Four-Photon Excitation of 2,2'-Dimethyl--terphenyl.

We report the emission spectra, intensity decays, and anisotropy decay of 2,2'-dimethyl--terphenyl (DMT) with four-photon excitation. When excited with a fs Ti:Sapphire laser the emission intensity of DMT was found to depend on the third power of the incident intensity for excitation of 783 nm, and on the fourth power of the incident intensity for excitation at 882 nm. Surprisingly, at the highest value incident power, the emission intensity for four-photon excitation was about 10-fold less than with three-photon excitation.

Radiative decay engineering. 2. Effects of Silver Island films on fluorescence intensity, lifetimes, and resonance energy transfer.

Metallic surfaces can have unusual effects on fluorophores such as increasing or decreasing the rates of radiative decay and the rates of resonance energy transfer (RET). In the present article we describe the effects of metallic silver island films on the emission spectra, lifetimes, and energy transfer for several fluorophores. The fluorophores are not covalently coupled to the silver islands so that there are a range of fluorophore-to-metal distances.

Intrinsic fluorescence from DNA can be enhanced by metallic particles.

High sensitivity detection of DNA is essential for genomics. The intrinsic fluorescence from DNA is very weak and almost all methods for detecting DNA rely on the use of extrinsic fluorescent probes. We show that the intrinsic emission from DNA can be enhanced many-fold by spatial proximity to silver island films.

On the effect of sodium dodecyl sulfate on the structure of beta-galactosidase from Escherichia coli. A fluorescence study.

An understanding of the structure-function relationship of proteins under different chemical-physical conditions is of fundamental importance for an understanding of their structure and function in cells. In this paper we report the effects of sodium dodecyl sulfate and temperature on the structure of beta-galactosidase from Escherichia coli, as monitored by fluorescence spectroscopy. The structure of the protein was studied in the temperature range of 10-60 degrees C in the absence and presence of sodium dodecyl sulfate by frequency-domain measurement of the intrinsic fluorescence intensity and anisotropy decays.

Multiphoton Ligand-Enhanced Excitation of Lanthanides.

We describe multiphoton excitation of the lanthanides europium (Eu) and terbium (Tb) when these ions are complexed with nucleic acids, proteins, and fluorescent chelators. In all cases excitation occurs by multiphoton absorption of the sensitizers. For the nucleotide GDP and an oligonucleotide with several guanines, the sensitized emission of Tb excited at 776 nm indicated a three-photon process.

Multiphoton excitation of lanthanides.

Without requiring chelation by sensitizers, a method to detect lanthanides by multiphoton excitation is reported. This suggests the use of lanthanides as calcium analogues in multiphoton microscopy. The spectrum shows the normalized two-photon excitation emission spectra (λex = 796 nm) of Eu(3+) with a resolution of about 8 nm.

Calcium-induced flexibility changes in the troponin C-troponin I complex.

The contraction of vertebrate striated muscle is modulated by Ca(2+) binding to the regulatory protein troponin C (TnC). Ca(2+) binding causes conformational changes in TnC which alter its interaction with the inhibitory protein troponin I (TnI), initiating the regulatory process. We have used the frequency domain method of fluorescence resonance energy transfer (FRET) to measure distances and distance distributions between specific sites in the TnC-TnI complex in the presence and absence of Ca(2+) or Mg(2+).

Donor fluorescence decay analysis for energy transfer in double-helical DNA with various acceptor concentrations.

We studied fluorescence resonance energy transfer between donors and acceptors bound to double-helical DNA. The donor Hoechst 33258 binds to the minor groove of DNA and the acceptor propidium iodide (PI) is an intercalator. The time-resolved donor decays were measured in the frequency domain.

A protein biosensor for lactate.

Blood lactate is a clinically valuable diagnostic indicator. In this preliminary report we describe a protein biosensor for L-lactate based on beef heart lactate dehydrogenase (LDH). LDH was noncovalently labeled with 8-anilino-1-naphthalene sulfonic acid (ANS).

A thermophilic apoglucose dehydrogenase as nonconsuming glucose sensor.

Blood glucose is a clinically important analytes for diabetic health care. In this preliminary report we describe a protein biosensor for d-glucose based on a thermostable glucose dehydrogenase. The glucose dehydrogenase was noncovalently labeled with 8-anilino-1-naphthalene sulfonic acid (ANS).

Novel fluorescence sensing methods for high throughput screening.

We describe two new methods of fluorescence sensing for use in high throughput screening (HTS). Modulation sensing transforms analyte-dependent intensity changes into a change in the low-frequency modulation signal. Polarization sensing transforms an intensity change into a change in polarization.

High Throughput Screening with Multiphoton Excitation.

Fluorescence detection is extensively used in high throughput screening. In HTS there is a continuous migration toward higher density plates and smaller sample volumes. In the present report we describe the advantages of two-photon or multiphoton excitation for HTS.

Time-resolved spectral observations of cadmium-enriched cadmium sulfide nanoparticles and the effects of DNA oligomer binding.

We measured the steady-state and time-resolved fluorescence spectral properties of cadmium-enriched nanoparticles (CdS-Cd2+). These particles displayed two emission maxima, at 460 and 580 nm. The emission spectra were independent of excitation wavelength.

Polarization-based oxygen sensor.

A new approach to oxygen sensing based on the luminescence polarization observed from a novel type of sensor is described. The oxygen sensor consists of an oxygen-sensitive silicone film containing tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) chloride [Ru(dpp)3Cl2] and an oxygen-insensitive film of Styryl 7 in poly(vinyl alcohol). Polarizers are used to select orthogonally polarized emission components from Ru(dpp)3Cl2 and Styryl 7.

Effects of diffusion on energy transfer in solution using a microsecond decay time rhenium metal-ligand complex as the donor.

We used resonance energy transfer and frequency-domain fluorometry to measure slow donor to acceptor diffusion in viscous media. The frequency-domain RET data were analyzed using a new numerical algorithm for predicting the donor intensity decay in the presence of diffusion occurring within the donor decay time. By the use of a rhenium metal-ligand complex as a microsecond decay time donor we were able to measure mutual donor-to-acceptor diffusion coefficients as low as 2 × 10 cm/s.

Perturbation of Conformational Dynamics of from by Temperature and Sodium Dodecyl Sulfate.

is a protein found in cuticlin, the insoluble residue of the cuticles of the nematode . It contains the CUT-1-like domain which is shared by members of a novel family of components of extracellular matrices. The monomeric form of contains a single tryptophan residue.

End-to-end diffusion on the microsecond timescale measured with resonance energy transfer from a long-lifetime rhenium metal-ligand complex.

We measured the end-to-end diffusion coefficient of an alkyl chain-linked donor-acceptor pair using the time-resolved frequency-domain decay of the donor. The donor was a rhenium metal-ligand complex with a mean decay time ranging from 2.1 to 7.

Background suppression in frequency-domain fluorometry.

Gated detection is often used in time-domain measurements of long-lived fluorophores for suppression of interfering short-lived autofluorescence. However, no direct method has been available for gated detection and background suppression when using frequency-domain fluorometry. We describe a direct method for real-time suppression of autofluorescence in frequency-domain fluorometry.

Intensity measurements in scattering media.

We describe a new method which allows quantitative measurements of fluorescence intensity in highly scattering media. The measurement principle is based on observing the emission from both the fluorophore of interest with a nanosecond decay time and of a reference fluorophore which displays a much longer microsecond lifetime. The reference fluorophore is placed on rather than in the sample to mimic a sensing device with the long lifetime reference held against the skin.

Resonance energy transfer study using a rhenium metal-ligand lipid conjugate as the donor in a model membrane.

We measured steady state and time-resolved resonance energy transfer between donors and acceptors in model membranes. The donor was a long lifetime rhenium-lipid complex, which displayed a mean lifetime of 1 microsecond and lifetime components as long as 3 microseconds in the labeled DOPC membranes. The transfer efficiencies were found to be substantially larger than those predicted without consideration of lateral diffusion.

The beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: enzyme activity and conformational dynamics at temperatures above 100 degrees C.

Enzymes from thermophilic organisms are stable and active at temperatures which rapidly denature mesophilic proteins. However, there is not yet a complete understanding of the structural basis of their thermostability and thermoactivity since for each protein there seems to exist special networks of interactions that make it stable under the desired conditions. Here we have investigated the activity and conformational dynamics above 100 degrees C of the beta-glycosidase isolated from the hyperthermophilic archaeon Sulfolobus solfataricus.

Polarization-based sensing of glucose using an oriented reference film.

We describe a new approach to glucose sensing using polarization measurements in the presence of a stretch-oriented reference film. The method relies on measurement of the polarized emission from the reference film and of a fluorophore which changes intensity in response to glucose. A glucose-sensitive fluorescent signal was provided by the glucose/galactose binding protein from E.