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The evaluation of lateral diffusion coefficients of membrane components by the technique of fluorescence recovery after photobleaching (FRAP) is often complicated by uncertainties in the values of the intensities F(O), immediately after bleaching, and F(infinity), after full recovery. These uncertainties arise from instrumental settling time immediately after bleaching and from cell, tissue, microscope, or laser beam movements at the long times required to measure F(infinity). We have developed a method for precise analysis of FRAP data that minimizes these problems. The method is based on the observation that a plot of the reciprocal function R(tau) = F(infinity)/[F(infinity)-F(tau)] is linear over a large time range when (a) the laser beam has a Gaussian profile, (b) recovery involves a single diffusion coefficient, and (c) there is no membrane flow. Moreover, the ratio of intercept to slope of the linear plot is equal to tau 1/2, the time required for the bleached fluorescence to rise to 50% of the full recovery value, F(infinity). The lateral diffusion coefficient D is related to tau 1/2 by tau 1/2 = beta w2/4D where beta is a defined parameter and w is the effective radius of the focused laser beam. These results are shown to indicate that the recovery of fluorescence F(tau) can be represented over a large range of percent bleach, and recovery time tau by the relatively simple expression F(tau) = [ F(o) + F(infinity) (tau/tau 1/2)]/[1 + tau/tau 1/2)]. FRAP data can therefore be easily evaluated by a nonlinear regression analysis with this equation or by a linear fit to the reciprocal function R(tau). It is shown that any error in F(infinity) can be easily detected in a plot of R(tau) vs. tau which deviates significantly from a straight line when F(infinity) is in error by as little as 5%. A scheme for evaluating D by linear analysis is presented. It is also shown that the linear reciprocal plot provides a simple method for detecting flow or multiple diffusion coefficients and for establishing conditions (data precision, differences in multiple diffusion coefficients, magnitude of flow rate compared to lateral diffusion) under which flow or multiple diffusion coefficients can be detected. These aspects are discussed in some detail.

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We have developed a new detection technology that uses resonance light scattering (RLS) particles as labels for analyte detection in a wide range of formats including immuno and DNA probe type of assays in solution, solid phase, cells, and tissues. When a suspension of nano sized gold or silver particles is illuminated with a ®ne beam of white light, the scattered light has a clear (not cloudy) color that depends on composition and particle size. This scattered light can be used as the signal for ultrasensitive analyte detection. The advantages of gold particles as detection labels are that (a) their light producing power is equivalent to more than 500,000¯uorescein molecules, (b) they can be detected at concentrations as low as 10 À15 M in suspension by eye and a simple illuminator, (c) they do not photobleach, (d) individual particles can be seen in a simple student microscope with dark ®eld illumination, (e) color of scattered light can be changed by changing particle size or composition for multicolor multiplexing, and (f) they can be conjugated with antibodies, DNA probes, ligands, and protein receptors for speci®c analyte detection. These advantages allow for ultra-senstive analyte detection with easiness of use and simple and relatively inexpensive instrumentation. We have shown that our RLS technology can indeed be used for ultra-sensitive detection in a wide range of applications including immuno and DNA probe assays in solution and solid phases, detection of cell surface components and in situ hybridization in cells and tissues. Most of the assay formats described in this article can be adapted for drug fast throughput screening.

Hemoglobin quenching of the fluorescence intensity of 12-(9-anthroyl)stearic acid (AS) embedded in the red blood cell membrane occurs through an energy transfer mechanism and can be used to measure the binding of hemoglobin to the membrane. The binding of hemoglobin to red cell membranes was found to be reversible and electrostatic in nature. Using a theory of energy transfer based on Förster formulation, the quantitative data for the binding were derived. The number of binding sites was found to be 1.4 +/- 0.2 X 10(6) molecules per cell and the binding constant was 0.85 X 10(8) M-1.

Fluorescein is a complex fluorophore that can exist in one or more of four different prototropic forms (cation, neutral, dianion, and monoanion) depending on pH. In the pH range 6-10, only the dianion and monanion forms are important. In a previous article, we showed by steady-state fluorescein measurements that an excited fluorescein molecule displays excited-state proton transfer reactions which interconvert the monoanion and dianion forms. However, we found that these reactions can occur only in the presence of a suitable proton donor-acceptor buffer such as phosphate buffer. Assuming that, at 1 M phosphate buffer concentration, the excited-state proton exchange reaction of fluorescein rapidly equilibrates during the lifetime of fluorescein, we were able to fit quantitatively steady-state fluorescence intensity vs pH titration graphs to a relatively simple reaction model. In this article, we use nanosecond emission (decay time) methods to study the excitedstate proton reactions of fluorescein in the pH range 6-10 and in the presence of a phosphate buffer concentration. Fluorescein is a challenging fluorophore for the study of excited-state proton reactions because of the strong overlap of the absorption and emission spectra of the monoanion and dianion forms of fluorescein. However by recording nanosecond emission graphs and using methods of analysis of high precision, we have been able to test kinetic mechanisms and evaluate the specific rate constants for the excited-state proton reactions as well as the lifetimes of the monoanion and dianion. Using these values for lifetimes and rate constants, we discuss the process of equilibration in the excited-state and derive expressions which allow us to predict how quickly the excited-state reactions can reach equilibrium. Moreover, we use the above kinetic and spectral parameters to calculate steady-state fluorescence intensity F S vs pH at 1 M phosphate buffer concentration and compare this theoretically calculated graph with the experimental graph.