––that is, effects upon the orbitals or states of the fluorophore in the presence of any static fields associated with the surface rather, the effect is classical optical––that is, effects on the electromagetic field generated by a classical oscillating dipole in the presence of an interface among any media with dissimilar refractive indices.