Fluorescence resonance energy transfer (FRET) from donor to acceptor molecules is one of the most powerful techniques for monitoring structure and dynamics. This is because FRET has a strong spatial dependence with angstroms resolution. This dependence includes the simplest case of a random distribution of acceptors for which an analytical solution exists for the fluorescence impulse response I(t). However, in general the acceptor distribution function p(r) is not random and a unique solution cannot be found for I(t). In many important applications of FRET eg in proteins, the simple random treatment is quite inappropriate and yet the information concerning conformation changes is preserved in p(r). One approach, which as been applied to the problem of determining p(r), is to make some assumptions as to its form eg Gaussian and then try to use this to describe I(t).

Recently, there has been substantial interest in reducing the levels of toxic heavy metals in wastewater effluents from activities such as shipyards. Of particular interest is copper, which comprises tens of percent by weight of the hundreds of pounds of antifouling paint coating the bottom of a large vessel, but which is toxic to commercially important shellfish at sub-part per billion levels. As a result wastewater effluents must be monitored closely with sensor(s) capable of rapidly and accurately detecting excess copper in time to prevent release. We have pursued a fluorescence-based biosensing approach to obtain sub-ppb sensitivity for Cu(II) and immunity from interference from other cations abundant in sea water, such as Ca, Mg, and Sr. Our approach uses a protein, apocarbonic anhydrase II, as a very sensitive and selective ligand for Cu(II) which transduces the (reversible) binding of the metal as a change in fluorescence intensity, lifetime, or anisotropy, the first two of which may be conveniently measured through optical fiber. Thus we have been able to measure sub-ppb levels of Cu added to sea water, and to characterize the speciation of the Cu(II) to some degree, due to the presence of other ligands.

We developed a new method of glucose sensing using inactive forms of glucose oxidase from Aspergillus niger and glucose dehydrogenase from the thermophilic microorganism Thermoplasma acidophilum. Glucose oxidase was rendered inactive by removal of the FAD cofactor. The resulting apo- glucose oxidase still binds glucose as observed from a decrease in its intrinsic tryptophan fluorescence. 8- Anilino-1-naphthalene sulfonic acid (ANS) was found to bind spontaneously to apo-glucose oxidase as seen from an enhancement of the ANS fluorescence. The steady state intensity of the bound ANS decreased 25% upon binding of glucose, and the mean lifetime of the bound ANS decreased about 40%. These spectral changes occurred with a midpoint from 10 to 20 mM glucose, which is comparable to the K_o of holo-glucose oxidase. These results suggest that apo- glucose oxidase can be used as a reversible non-consuming sensor for glucose.

The array biosensor has been developed for simultaneous analysis of multiple samples for multiple analytes. A patterned array of capture antibodies is immobilized on the surface of a planar waveguide and a sandwich immunoassay conducted using a cocktail of fluorescent tracer antibodies. Upon excitation of the fluorescent label using a 635 nm diode laser, a CCD camera detects the pattern of fluorescent antigen:antibody complexes on the sensor surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. The assays are fast, sensitive, and specific.

Enhanced metal cluster fluorescence absorption enables the transduction of molecular binding events into visually detectable signals. Biochips based on nanocluster-enhanced absorption were developed as a new technology for gaining complex bioanalytical information for use in Genomics, Proteomics and clinical diagnostics.

In this work we characterized seven dyes that are one-photon excited and fluoresce in the far-red region of the visible spectrum, five of which became available only recently, as to their suitability as labels on the red emission channel in a two-color FSC method. Spectroscopic properties and binding to albumin were studied. In addition, when one of these was used as the label in a first-pass high-throughput screen with single-color fluorescence polarization detection, its foreseen advantage of avoiding the excitation of interfering background autofluorescence from compounds in libraries was deemed valuable after a comparison of two complete high-throughput screens of a chemical compound library at Abbott.

Fluorescence has been investigated with respect to new methods for monitoring protein uptake by silica, with particular attention being given to haze forming proteins and foam proteins present in beer. These are of particular interest to the brewing industry as an important aspect of the brewing process is the prevention of chill haze formation. This is necessary in order to maintain the clarity of the beer and to extend the shelf life. Chill haze, which is a result of the interaction of certain proteins with some polyphenols, can be prevented by the removal of one or both of these constituents.

Analytical systems based on immunochemistry are largely used in medical diagnostics and in biotechnology. There is a significant pressure to develop the present assay formats to become easier to use, faster, and less reagent consuming. Further developments towards high density array--like multianalyte measurement systems would be valuable. To this aim we have studied the applicability of fluorescence resonance energy transfer and time-resolved fluorescence resonance energy transfer in immunoassays on microspots and in microwells. We have used engineered recombinant antibodies detecting the pentameric protein CRP as a model analyte system, and tested different assay formats. We describe also the construction of a time-resolved scanning epifluorometer with which we could measure the FRET interaction between the slow fluorescence decay from europium chelates and its energy transfer to the rapidly decaying fluorophore Cy5.

A novel spectrofluorometer equipped with two photo-detectors has been developed based on a conventional spectrofluorometer with a view to measuring both bioluminescence and its related fluorescence originating from bacterial luciferase reaction. The configuration of the two monochromator-photomultiplier systems is to be opposite to each other.

Instilled particle burdens of uniform green-yellow fluorescent microspheres phagocytized by rat alveolar (lung) macrophages and cell viability, as indexed by propidium iodide uptake (red fluorescence), were assessed using flow cytometry. Since the spectral emission from phagocytized microspheres partially overlapped the propidium iodide fluorescence and interfered with the conventional flow cytometric measurement of damaged/dead cells without subtractive compensation, this caused errors when estimating the percentage of non-viable, propidium iodide positive, phagocytic macrophages. The interference was eliminated by employing phase-sensitive detection in the red fluorescence measurement channel based on differences in lifetimes between the fluorescent microspheres and propidium iodide. In addition, intrinsic cellular autofluorescence, whose fluorescence lifetime is approximately the same as the phagocytized microspheres, also was eliminated in the measurement process. Since there was no detectable spectral interference of propidium iodide in the green fluorescence (particle phagocytosis) measurement channel, conventional fluorescence detection was employed.

We present the Lambert Instruments Fluorescence Lifetime Attachment LIFA. LIFA enables easy to use and affordable microscopy and macroscopic FLIM. The system implements the homodyne detection scheme for measuring the fluorescence lifetime in each pixel of the image. The microscopy system features an ultra bright LED illuminator, the LI-(mu) Cam intensified CCD camera a high frequency signal generator. The illuminator replaces the excitation light source of a standard fluorescence microscopy, while the LI-(mu) CAM intensified CCD camera is attached to the photo-port. Both the illuminator and the intensifier are modulated at a frequency up to 100 MHz at a series of phase differences. The lifetime image is calculated from the series of images on a personal computer.

The Fluorescence Lifetime Module (LIMO) from Nikon is a versatile device for time domain Fluorescence Lifetime Imaging. The high efficiency and high sample speed make LIMO ideal for the fast acquisition of fluorescence lifetime images using a confocal scanning laser microscope or multi- photon excitation microscope. We employed LIMO to acquire fluorescence lifetime images with the Nikon PCM 2000 CLSM.

We investigated the time-resolved fluorescence spectra of tissue structural proteins elastin and collagen I for different excitation wavelengths to assess the usefulness of multispectral excitation for characterization of fluorescent structural proteins in tissue. Laser excitation pulses (4 ns FWHM) at wavelengths between 337 nm and 422 nm were directed to the samples with a multifiber fluorescence probe. The fluorescence emission was measured with a photomultiplier and deconvoluted from the emission pulse with an algorithm based on the Laguerre expansion of kernels technique. A multiexponential approximation of the intrinsic fluorescence decay was computed using a global approach in which the decay constants were held fixed across wavelengths of emission.

In this paper thyroid samples have been analyzed by fluorescent technique and characterization of the spectral response has been performed by studying both emission and excitation fluorescence spectra. The measurements have been performed by using a double monochromator spectrofluorometer. The nature of the medium containing the tissue sample has resulted to be of great importance in eliminating spurious effects not related to the sample itself. Observations fulfilled on a number of samples will be reported and comparison between healthy tissue and tumor tissue will be discussed.

The genetically-modified binding proteins calmodulin, the phosphate binding protein, the sulfate binding protein, and the galactose/glucose binding protein have been successfully employed as biosensing elements for the detection of phenothiazines, phosphate, sulfate, and glucose, respectively. Mutant proteins containing unique cysteine residues were utilized in the site-specific labeling of environment-sensitive fluorescent probes. Changes in the environment of the probes upon ligand-induced conformational changes of the proteins result in changes in fluorescence intensity.

We demonstrate a new application of multiphoton excitation of fluorescence; the measurement of silica particle growth during sol-gel polymerization. Recently we have reported the use of fluorescence anisotropy as an alternative approach to nm particle sizing using conventional techniques such as small angle light, neutron or x-ray scattering. Advantages of our approach include near angstrom resolution, minimal interference from the gel network as well as the additional benefits of providing microviscosity and hydrodynamic information. Fluorescence anisotropy applied to particle sizing in colloids in general is still in its infancy, but in this paper we show using probes fluorescein and rhodamine 6G, under conditions unsuitable for one-photon excitation, can be successful with multiphoton excitation.

Dopamine hydrochloric acid salt in aqueous solution was excited at 266 nm Al₂O₃:Ti laser and the sufficient fluorescence emission peaking at 330 nm was detected with a streak camera. The fluorescence decay curve was fitted by 1- exponential functions, with the lifetime of approximately 0.80 ns. The influence of deep-UV laser excitation on cells is also discussed for the direct observation of dopamine in the living cells. In addition, it is needed to detect the dopamine fluorescence in the living cell sensitively, and separately from emission of other fluorescent species. When instrumental arrangement and time-resolved spectral analysis can make it possible to solve such problems, direct visualization of the secretion process of individual cells will be achieved by the laser-induced native fluorescence imaging microscopy, without using any additional fluorescent probes. This quantitative imaging technique will provide a useful noninvasive approach for the study of dynamic cellular changes and the understanding of the molecular mechanisms of information transporting processes.